Abstract: Over the last decade, researchers in our laboratory have engineered and developed several series of genetically encoded voltage-sensitive fluorescent proteins (VSFPs) by molecular fusion of a voltage-sensing domain operand with different fluorescent reporter proteins. These genetically encoded VSFPs have been shown to provide a reliable optical report of membrane potential from targeted neurons and muscle cells in culture or in living animals. However, these various reporters also exhibit discrepancies in both their voltage-sensing and targeting properties that are essentially related to the intrinsic characteristics of the fluorescent reporter proteins. It is therefore important carefully to select the sensor that is most appropriate for the particular question being investigated experimentally. Here we examine the current state of this subfield of optogenetics, address current limitations and challenges, and discuss what is likely to be feasible in the near future.
Abstract: Cerebellar Purkinje neurones (PNs) express high levels of the plasma membrane calcium ATPase, PMCA2, a transporter protein critical for the clearance of calcium from excitable cells. Genetic deletion of one PMCA2 encoding gene in heterozygous PMCA2 knock-out (PMCA2(+/-) mice enabled us to determine how PMCA2 influences PN calcium regulation without the complication of the severe morphological changes associated with complete PMCA2 knock-out (PMCA2(-/-) in these cells. The PMCA2(+/-) cerebellum expressed half the normal levels of PMCA2 and this nearly doubled the time taken for PN dendritic calcium transients to recover (mean fast and slow recovery times increased from 70 ms to 110 ms and from 600 ms to 1100 ms). The slower calcium recovery had distinct consequences for PMCA2(+/-) PN physiology. The PNs exhibited weaker climbing fibre responses, prolonged outward Ca(2+)-dependent K(+) current (mean fast and slow recovery times increased from 136 ms to 192 ms and from 595 ms to 1423 ms) and a slower mean frequency of action potential firing (7.4 Hz compared with 15.8 Hz). Our findings were consistent with prolonged calcium accumulation in the cytosol of PMCA2(+/-) Purkinje neurones. Although PMCA2(+/-) mice exhibited outwardly normal behaviour and little change in their gait pattern, when challenged to run on a narrow beam they exhibited clear deficits in hindlimb coordination. Training improved the motor performance of both PMCA2(+/-) and wild-type mice, although PMCA2(+/-) mice were always impaired. We conclude that reduced calcium clearance perturbs calcium dynamics in PN dendrites and that this is sufficient to disrupt the accuracy of cerebellar processing and motor coordination.
Abstract: Cortical information processing relies on synaptic interactions between diverse classes of neurons with distinct electrophysiological and connection properties. Uncovering the operational principles of these elaborate circuits requires the probing of electrical activity from selected populations of defined neurons. Here we show that genetically encoded voltage-sensitive fluorescent proteins (VSFPs) provide an optical voltage report from targeted neurons in culture, acute brain slices and living mice. By expressing VSFPs in pyramidal cells of mouse somatosensory cortex, we also demonstrate that these probes can report cortical electrical responses to single sensory stimuli in vivo. These protein-based voltage probes will facilitate the analysis of cortical circuits in genetically defined cell populations and are hence a valuable addition to the optogenetic toolbox.
Abstract: A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.
Abstract: Cerebellar Purkinje neurons receive synaptic inputs from three different sources: the excitatory parallel fibre and climbing fibre synapses as well as the inhibitory synapses from molecular layer stellate and basket cells. These three synaptic systems use distinct mechanisms in order to generate Ca(2+) signals that are specialized for specific modes of neurotransmitter release and post-synaptic signal integration. In this review, we first describe the repertoire of Ca(2+) regulatory mechanisms that generate and regulate the amplitude and timing of Ca(2+) fluxes during synaptic transmission and then explore how these mechanisms interact to generate the unique functional properties of each of the Purkinje neuron synapses.
Abstract: The deep cerebellar nuclei (DCN) are a major hub in the cerebellar circuitry but the functional classification of their neurons is incomplete. We have previously characterized three cell groups in the lateral cerebellar nucleus: large non-GABAergic neurons and two groups of smaller neurons, one of which express green fluorescence protein (GFP) in a GAD67/GFP mouse line and is therefore GABAergic. However, as a substantial number of glycinergic and glycine/GABA co-expressing neurons have been described in the DCN, this classification needed to be refined by considering glycinergic neurons. To this end we took advantage of a glycine transporter isoform 2 (GlyT2)-eGFP mouse line that allows identification of GlyT2-expressing, presumably glycinergic neurons in living cerebellar slices and compared their electrophysiological properties with previously described DCN neuron populations. We found two electrophysiologically and morphologically distinct sets of GlyT2-expressing neurons in the lateral cerebellar nucleus. One of them showed electrophysiological similarity to the previously characterized GABAergic cell group. The second GlyT2+ cell population, however, differed from all other so far described neuron types in DCN in that the cells (1) are intrinsically silent in slices and only fire action potentials upon depolarizing current injection and (2) have a projecting axon that was often seen to leave the DCN and project in the direction of the cerebellar cortex. Presence of this so far undescribed DCN neuron population in the lateral nucleus suggests a direct inhibitory pathway from the DCN to the cerebellar cortex.
Abstract: This mini-symposium aims to provide an integrated perspective on recent developments in optogenetics. Research in this emerging field combines optical methods with targeted expression of genetically encoded, protein-based probes to achieve experimental manipulation and measurement of neural systems with superior temporal and spatial resolution. The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes. The first generation of genetically encoded calcium reporters, fluorescent proteins, and neural activators has already had a great impact on neuroscience. Now, a second generation of voltage reporters, neural silencers, and functionally extended fluorescent proteins hold great promise for continuing this revolution. In this review, we will evaluate and highlight the limitations of presently available optogenic tools and discuss where these technologies and their applications are headed in the future.
Abstract: The deep cerebellar nuclei (DCN) are at the center of the cerebellum not only anatomically but also functionally. Classical anatomical studies have described different types of DCN neurons according to their expression of various marker proteins, but only recently have we begun to characterize these different cell types according to their electrophysiological properties. These efforts have benefited greatly from the availability of transgenic mouse lines that express green fluorescent protein under the control of the glutamic acid decarboxylase (GAD67) and glycine transporter (GlyT2) promoters, which are markers for GABAergic and glycinergic neurons, respectively. These studies have identified several types of neurons within the lateral cerebellar nuclei, each of which exhibits distinct active membrane properties. In addition to their differential use of neurotransmitters (glutamate, GABA, or glycine), these cell types also receive and provide synaptic information from different sources and to different targets.
Abstract: The cerebellum expresses one of the highest levels of the plasma membrane Ca(2+) ATPase, isoform 2 in the mammalian brain. This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex; i.e. the Purkinje neurons (PNs). Here we review recent evidence, including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2 (PMCA2) knockout mouse, to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour. These studies have also revealed that deletion of PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development, they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.
Abstract: Genetic deletion of the plasma membrane calcium ATPase type 2 (PMCA2), a calcium transporter protein, is associated with an overtly ataxic phenotype in mice. PMCA2 is expressed at high levels in cerebellar Purkinje neurons (PNs) where functional integrity is essential for normal cerebellar function. Indeed, loss of PN function accompanies cerebellar ataxia in humans and mouse models. In the ataxic PMCA2 knockout (PMCA2-/-) mouse the ability of the PNs to control their cytosolic calcium levels was severely impaired; basal calcium levels were high and calcium recovery kinetics slow. Whole cell patch clamp recordings from PMCA2-/- PNs revealed that they possessed hyperpolarised membrane potentials, reduced frequency and increased irregularity of spontaneous action potential firing, curtailed complex spikes and sustained calcium-dependent outward K+ currents. We propose that these alterations limit pathological excursions in PN cytosolic calcium as an aid to survival but that they are insufficient to prevent loss of functional cerebellar output.
Abstract: Plasticity at synapses between parallel fiber (PF) and Purkinje neurons (PN) is widely accepted as a cellular model for certain forms of cerebellar learning. Although PF-PN synapses are known to express bidirectional long-term plasticity at the postsynaptic site, long-term plasticity at the presynaptic site is currently limited to potentiation of the synapses. In this paper, we report on presynaptically expressed PF long-term depression (preLTD) that is observed when presynaptically expressed PF long-term potentiation (preLTP) is pharmacologically prevented. PF preLTD is most efficiently induced by 4 Hz PF stimulation and requires activation of cannabinoid CB1 receptors. Our results indicate that, during preLTD induction, endocannabinoids are released in an NMDA receptor-dependent, but not mGlu1 receptor-dependent, fashion. We conclude that bidirectional plasticity mechanisms exist for both presynaptic and postsynaptic components of cerebellar learning.
Abstract: The N-terminus of Ciona intestinalis (Ci-VSP) is a voltage-sensing domain (VSD) controlling the activity of a phosphatase domain on the C terminus. By replacing the phosphatase domain with a tandem of fluorescent proteins, CFP and YFP, a family of fluorescence resonance energy transfer-based, genetically encoded voltage-sensing fluorescent protein (VSFP) was created. VSFP2.3, one of the latest versions of this family, showed large changes in YFP emission upon changes in membrane potential with CFP excitation when expressed in Xenopus laevis oocytes. The time course of the fluorescence has two components: the fast component correlates with the time course of sensing current produced by the charge movement, while the slow component is at least one order-of-magnitude slower than the sensing current. This suggests that the tandem of fluorescent proteins reports a secondary conformational transition of the VSD which resembles the relaxation of the VSD of Ci-VSP described in detail for the Ci-VSP. This observation indicates that the relaxation of the VSD of VSFP2.3 is a global conformational change that encompasses the entire S4 segment.
Abstract: Olfactory glomeruli are the loci where the first odor-representation map emerges. The glomerular layer comprises exquisite local synaptic circuits for the processing of olfactory coding patterns immediately after their emergence. To understand how an odor map is transferred from afferent terminals to postsynaptic dendrites, it is essential to directly monitor the odor-evoked glomerular postsynaptic activity patterns. Here we report the use of a transgenic mouse expressing a Ca(2+)-sensitive green fluorescence protein (GCaMP2) under a Kv3.1 potassium-channel promoter. Immunostaining revealed that GCaMP2 was specifically expressed in mitral and tufted cells and a subpopulation of juxtaglomerular cells but not in olfactory nerve terminals. Both in vitro and in vivo imaging combined with glutamate receptor pharmacology confirmed that odor maps reported by GCaMP2 were of a postsynaptic origin. These mice thus provided an unprecedented opportunity to analyze the spatial activity pattern reflecting purely postsynaptic olfactory codes. The odor-evoked GCaMP2 signal had both focal and diffuse spatial components. The focalized hot spots corresponded to individually activated glomeruli. In GCaMP2-reported postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both individual glomerular response amplitude and the total number of activated glomeruli. Furthermore, the GCaMP2 response displayed a fast time course that enabled us to analyze the temporal dynamics of odor maps over consecutive sniff cycles. In summary, with cell-specific targeting of a genetically encoded Ca(2+) indicator, we have successfully isolated and characterized an intermediate level of odor representation between olfactory nerve input and principal mitral/tufted cell output.
Abstract: Fluorescent protein voltage sensors are recombinant proteins that are designed as genetically encoded cellular probes of membrane potential using mechanisms of voltage-dependent modulation of fluorescence. Several such proteins, including VSFP2.3 and VSFP3.1, were recently reported with reliable function in mammalian cells. They were designed as molecular fusions of the voltage sensor of Ciona intestinalis voltage sensor containing phosphatase with a fluorescence reporter domain. Expression of these proteins in cell membranes is accompanied by additional dynamic membrane capacitance, or "sensing capacitance", with feedback effect on the native electro-responsiveness of targeted cells. We used recordings of sensing currents and fluorescence responses of VSFP2.3 and of VSFP3.1 to derive kinetic models of the voltage-dependent signaling of these proteins. Using computational neuron simulations, we quantitatively investigated the perturbing effects of sensing capacitance on the input/output relationship in two central neuron models, a cerebellar Purkinje and a layer 5 pyramidal neuron. Probe-induced sensing capacitance manifested as time shifts of action potentials and increased synaptic input thresholds for somatic action potential initiation with linear dependence on the membrane density of the probe. Whereas the fluorescence signal/noise grows with the square root of the surface density of the probe, the growth of sensing capacitance is linear. We analyzed the trade-off between minimization of sensing capacitance and signal/noise of the optical read-out depending on kinetic properties and cellular distribution of the probe. The simulation results suggest ways to reduce capacitive effects at a given level of signal/noise. Yet, the simulations indicate that significant improvement of existing probes will still be required to report action potentials in individual neurons in mammalian brain tissue in single trials.
Abstract: Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP) reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs), referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.
Abstract: BACKGROUND: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin. RESULTS: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons. CONCLUSION: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.
Abstract: The relatively simple and highly modular circuitry of the cerebellum raised expectations decades ago that a realistic computational model of cerebellar circuit operations would be feasible, and prove insightful for unraveling cerebellar information processing. To this end, the biophysical properties of most cerebellar cell types and their synaptic connections have been well characterized and integrated into realistic single cell models. Furthermore, large scale models of cerebellar circuits that extrapolate from single cell properties to circuit dynamics have been constructed. While the development of single cell models have been constrained by microelectrode recordings, guidance and validation as these models are scaled up to study network interactions requires an experimental methodology capable of monitoring cerebellar dynamics at the population level. Here we review the potential of optical imaging techniques to serve this purpose.
Abstract: Electrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically encoded voltage-sensitive fluorescent proteins by molecular fusion of the voltage-sensing domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) to red-shifted fluorescent protein operands. We show how these indicator proteins convert voltage-dependent structural rearrangements into a modulation of fluorescence output and demonstrate their applicability for optical recording of individual or simultaneous electrical signals in cultured hippocampal neurons at single-cell resolution without temporal averaging.
Abstract: Genetically-encoded optical probes for membrane potential hold the promise of monitoring electrical signaling of electrically active cells such as specific neuronal populations in intact brain tissue. The most advanced class of these probes was generated by molecular fusion of the voltage sensing domain (VSD) of Ci-VSP with a fluorescent protein (FP) pair. We quantitatively compared the three most advanced versions of these probes (two previously reported and one new variant), each involving a spectrally distinct tandem of FPs. Despite these different FP tandems and dissimilarities within the amino acid sequence linking the VSD to the FPs, the amplitude and kinetics of voltage dependent fluorescence changes were surprisingly similar. However, each of these fluorescent probes has specific merits when considering different potential applications.
Abstract: Genetically encoded fluorescent protein (FP) voltage sensors are promising tools for optical monitoring of the electrical activity of cells. Over the last decade, several designs of fusion proteins have been explored and some of them have proven to be sensitive enough to record membrane voltage transients from single mammalian cells. Most prominent are the families of voltage sensitive fluorescent proteins (VSFPs) that utilize the voltage sensor domain (VSD) of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP). The voltage sensitivity of the fluorescence readout of these previously reported membrane potential indicators is achieved either via a change in the efficiency of fluorescence resonance energy transfer between two FP spectral variants or via modulation in the fluorescence intensity of a single FP. Here, we report our exploration on a third VSFP design principle based on circularly permuted fluorescent protein (cpFP) variants. Using circularly permuted EGFP derived from GCaMP2 and two newly generated circularly permuted variants of the far-red emitting protein named mKate, we generated and characterized a series of voltage-sensitive probes wherein the cpFPs were fused to the VSD of Ci-VSP. The most promising variants were based on circularly permuted mKate with new N- and C-termini given by residues 180 and 182. Even so their voltage sensitivity was relatively modest, they constitute a proof of principle for this novel protein design.
Abstract: Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state. The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission. Here, we report experiments designed to investigate mGluR kinetics in real-time. We inserted either CFP or YFP into the second intracellular loop of mGluR1beta. When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC(50) of 7.5 microM. The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators. These results suggest that the FRET response reports active conformations of mGluR1 dimers. The solution exchange at the cell membrane was optimized for voltage-clamped cells by recording the current induced by co-application of 30 mM potassium. When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (approximately 50 ms). During long-lasting applications, no desensitization was observed. In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms. Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC(50) and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission.
Abstract: Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.
Abstract: The deep cerebellar nuclei (DCN) are the final integrative units of the cerebellar network. The strongest single afferent to the DCN is formed by GABAergic Purkinje neuron axons whose synapses constitute the majority of all synapses in the DCN, with their action strongly regulating the intrinsic activity of their target neurons. Although this is well established, it remains unclear whether all DCN cell groups receive a functionally similar inhibitory input. We previously characterized three types of mouse DCN neurons based on the expression of glutamic acid decarboxylase isoform 67 (GAD67), their active membrane properties and morphological features. Here we describe the GABAergic synapses in these cell groups and show that spontaneous GABAergic synaptic activity can be seen in all three cell types. Since the majority of DCN neurons fire action potentials spontaneously at high frequencies both in vivo and in vitro, we expected that spontaneous GABAergic synaptic activities mediated by intra-DCN synaptic connections could be uncovered by their sensitivity to TTX. However, TTX had little effect on spontaneous synaptic activity. It seems, therefore that functional GABAergic connectivity within the DCN is sparse and/or weak at least under our experimental conditions. Even though present in all cell types, the spontaneous GABAergic events showed significant differences between the cell types. The synaptic currents in GABAergic cells had lower amplitude, lower frequency and slower kinetics than those of non-GABAergic cells. These differences could not be sufficiently explained by considering only cell size differences or a differential GABA(A)-receptor alpha-subunit composition. Rather, the main differentiating factor appears to be the dendritic localization of GABAergic synapses in the GABAergic cells.
Abstract: Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.
Abstract: Interest in non-invasive methods for optical probing of neuronal electrical activity has been ongoing for several decades and methods for imaging the activity of single or multiple individual neurons in networks composed of thousands of neurons have been developed. Most widely used are techniques that use organic chemistry-based dyes as indicators of calcium and membrane potential. More recently a new generation of probes, genetically encoded fluorescent protein sensors, have emerged for use by physiologists studying the operation of neuronal circuits. In this review we describe the advance of these emerging optical techniques and compare them with more conventional approaches.
Abstract: Metabotropic glutamate receptors, in contrast to ionotropic glutamate receptors, do not form ion channels but instead affect intracellular chemical messenger systems. They couple via GTP-binding proteins ("G-proteins") to a variety of effectors such as ion channels and thus give glutamate, the major excitatory transmitter in the CNS, the ability to modulate processes involved in excitatory synaptic transmission. Therefore, excitatory synaptic transmission is regulated not only by the conventional GABAergic but also by the glutamatergic mechanisms themselves. Many metabotropic glutamate receptors are localized outside the immediate vicinity of transmitter release sites, thereby setting specific requirements for their activation, such as cooperation between synapses, burst activity, and glial involvement in the regulation of ambient glutamate levels.
Abstract: Very fast oscillations (VFO; > 75 Hz) occur transiently in vivo, in the cerebellum of mice genetically modified to model Angelman syndrome, and in a mouse model of fetal alcohol syndrome. We recently reported VFO in slices of mouse cerebellar cortex (Crus I and II of ansiform and paramedian lobules), either in association with gamma oscillations (approximately 40 Hz, evoked by nicotine) or in isolation [evoked by nicotine in combination with gamma-aminobutyric acid (GABA)(A) receptor blockade]. The experimental data suggest a role for electrical coupling between Purkinje cells (blockade of VFO by drugs reducing gap junction conductance and spikelets in some Purkinje cells); and the data suggest the specific involvement of Purkinje cell axons (because of field oscillation maxima in the granular layer). We show here that a detailed network model (1000 multicompartment Purkinje cells) replicates the experimental data when gap junctions are located on the proximal axons of Purkinje cells, provided sufficient spontaneous firing is present. Unlike other VFO models, most somatic spikelets do not correspond to axonal spikes in the parent axon, but reflect spikes in electrically coupled axons. The model predicts gating of VFO frequency by g(Na) inactivation, and experiments prolonging this inactivation time constant, with beta-pompilidotoxin, are consistent with this prediction. The model also predicts that cerebellar VFO can be explained as an electrically coupled system of axons that are not intrinsic oscillators: the electrically uncoupled cells do not individually oscillate (in the model) and axonal firing rates are much lower in the uncoupled state than in the coupled state.
Abstract: Both cerebellum and neocortex receive input from the somatosensory system. Interaction between these regions has been proposed to underpin the correct selection and execution of motor commands, but it is not clear how such interactions occur. In neocortex, inputs give rise to population rhythms, providing a spatiotemporal coding strategy for inputs and consequent outputs. Here, we show that similar patterns of rhythm generation occur in cerebellum during nicotinic receptor subtype activation. Both gamma oscillations (30-80 Hz) and very fast oscillations (VFOs, 80-160 Hz) were generated by intrinsic cerebellar cortical circuitry in the absence of functional glutamatergic connections. As in neocortex, gamma rhythms were dependent on GABA(A) receptor-mediated inhibition, whereas VFOs required only nonsynaptically connected intercellular networks. The ability of cerebellar cortex to generate population rhythms within the same frequency bands as neocortex suggests that they act as a common spatiotemporal code within which corticocerebellar dialog may occur.
Abstract: Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.
Abstract: Huntington's disease (HD) is a fatal neurodegenerative disorder. Despite a tremendous effort to develop therapeutic tools in several HD models, there is no effective cure at present. Acidosis has been observed previously in cellular and in in vivo models as well as in the brains of HD patients. Here we challenged HD models with amiloride (Ami) derivative benzamil (Ben), a chemical agent used to rescue acid-sensing ion channel (ASIC)-dependent acidotoxicity, to examine whether chronic acidosis is an important part of the HD pathomechanism and whether these drugs could be used as novel therapeutic agents. Ben markedly reduced the huntingtin-polyglutamine (htt-polyQ) aggregation in an inducible cellular system, and the therapeutic value of Ben was successfully recapitulated in the R6/2 animal model of HD. To reveal the mechanism of action, Ben was found to be able to alleviate the inhibition of the ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. More importantly, we were able to demonstrate that blocking the expression of a specific isoform of ASIC (asic1a), one of the many molecular targets of Ben, led to an enhancement of UPS activity and this blockade also decreased htt-polyQ aggregation in the striatum of R6/2 mice. In conclusion, we believe that chemical compounds that target ASIC1a or pharmacological alleviation of UPS inhibition would be an effective and promising approach to combat HD and other polyQ-related disorders.
Abstract: Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.
Abstract: The validation of the selective, potent and systemically active non-competitive mGlu1 antagonists YM-298198 and JNJ16259685 in a physiological functional assay will facilitate elucidation of this receptor's role in brain function and as a potential drug target.
Abstract: In the brain, neuronal activation triggers an increase in cerebral blood flow (CBF). Here, we use two animal models and several techniques (two-photon imaging of CBF and neuronal calcium dynamics, intracellular and extracellular recordings, local pharmacology) to analyze the relationship between neuronal activity and local CBF during odor stimulation in the rodent olfactory bulb. Application of glutamate receptor antagonists or tetrodotoxin directly into single rat olfactory glomeruli blocked postsynaptic responses but did not affect the local odor-evoked CBF increases. This suggests that in our experimental conditions, odor always activates more than one glomerulus and that silencing one of a few clustered glomeruli does not affect the vascular response. To block synaptic transmission more widely, we then superfused glutamate antagonists over the surface of the olfactory bulb in transgenic G-CaMP2 mice. This was for two reasons: (1) mice have a thin olfactory nerve layer compared to rats and this will favor drug access to the glomerular layer, and (2) transgenic G-CaMP2 mice express the fluorescent calcium sensor protein G-CaMP2 in mitral cells. In G-CaMP2 mice, odor-evoked, odor-specific, and concentration-dependent calcium increases in glomeruli. Superfusion of glutamate receptor antagonists blocked odor-evoked postsynaptic calcium signals and CBF responses. We conclude that activation of postsynaptic glutamate receptors and rises in dendritic calcium are major steps for neurovascular coupling in olfactory bulb glomeruli.
Abstract: Cerebellar Purkinje cells (PCs) receive synaptic input from numerous parallel fibers (PFs) and from a single climbing fiber (CF). At both types of synapses, fast synaptic transmission is mediated by AMPA receptors, while at PF synapses burst activity can additionally recruit metabotropic glutamate receptors (mGluRs) that mediate a slow depolarizing potential. Here, we show that mGluR-activated slow potentials can be evoked throughout the dendrite by CF-evoked complex spike firing in the presence of an mGluR agonist. The CF-triggered mGluR potential was not only blocked by an mGluR antagonist but also when the CF-induced Ca(2+) transient was blocked by an AMPA receptor antagonist, suggesting the possibility that the slow potential can be activated by the simultaneous occurrence of agonist binding at mGluRs and a CF-evoked Ca(2+) transient. In turn, these CF-triggered slow mGluR potentials enhance the complex spike-associated calcium signals throughout the dendrite. Moreover, they provide a mechanism by which CFs can modulate the simple spike frequency of PCs.
Abstract: BACKGROUND: Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. RESULTS: Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. CONCLUSIONS AND SIGNIFICANCE: This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously.
Abstract: Plasma membrane Ca2+ ATPase 2 (PMCA2) is a fast, highly effective mechanism to control resting cytosolic Ca2+ and Ca2+ excursions in neurons and other excitable cells. The strong expression of PMCA2 in the cerebellum and the cerebellar behavioral deficits presented by PMCA2-/- knock-out mice all point to its importance for cerebellar circuit dynamics. Here, we provide direct functional evidence for the influence of presynaptic PMCA2-mediated Ca2+ extrusion for short-term plasticity at cerebellar parallel fiber to Purkinje neuron synapses. Dramatic structural alterations to the Purkinje neurons in the absence of PMCA2 also suggest a strong influence of this fast PMCA2 isoform for development and maintenance of cerebellar function.
Abstract: Genetically encoded fluorescent calcium indicator proteins provide the potential to monitor activity from genetically specified target cells without a need for single cell resolution. Here we report the use of transgenic mice expressing the fluorescent calcium indicator protein GCaMP2 in cerebellar granule cells to image parallel fiber activity transcranially in vivo. We demonstrated reliable measurements of calcium transients from beams of parallel fibers in response to electrical stimulation in the molecular layer through the intact skull. These parallel fiber calcium transients differed from intrinsic postsynaptic autofluorescence signals in their faster kinetics and resistance to blockers of synaptic transmission. Finally, we used 2P laser-scanning microscopy to demonstrate reliable measurements of calcium transients from beams of parallel fibers at high spatial resolution in living mice. We expect that genetically targeted fluorescent calcium indicator proteins along with optical imaging techniques will be instrumental for the construction of macroscopic and microscopic maps of the function of specific brain circuits.
Abstract: Plasma membrane calcium ATPase isoforms (PMCAs) are expressed in a wide variety of tissues where cell-specific expression provides ample opportunity for functional diversity amongst these transporters. The PMCAs use energy derived from ATP to extrude submicromolar concentrations of intracellular Ca2+ ([Ca2+]i) out of the cell. Their high affinity for Ca2+ and the speed with which they remove [Ca2+]i depends upon splicing at their carboxy (C)-terminal site. Here we provide biochemical and functional evidence that a brain-specific, C-terminal truncated and therefore fast variant of PMCA2, PMCA2a, has a role at hippocampal CA3 synapses. PMCA2a was enriched in forebrain synaptosomes, and in hippocampal CA3 it colocalized with the presynaptic marker proteins synaptophysin and the vesicular glutamate transporter 1, but not with the postsynaptic density protein PSD-95. PMCA2a also did not colocalize with glutamic acid decarboxylase-65, a marker of GABA-ergic terminals, although it did localize to a small extent with parvalbumin-positive presumed inhibitory terminals. Pharmacological inhibition of PMCA increased the frequency but not the amplitude of mEPSCs with little effect on mIPSCs or paired-pulse depression of evoked IPSCs. However, inhibition of PMCA activity did enhance the amplitude and slowed the recovery of paired-pulse facilitation (PPF) of evoked EPSCs. These results indicated that fast PMCA2a-mediated clearance of [Ca2+]i from presynaptic excitatory terminals regulated excitatory synaptic transmission within hippocampal CA3.
Abstract: Plasticity of synaptic transmission between parallel fiber (PF) and Purkinje neurons (PNs) is widely accepted as a cellular model for certain forms of cerebellar learning. Whereas the signaling cascades involved in postsynaptically expressed bidirectional long-term changes at PF-PN synapses are well investigated, data on presynaptically expressed long-term potentiation (LTP) are incomplete and controversial. Here we used transgenic mice that express a fluorescent protein Ca2+ sensor in PFs to demonstrate LTP of PF presynaptic Ca2+ transients after PF stimulation with 120 pulses at 4 Hz. Potentiation of the presynaptic Ca2+ transients correlated with the expression of simultaneously recorded LTP of PF-PN synaptic transmission and was suppressed by a protein kinase A inhibitor. Moreover, this presynaptically expressed form of LTP clearly required activation of an NMDA receptor/nitric oxide pathway, in contrast with the majority of previous reports. Blockade of NMDA receptors did not affect the PF Ca2+ transients induced during 4 Hz stimulation, indicating that the NMDA receptors required for the induction of presynaptic PF LTP are not localized in PFs.
Abstract: The deep cerebellar nuclei (DCN) integrate inputs from the brain stem, the inferior olive, and the spinal cord with Purkinje cell output from cerebellar cortex and provide the major output of the cerebellum. Despite their crucial function in motor control and learning, the various populations of neurons in the DCN are poorly defined and characterized. Importantly, differences in electrophysiological properties between glutamatergic and GABAergic cells of the DCN have been largely elusive. Here, we used glutamate decarboxylase (GAD) 67-green fluorescent protein (GFP) knock-in mice to unambiguously identify GABAergic (GAD-positive) and non-GABAergic (GAD-negative, most likely glutamatergic) neurons of the DCN. Morphological analysis of DCN neurons patch-clamped with biocytin-containing electrodes revealed a significant overlap in the distributions of the soma sizes of GAD-positive and GAD-negative cells. Compared with GAD-negative DCN neurons, GAD-positive DCN neurons fire broader action potentials, display stronger frequency accommodation, and do not reach as high firing frequencies during depolarizing current injections. Furthermore, GAD-positive cells display slower spontaneous firing rates and have a more shallow frequency-to-current relationship than the GAD-negative cells but exhibit a longer-lasting rebound depolarization and associated spiking after a transient hyperpolarization. In contrast to the rather homogeneous population of GAD-positive cells, the GAD-negative cells were found to consist of two distinct populations as defined by cell size and electrophysiological features. We conclude that GABAergic DCN neurons are specialized to convey phasic spike rate information, whereas tonic spike rate is more faithfully relayed by the large non-GABAergic cells.
Abstract: Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.
Abstract: The voltage-gated potassium channels Kv3.1 and Kv3.3 are expressed in several distinct neuronal subpopulations in brain areas known to be involved in motor control such as cortex, basal ganglia and cerebellum. Depending on the lack of Kv3.1 or Kv3.3 channel subunits, mutant mice show different Kv3-null allele-dependent behavioral alterations that include constitutive hyperactivity, sleep loss, impaired motor performance and, in the case of the Kv3.1/Kv3.3 double mutant, also severe ataxia, tremor and myoclonus (Espinosa et al. 2001, J Neurosci 21, 6657-6665, Genes, Brain Behav 3, 90-100). The lack of Kv3.1 channel subunits is mainly responsible for the constitutively increased locomotor activity and for sleep loss, whereas the absence of Kv3.3 subunits affects cerebellar function, in particular Purkinje cell discharges and olivocerebellar system properties (McMahon et al. 2004, Eur J Neurosci 19, 3317-3327). Here, we describe two sensitive and non-invasive tests to reliably quantify normal and abnormal motor functions, and we apply these tests to characterize motor dysfunction in Kv3-mutant mice. In contrast to wildtype and Kv3.1-single mutants, Kv3.3-single mutants and Kv3 mutants lacking three and four Kv3 alleles display Kv3-null allele-dependent gait alterations. Although the Kv3-null allele-dependent gait changes correlate with reduced motor performance, they appear to not affect the training-induced improvement of motor performance. These findings suggest that altered cerebellar physiology in the absence of Kv3.3 channels is responsible for impaired motor task execution but not motor task learning.
Abstract: Purkinje neurons spontaneously generate action potentials in the absence of synaptic drive and thereby exert a tonic, yet plastic, input to their target cells in the deep cerebellar nuclei. Purkinje neurons express two ionic currents with biophysical properties that are specialized for high-frequency firing: resurgent sodium currents and potassium currents mediated by Kv3.3. How these ionic currents determine the intrinsic activity of Purkinje neurons has only partially been understood. Purkinje neurons from mutant mice lacking Kv3.3 have a reduced rate of spontaneous firing. Dynamic-clamp recordings demonstrated that normal firing rates are rescued by inserting artificial Kv3 currents into Kv3.3 knock-out Purkinje neurons. Numerical simulations indicated that Kv3.3 increases the spontaneous firing rate via cooperation with resurgent sodium currents. We conclude that the rate of spontaneous action potential firing of Purkinje neurons is controlled by the interaction of Kv3.3 potassium currents and resurgent sodium currents.
Abstract: Fast synaptic transmission between olfactory receptor neurons and mitral cells (MCs) is mediated through AMPA and NMDA ionotropic glutamate receptors. MCs also express high levels of metabotropic glutamate receptor 1 (mGluR1) whose functional significance is less understood. Here we characterized a slow mGluR1-mediated potential that was evoked by high-frequency (100-Hz) olfactory nerve (ON) stimulation in the presence of NBQX and D-APV, blockers of ionotropic glutamate receptors, and that was associated with a local Ca2+ transient in the MC dendritic tuft. High-frequency ON stimulation in the presence of NBQX and D-APV also evoked a slow, nearly 2-Hz oscillation of MC membrane potential that was abolished by the mGluR1 antagonist LY367385 (50 microM). Both mGluR slow potential and slow oscillation persisted in the presence of gabazine (10 microM), a GABA(A) receptor antagonist, and intracellular QX-314 (10 mM), a Na+ channel blocker. In contrast to a slow mGluR1 potential in cerebellar Purkinje neurons, the MC mGluR1 potential was not depressed by SKF96365 (< or =250 microM) and thus is likely not mediated by TRPC1 cation channels, nor was it potentiated by an elevation of intracellular Ca2+ level. Imaging with the Na+ indicator SBFI revealed a Na+ transient in the MC dendrite accompanying the mGluR1 slow potential. We conclude that the MC mGluR1 potential triggered by glutamate released from the ON supports oscillations and synchronizations of MCs associated within one glomerulus.
Abstract: During the past few decades, optical methods for imaging activity in networks composed of thousands of neurons have been developed. These techniques rely mainly on organic-chemistry-based dyes as indicators of Ca(2+) and membrane potential. However, recently a new generation of probes, genetically encoded fluorescent protein sensors, has emerged for use by physiologists studying the operation of neuronal circuits. We critically review the development of these new probes, and analyze objectives and experimental conditions in which classical probes are likely to prevail and where the fluorescent protein sensors will open paths to previously unexplored territories of functional neuroimaging.
Abstract: Olfactory receptor neuron axons form the olfactory nerve (ON) and project to the glomerular layer of the olfactory bulb, where they form excitatory synapses with terminal arborizations of the mitral cell (MC) tufted primary dendrite. Clusters of MC dendritic tufts define olfactory glomeruli, where they involve in complex synaptic interactions. The computational function of these cellular interactions is not clear. We used patch-clamp electrophysiology combined with whole field or two-photon Ca2+ imaging to study ON stimulation-induced Ca2+ signaling at the level of individual terminal branches of the MC primary dendrite in mice. ON-evoked subthreshold excitatory postsnaptic potentials induced Ca2+ transients in the MC tuft dendrites that were spatially inhomogeneous, exhibiting discrete "hot spots." In contrast, Ca2+ transients induced by backpropagating action potentials occurred throughout the dendritic tuft, being larger in the thin terminal dendrites than in the base of the tuft. Single ON stimulation-induced Ca2+ transients were depressed by the NMDA receptor antagonist D-aminophosphonovaleric acid (D-APV), increased with increasing stimulation intensity, and typically showed a prolonged rising phase. The synaptically induced Ca2+ signals reflect, at least in part, dendrodendritic interactions that support intraglomerular coupling of MCs and generation of an output that is common to all MCs associated with one glomerulus.
Abstract: The synapses formed by the olfactory nerve (ON) convey sensory information to olfactory glomeruli, the first stage of central odor processing. Morphological and behavioral studies suggest that glomerular odor processing is plastic in neonate rodents. However, long-term synaptic plasticity, a cellular correlate of functional and structural plasticity, has not yet been demonstrated in this system. Here, we report that ON-->mitral cell (MC) synapses of 5- to 8-d-old mice express long-term depression (LTD) after brief low-frequency ON stimulation. Pharmacological techniques and imaging of presynaptic calcium signals demonstrate that ON-MC LTD is expressed presynaptically and requires the activation of metabotropic glutamate receptors but does not require fast synaptic transmission. LTD at the ON--> MC synapse is potentially relevant for the establishment, maintenance, and experience-dependent refinement of odor maps in the olfactory bulb.
Abstract: Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5' regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-D-aspartate receptors.
Abstract: Synapses formed by the olfactory nerve (ON) provide the source of excitatory synaptic input onto mitral cells (MC) in the olfactory bulb. These synapses, which relay odor-specific inputs, are confined to the distally tufted single primary dendrites of MCs, the first stage of central olfactory processing. beta-adrenergic modulation of electrical and chemical signaling at these synapses may be involved in early odor preference learning. To investigate this possibility, we combined electrophysiological recordings with calcium imaging in olfactory bulb slices prepared from neonatal rats and mice. Activation of ON-MC synapses induced postsynaptic potentials, which were associated with large postsynaptic calcium transients. Neither electrical nor calcium responses were affected by beta-adrenergic agonists or antagonist. Immunocytochemical analysis of MCs and their tufted dendrites revealed clear immunoreactivity with antibodies against alpha1A (Cav2.1, P/Q-type) and alpha1B (Cav2.2, N-type), but not against alpha1C (Cav1.2, L-type) or alpha1D (Cav1.3, L-type) calcium channel subunits. Moreover, nimodipine, a blocker of L-type calcium channels, had no effect on either electrical or calcium signaling at ON-MC synapses. In contrast to previous evidence, we concluded that in neonatal rats and mice (P5-P8), mitral cells do not express significant amounts of L-type calcium channels, the calcium channel type that is often targeted by beta-adrenergic modulation. The absence of beta-adrenergic modulation on either electrical or calcium signaling at ON-MC synapses of neonatal rats and mice excludes the involvement of this mechanism in early odor preference learning.
Abstract: Long term potentiation (LTP) at hippocampal Schaffer collateral-CA1 synapses involves an early and a late phase, where only the latter is sensitive to protein synthesis inhibitors. Here we characterized the dynamics of protein synthesis associated with the induction of L-LTP using a transgenic mouse model in which a cAMP responsive element (CRE)-regulated promoter drives production of an enhanced yellow fluorescent protein (eYFP). We found that eYFP fluorescence increased after less than 30 min following L-LTP induction. Application of transcription and translation suppressors and the NMDA receptor antagonist D-AP5 inhibited the L-LTP and prevented the rise in eYFP levels. The early-phase of LTP was not affected by inhibiting protein synthesis.
Abstract: The neuronal components of cortical circuits have been characterized on the basis of their morphological and functional properties, and further refined by correlation of marker proteins with particular cell types. This latter approach has been very fruitful for GABA-containing neurons, but comparable diagnostic markers for subpopulations of excitatory pyramidal cells have been more elusive. An emerging new approach consists of transgenic mice that express fluorescent proteins under the control of promoters that are active in specific cell types. Here, we analyzed a line of transgenic mice that carries a transgene consisting of regulatory sequences of the potassium channel Kv3.1 and enhanced yellow fluorescent protein (EYFP). In these mice, a set of neurons in neocortical layer 5 expresses high levels of the transgenic marker protein. EYFP-expressing, and nonexpressing layer 5 cells were easily identified in living tissue under conditions suitable for patch-clamp electrophysiology. By using immunolabeling, retrograde Fast Blue labeling and electrophysiological recordings with biocytin injections, we identified the fluorescent neurons as a population of pyramidal cells with distinct morphological and electrophysiological properties when compared with nonfluorescent neighboring layer 5 pyramidal cells. The most prominent morphological difference between these two populations was a much smaller number of apical oblique dendrites in EYFP-positive as compared with the EYFP-negative cells. The most prominent electrophysiological feature was a steady spike frequency adaptation in EYFP-positive cells, whereas EYFP-negative cells responded to a depolarizing current injection with a closely spaced spike doublet followed by constant frequency firing. The in vivo labeled transgenic mice provide an experimental tool for further functional differentiation of these populations of layer 5 pyramidal cells.
Abstract: We report an activity-induced green fluorescence signal observed when mouse cerebellar slices were illuminated with blue light and parallel fibre-Purkinje cell synapses were activated. The optical signal consisted of an initial increase in fluorescence that peaked within 1-2 s after the onset of stimulation, followed by a long lasting (40 s) transient decrease in fluorescence. Single or tetanic electrical stimuli applied to the molecular layer elicited 'beam-shaped' fluorescence changes along the trajectory of parallel fibres. These signals reported activation of Purkinje cells as they were depressed by antagonists of ionotropic and metabotropic glutamate receptors at Purkinje cells and correlated with Purkinje cell spiking activity. Optical responses induced by direct pharmacological activation of glutamate receptors were reduced by a calcium-free extracellular medium, consistent with the hypothesis that they reflect metabolic activity due to an increased intracellular calcium load associated with neuronal activation. We used these intrinsic fluorescence signals to address the question of whether granule cells excite Purkinje cells only locally via the ascending branches of their axons, or more widespread along the parallel fibre trajectory. White matter stimulation of the mossy fibres also elicited a beam-like fluorescence change along the trajectory of parallel fibres. Simultaneous imaging and extracellular recording demonstrated the association between the beam-like fluorescence signal and Purkinje cell spiking. This non-invasive imaging technique supports the notion that parallel fibre activity, evoked either locally or through the mossy fibre-granule cell pathway, can activate postsynaptic Purkinje cells along more than 3 mm of the parallel fibre trajectory.
Abstract: Double-mutant mice (DKO) lacking the two voltage-gated K(+) channels Kv3.1 and Kv3.3 display a series of phenotypic alterations that include ataxia, myoclonus, tremor and alcohol hypersensitivity. The prominent cerebellar expression of mRNAs encoding Kv3.1 and Kv3.3 subunits raised the question as to whether altered electrical activity resulting from the lack of these K(+) channels might be related to the dramatic motor changes. We used the tremorogenic agent harmaline to probe mutant mice lacking different K(+) channel alleles for altered olivocerebellar circuit properties. Harmaline induced the characteristic 13-Hz tremor in wildtype mice (WT); however, no tremor was observed in DKO suggesting that the ensemble properties of the olivocerebellar circuitry are altered in the absence of Kv3.1 and Kv3.3 subunits. Harmaline induced tremor in Kv3.1-single mutants, but it was of smaller amplitude and at a lower frequency indicating the participation of Kv3.1 subunits in normal olivocerebellar system function. In contrast, harmaline tremor was virtually absent in Kv3.3-single mutants indicating an essential role for Kv3.3 subunits in tremor induction by harmaline. Immunohistochemical staining for Kv3.3 showed clear expression in the somata and proximal dendrites of Purkinje cells and in their axonal projections to the deep cerebellar nuclei (DCN). In DCN, both Kv3.1 and Kv3.3 subunits are expressed. Action potential duration is increased by approximately 100% in Purkinje cells from Kv3.3-single mutants compared to WT or Kv3.1-single mutants. We conclude that Kv3.3 channel subunits are essential for the olivocerebellar system to generate and sustain normal harmaline tremor whereas Kv3.1 subunits influence tremor amplitude and frequency.
Abstract: The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.
Abstract: Optical imaging of electrical activity using voltage-sensitive dyes has been envisaged for many years as a powerful method to investigate multineuronal representation of information processing in brain tissue. This article describes the advent of novel genetically targeted voltage-sensitive fluorescent proteins. This new class of membrane voltage sensors overcomes previous limitations related to the nonselective staining of membranes associated with conventional voltage-sensitive dyes. Here, we discuss the methodology, applications, and potential advantages of this novel technique.
Abstract: A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.
Abstract: Micelacking both Kv3.1 and both Kv3.3 K+ channel alleles display severe motor deficits such as tremor, myoclonus, and ataxic gait. Micelacking one to three alleles at the Kv3.1 and Kv3.3 loci exhibit in an allele dose-dependent manner a modest degree of ataxia. Cerebellar granule cells coexpress Kv3.1 and Kv3.3 K+ channels and are therefore candidate neurons that might be involved in these behavioral deficits. Hence, we investigated the synaptic mechanisms of transmission in the parallel fiber-Purkinje cell system. Action potentials of parallel fibers were broader in mice lacking both Kv3.1 and both Kv3.3 alleles and in mice lacking both Kv3.1 and a single Kv3.3 allele compared with those of wild-type mice. The transmission of high-frequency trains of action potentials was only impaired at 200 Hz but not at 100 Hz in mice lacking both Kv3.1 and Kv3.3 genes. However, paired-pulse facilitation (PPF) at parallel fiber-Purkinje cell synapses was dramatically reduced in a gene dose-dependent manner in mice lacking Kv3.1 or Kv3.3 alleles. Normal PPF could be restored by reducing the extracellular Ca2+ concentration indicating that increased activity-dependent presynaptic Ca2+ influx, at least in part caused the altered PPF in mutant mice. Induction of metabotropic glutamate receptor-mediated EPSCs was facilitated, whereas longterm depression was not impaired but rather facilitated in Kv3.1/Kv3.3 double-knockout mice. These results demonstrate the importance of Kv3 potassium channels in regulating the dynamics of synaptic transmission at the parallel fiber-Purkinje cell synapse and suggest a correlation between short-term plasticity at the parallel fiber-Purkinje cell synapse and motor performance.
Abstract: Maturation of specific neuronal connections in the mature nervous system includes elimination of redundant synapses formed earlier during development. In the cerebellum of adult animals, each Purkinje cell (PC) is innervated by a single climbing fiber (CF). In early postnatal development each PC is innervated by multiple CFs and elimination of synapses formed by supernumerary CFs occurs until monoinnervation is established at around postnatal day 20 (P20) in mice. It is not clear whether multiple CFs, or only a single CF, translocate from the cell body of immature PCs to the developing dendrite and, in case several CFs translocate, whether they share or segregate their innervation fields. To localize CF innervation fields, we imaged changes in postsynaptic sodium concentration resulting from CF-mediated postsynaptic currents. We found that more than one CF translocates from an innervation field on the cell body of the PC to the developing dendrite and that these CFs share rather than segregate their innervation fields. We concluded that both the soma and the proximal dendrite of the PC are territories of competition for the developing CFs and that the overlapping of their termination fields may be the prerequisite for a local process of elimination of all but one CF, as previously demonstrated in the developing neuromuscular junction.
Abstract: At the cerebellar parallel fiber-Purkinje cell synapse, isolated presynaptic activity induces fast excitatory postsynaptic currents via ionotropic glutamate receptors while repetitive, high-frequency, presynaptic activity can also induce a slow excitatory postsynaptic current that is mediated by metabotropic glutamate receptors (mGluR1-EPSC). Here we investigated the involvement of glutamate uptake in the expression of the mGluR1-EPSC. Inhibitors of glutamate uptake led to a large increase of the mGluR1-EPSC. D-aspartate (0.4 mM) and L(-)-threo-3-hydroxyaspartate (0.4 mM) increased the mGluR1-EPSC approximately 4.5 and approximately 9-fold, respectively, while dihydrokainic acid (1 mM), had no significant effect on the mGluR1-EPSC. D-aspartate (0.4 mM) shifted the concentration-response curve of the depression of the mGluR1-EPSC by the low-affinity mGluR1 antagonist (S)-a-Methyl-4-carboxyphenylglycine [(S)-MCPG] to higher concentrations and decreased the stimulus intensity and the number of necessary stimuli to evoke an mGluR1-EPSC. Depression of the mGluR1-EPSC by rapid pressure application of (S)-MCPG at varying time intervals after tetanic stimulation of the parallel fibers indicated that the glutamate concentration in the peri- and extrasynaptic space decayed with time constants of 36 and 316 ms under control conditions and with inhibition of glutamate uptake, respectively. These results show that expression of the slow mGluR-mediated excitatory postsynaptic current is controlled by glutamate transporter activity. Thus in contrast to fast glutamatergic synaptic transmission, metabotropic glutamate receptor-mediated transmission is critically dependent on the activity and capacity of glutamate uptake.
Abstract: Early olfactory preference learning in rat pups occurs when novel odors are paired with reinforcing tactile stimulation that activate the noradrenergic locus coeruleus. Pairing of odor and a noradrenergic agonist in the olfactory bulb is both necessary and sufficient for odor preference learning. This suggests the memory change occurs in the olfactory bulb. Previous electrophysiological experiments demonstrated that odor preference training induces an increase in the field excitatory postsynaptic potential to olfactory nerve input and an alteration, after training, in glomerular [14C]2- deoxyglucose uptake and in single-unit responses of principal cells. We investigate here whether, 24 h after olfactory preference training, there is an alteration in intrinsic optical signals at the glomerular level. Six-day-old rat pups were trained, as previously, for a peppermint odor preference. Trained pups and control littermates were subjected to imaging of odor-induced intrinsic optical signals 1 day after the training session. Trained pups exhibited significantly larger responses to the peppermint compared with untrained littermates previously exposed to the same odor. The response of trained pups to a control odor (amyl acetate) was, however, not significantly different from that of untrained littermates. These observations demonstrate that odor preference memory can be read-out by optical imaging techniques.
Abstract: Glial cells are traditionally regarded as elements for structural support and ionic homeostasis, but have recently attracted attention as putative integral elements of the machinery involved in synaptic transmission and plasticity. Here, we demonstrate that calcium-binding protein S100B, which is synthesized in considerable amounts in astrocytes (a major glial cell subtype), modulates long-term synaptic plasticity. Mutant mice devoid of S100B developed normally and had no detectable abnormalities in the cytoarchitecture of the brain. These mutant mice, however, had strengthened synaptic plasticity as identified by enhanced long-term potentiation (LTP) in the hippocampal CA1 region. Perfusion of hippocampal slices with recombinant S100B proteins reversed the levels of LTP in the mutant slices to those of the wild-type slices, indicating that S100B might act extracellularly. In addition to enhanced LTP, mutant mice had enhanced spatial memory in the Morris water maze test and enhanced fear memory in the contextual fear conditioning. The results indicate that S100B is a glial modulator of neuronal synaptic plasticity and strengthen the notion that glial-neuronal interaction is important for information processing in the brain.
Abstract: Over the last two decades, glutamate has been established as the main excitatory neurotransmitter in the mammalian brain. Glutamate released from synapses activates ion channel-forming receptors at postsynaptic cells and consequently mediates fast postsynaptic potentials. These receptors are termed ionotropic glutamate receptors (iGluRs). The subsequent discovery of metabotropic glutamate receptors (mGluRs) revealed that glutamate can also mediate slow synaptic potentials, modulate ion channels, and directly couple to GTP binding proteins. In contrast to the iGluRs, the mGluRs possess seven transmembrane domains and a large intracellular C-terminus that involves interactions with a variety of other intracellular signaling systems. Eight functionally distinct mGluR subtypes are known to be localized to specific neuron types at presynaptic and/or postsynaptic membranes. Their physiological functions involve the generation of slow excitatory and inhibitory synaptic potentials, modulation of synaptic transmission, synaptic integration, and plasticity. The classical role of glutamate as a fast excitatory synaptic transmitter was largely extended by mGluRs acting as a neuromodulator and even as an activator of inhibitory mechanisms at certain synapses.
Abstract: During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.
Abstract: Metabotropic glutamate receptors (mGluRs) are a family of proteins that have seven transmembrane segments and that couple to G proteins. They differ from ionotropic glutamate receptors in that they do not form ion channels but instead affect intracellular chemical messenger systems. Eight genes coding for different subtypes of mGluRs have been identified to date and numbered accordingly in the order in which the cDNAs were cloned. Based on their principal signal-transduction capabilities in recombinant expression systems and sequence similarities, the family of mGluR subtypes is subdivided into three groups. Group 1 mGluRs (consisting of mGluR1 and 5) functionally couple to phospholipase C and affect the IP3/Ca2+ signaling pathway. The subtypes of group 2 (mGluR2 and 3) and group 3 (mGluR4, 6 7 and 8) inhibit adenylate cyclase and, thereby, mediate a decrease in cAMP concentration. All mGluR subtypes are found in the cerebellar cortex with the exception of mGluR6 which is exclusively expressed in the retina. At the parallel fiber-Purkinje cell synapses mGluR1 is localized in the peri- and extra-synaptic membrane of Purkinje cells. The main focus of this review deals with the functions of this postsynaptically localized mGluR1. These functions include (i) mediation of an inward current and a slow excitatory postsynaptic potential, and (ii) a role in induction of parallel fiber-Purkinje cell long-term depression. We discuss the mechanism underlying the mGluR1-mediated postsynaptic current as well as current theories on the role of mGluR1 in parallel fiber-Purkinje cell long-term depression.
Abstract: The metabotropic glutamate receptor 1 (mGluR(1)) plays a fundamental role in postnatal development and plasticity of ionotropic glutamate receptor-mediated synaptic excitation of cerebellar Purkinje cells. Synaptic activation of mGluR(1) by brief tetanic stimulation of parallel fibers evokes a slow excitatory postsynaptic current and an elevation of intracellular calcium concentration ([Ca2+](i)) in Purkinje cells. The mechanism underlying these responses has not been identified yet. Here we investigated the responses to synaptic and direct activation of mGluR(1) using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca2+](i) in mouse Purkinje cells. Following pharmacological block of ionotropic glutamate receptors, two to six stimuli applied to parallel fibers at 100 Hz evoked a slow inward current that was associated with an elevation of [Ca2+](i). Both the inward current and the rise in [Ca2+](i) increased in size with increasing number of pulses albeit with no clear difference between the minimal number of pulses required to evoke these responses. Application of the mGluR(1) agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) by means of short-lasting (5-100 ms) pressure pulses delivered through an agonist-containing pipette positioned over the Purkinje cell dendrite, evoked responses resembling the synaptically induced inward current and elevation of [Ca2+](i). No increase in [Ca2+](i) was observed with inward currents of comparable amplitudes induced by the ionotropic glutamate receptor agonist AMPA. The 3,5-DHPG-induced inward current but not the associated increase in [Ca2+](i) was depressed when extracellular Na+ was replaced by choline, but, surprisingly, both responses were also depressed when bathing the tissue in a low calcium (0.125 mM) or calcium-free/EGTA solution. Thapsigargin (10 microM) and cyclopiazonic acid (30 microM), inhibitors of sarco-endoplasmic reticulum Ca2+-ATPase, had little effect on either the inward current or the elevation in [Ca2+](i) induced by 3,5-DHPG. Furthermore, the inward current induced by 3,5-DHPG was neither blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy] ethyl-1H-imidazole, an inhibitor of store operated calcium influx, nor by nimodipine or omega-agatoxin, blockers of voltage-gated calcium channels. These electrophysiological and Ca2+-imaging experiments suggest that the mGluR(1)-mediated inward current, although mainly carried by Na+, involves influx of Ca2+ from the extracellular space.
Abstract: Optical imaging of electrical activity has been suggested as a promising approach to investigate the multineuronal representation of information processing in brain tissue. While considerable progress has been made in the development of instrumentation suitable for high-speed imaging, intrinsic or extrinsic dye-mediated optical signals are often of limited use due to their slow response dynamics, low effective sensitivity, toxicity or undefined cellular origin. Protein-based and DNA-encoded voltage sensors could overcome these limitations. Here we report the design and generation of a voltage-sensitive fluorescent protein (VSFP) consisting of a voltage sensing domain of a potassium channel and a pair of cyan and yellow emitting mutants of green fluorescent protein (GFP). In response to a change in transmembrane voltage, the voltage sensor alters the amount of fluorescence resonance energy transfer (FRET) between the pair of GFP mutants. The optical signals respond in the millisecond time-scale of fast electrical signalling and are large enough to allow monitoring of voltage changes at the single cell level.
Abstract: New variants of green fluorescent protein (GFP) can be engineered by circular permutation of their amino acid sequence. We characterized a series of permuted enhanced GFP (PEGFP) with new termini introduced at N144-Y145 and linkers of 1, 3, 5 and 6 residues inserted between G232 and M1, as well as a variant with an extended 7-residues linker between K238 and M1. A minimum linker length of 3 residues was necessary for a functional chromophore to be formed, and linkers exceeding 4 residues yielded almost the same fluorescence quantum yield as enhanced GFP (EGFP). PEGFP exhibited dual-wavelength absorption and fluorescence excitation with peaks at 395 and 490 nm but single-wavelength emission at 512 nm. Fluorescence emission increased with increasing pH for all excitation wavelengths with a pKa of 7.7. Between the pH values of 6 and 8 optical absorption showed an isobestic point at 445 nm. PEGFP rapidly denatured in urea between 50 and 60 degrees C. Renaturation proceeded with a short (approximately 29 s) and a longer (> 150 s) time constant. Transient transfection of HEK293 and HeLa cells revealed the expression dynamics of PEGFP to be similar to that of EGFP. Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging.
Abstract: Optical recording of neuronal activities using voltage-sensitive dyes (VSDs) is a useful method for simultaneous multi-site recording. However, it has been rather difficult to distinguish optical signals from individual, identified cells. We applied the optical recording technique using a high-speed charge coupled device (CCD) imaging system to a teleost thalamic nucleus, corpus glomerulosum (CG) which has a well-defined histological organization and large postsynaptic dendrites. Patch-like dye (di-4-ANEPPS) signals were observed in the dendritic layer of the CG in response to afferent nerve stimulations. These responses were completely blocked by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) receptor antagonist, did not propagate, and the size of the patches were close to that of a single dendritic tip of the 'large cell'. Thus, we found that these patch-like VSD signals most likely represent postsynaptic potentials at individual dendritic tips of the large cells.
Abstract: To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.
Abstract: Cerebellar Purkinje cells express both ionotropic glutamate receptors and metabotropic glutamate receptors. Brief tetanic stimulation of parallel fibers in rat and mouse cerebellar slices evokes a slow excitatory postsynaptic current in Purkinje cells that is mediated by the mGluR1 subtype of metabotropic glutamate receptors. The effector system underlying this mGluR1 EPSC has not yet been identified. In the present study, we recorded the mGluR1 EPSC using the whole-cell patch-clamp technique in combination with microfluorometric recordings of the intracellular sodium concentration ([Na+]i) by means of the fluorescent sodium indicator SBFI. The mGluR1 EPSC was induced by local parallel fibre stimulation in the presence of the ionotropic glutamate receptor antagonists NBQX and D-APV and the GABAA receptor antagonists bicuculline or picrotoxin. The mGluR1 EPSC was associated with an increase in [Na+]i that was restricted to a specific portion of the dendritic tree. The mGluR1 EPSC as well as the increase in [Na+]i were inhibited by the mGluR antagonist S-MCPG. In the presence of NBQX, D-APV, pictrotoxin and TTX, bath application of the selective mGluR agonist 3,5-DHPG induced an elevation in [Na+]i which extended over the whole dendritic field of the Purkinje cell. This finding demonstrates that the mGluR1-mediated postsynaptic current leads to a significant influx of sodium into the dendritic cytoplasm of Purkinje cells and thereby provides a novel intracellular signalling mechanism that might be involved in mGluR1-dependent synaptic plasticity at this synapse.
Abstract: In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.
Abstract: G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.
Abstract: Western blot analysis of protein extracts from rat liver revealed the presence of the mGlu5 receptor, one of the G-protein-coupled receptors activated by glutamate (named "metabotropic glutamate receptors" or mGlu receptors). mGlu5 expression was particularly high in extracts from isolated hepatocytes, where levels were comparable with those seen in the rat cerebral cortex. The presence of mGlu5 receptors in hepatocytes was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) analysis, immunohistochemistry in neonate or adult rat liver, as well as by immunocytochemical analysis in HepG2 hepatoma cells, where the receptor appeared to be preferentially distributed in cell membranes. Interestingly, mGlu1 receptors (which are structurally and functionally homologous to mGlu5 receptors) were never found in rat liver or hepatocytes. In hepatocytes exposed to anoxic conditions for 90 minutes, glutamate, (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and quisqualate, which all activate mGlu5 receptors, accelerated the onset and increased the extent of cell damage, while 4-carboxy-3-hydroxyphenylglycine (4C3HPG), an agonist of mGlu2/3 receptors, was inactive. 2-methyl-6-(2-phenyl-1-ethynyl)-pyridine (MPEP), a novel, noncompetitive, highly selective mGlu5 receptor antagonist, not only abolished the toxic effect of 1S,3R-ACPD, but, unexpectedly, was protective by itself against anoxic damage. This suggests that hepatocytes express mGlu5 receptors and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes.
Abstract: Several metabotropic glutamate receptor (mGluR) subtypes have been identified in the cerebellar cortex that are targeted to different compartments in cerebellar cells. In this study, preembedding immunocytochemical methods for electron microscopy were used to investigate the subcellular distribution of the mGluR1b splice variant in the rat cerebellar cortex. Dendritic spines of Purkinje cells receiving parallel fiber synaptic terminals were immunoreactive for mGluR1b. With a preembedding immunogold method, approximately 25% of the mGluR1b immunolabeling was observed perisynaptically within 60 nm from the edge of the postsynaptic densities. Values of extrasynaptic gold particles beyond the first 60 nm were maintained at between 10 and 18% along the whole intracellular surface of the dendritic spine membranes of Purkinje cells. For comparison, the distribution of mGluR1a was studied. A predominant (approximately 37%) perisynaptic localization of mGluR1a was seen in dendritic spines of Purkinje cells, dropping the extrasynaptic labeling to 15% in the 60-120-nm bin from the edge of the postsynaptic specialization. Our results reveal that mGluR1b and mGluR1a are localized to the same subcellular compartments in Purkinje cells but that the densities of the perisynaptic and extrasynaptic pools were different for both isoforms. The compartmentalization of mGluR1b and mGluR1a might serve distinct requirements in cerebellar neurotransmission.
Abstract: This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.
Abstract: A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a-immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held.
Abstract: Metabotropic glutamate receptors modulate neuronal excitability via a multitude of mechanisms, and they have been implicated in the pathogenesis of neurodegenerative processes. Here we investigated the responses mediated by group I metabotropic glutamate receptors (mGluRs) in dopamine neurons of the rat substantia nigra pars compacta, using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca(2+)](i) and [Na(+)](i). The selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) was bath-applied (20 microM, 30 s to 2 min) or applied locally by means of short-lasting (2-4 s) pressure pulses, delivered through an agonist-containing pipette positioned close to the cell body of the neuron. 3,5-DHPG evoked an inward current characterized by a transient and a sustained component, the latter of which was uncovered only with long-lasting agonist applications. The fast component coincided with a transient elevation of [Ca(2+)](i), whereas the total current was associated with a rise in [Na(+)](i). These responses were not affected either by the superfusion of ionotropic excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphono-pentanoic acid (D-APV), nor by the sodium channel blocker tetrodotoxin (TTX). (S)-alpha-methyl-4-carboxyphenylglycine (S-MCPG) and the more selective mGluR1 antagonist 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt) depressed both 3,5-DHPG-induced inward current components and, although less effectively, the associated [Ca(2+)](i) elevations. On repeated agonist applications the inward current and the calcium transients both desensitized. The time constant of recovery from desensitization differed significantly between these two responses, being 67.4+/-4.4 s for the inward current and 28.6+/-2.7 s for the calcium response. Bathing the tissue in a calcium-free/EGTA medium or adding thapsigargin (1 microM) to the extracellular medium prevented the generation of the [Ca(2+)](i) transient, but did not prevent the activation of the inward current. These electrophysiological and fluorometric results show that the 3, 5-DHPG-induced inward current and the [Ca(2+)](i) elevations are mediated by independent pathways downstream the activation of mGluR1.
Abstract: We report in this study with a pre-embedding immunogold method, the clustering of the group III metabotropic glutamate receptor 4a (mGluR4a) along the presynaptic membrane of parallel fiber synaptic terminals in the cerebellar molecular layer. The mGluR4a clusters were homogeneously distributed and interspaced by about 60 nm. These results suggest a particular arrangement of mGluR4a which might help to a rapid and effective activation of this receptor by glutamate.
Abstract: Intracellular pH and membrane potential were measured during hypoxia and/or hypoglycaemia in cortical pyramidal neurones of a rat cortical slice preparation. Intracellular pH (pHi) was calculated from ratiometric microfluorometry of the pH-sensitive dye BCECF injected via sharp recording microelectrodes into the neurones. Transient (5 min) hypoxia induced a fall of pHi (7.12 +/- 0.03) of -0.72 +/- 0.11 pH units while transient (10 min) hypoglycaemia induced an increase of 0.37 +/- 0.09 pH units. Hypoglycaemia did not prevent the hypoxic acidification. Lowering extracellular Na+ induced a membrane hyperpolarization and alkalinization by 0.29 +/- 0.12 pH units but did not affect the development or recovery of the hypoxic acidification. The alkalinization during hypoglycaemia suggested that there is some anaerobic glycolysis under normoglycemic conditions. The hypoxic acidification, however, is unlikely to result from anaerobic glycolysis or reversal of Na(+)-dependent H+ extrusion.
Abstract: We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na+]i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na+]i. In dopamine neurons voltage-clamped at a holding potential of -60 mV elevations of [Na+]i were induced by long-lasting (45-60 s) voltage jumps to more negative membrane potentials (-90 to -120 mV) but not by corresponding voltage jumps to -30 mV. These hyperpolarization-induced elevations of [Na+]i were depressed during inhibition of I(h), a hyperpolarization-activated inward current, by Cs+. Hyperpolarization-induced elevations in [Na+]i might occur also in other cell types which express a powerful I(h) and might signal lack of postsynaptic activity.
Abstract: Cultured cerebellar granule cells grown in medium containing 10 mM K+ undergo apoptosis after 4-5 days in vitro (DIV), and, at that time, the activity of metabotropic glutamate (mGlu) receptors coupled to polyphosphoinositide (PI) hydrolysis begins to decline. In granule cells at 4 DIV, the mGlu receptor subtype mGlu5 was expressed at high levels. The expression of another PI-coupled mGlu receptor, the mGlu1a, was low at 4 DIV but increased during the following days. In cultures at 4-5 DIV, the few cells that already showed an apoptotic phenotype were devoid of mGlu5 receptors, but they all expressed mGlu1a receptors. The development of apoptosis was accelerated after treating the cultures with: (i) mGlu5 antisense oligonucleotides; (ii) the mixed mGlu receptor antagonist, (+)-alpha-methyl-4-carboxyphenylglycine; or (iii) the glutamate depleting enzyme, alanine aminotransferase. In contrast, an induced overexpression of mGlu5 receptors protected cultured granule cells against apoptotic death. We suggest that the activity of mGlu5 receptors supports cell survival, and a decline in the expression of mGlu5 receptors gives access to programmed cell death in cerebellar granule cells developing in primary cultures.
Abstract: The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (Vh -60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 +/- 15.2 (SE) pA (n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 +/- 10.6 pA (n = 18). Although the hypoOUT reversed at -83.7 +/- 3.8 mV (n = 18), the posthypoOUT did not reverse. The K+ATP-blocking sulphonylureas tolbutamide (100 microM) and glibenclamide (30 microM), significantly reduced the peak of the hypoOUT by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 microM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings (Vh -60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 microM), glibenclamide (10-30 microM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 microM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.
Abstract: The mGluR1 metabotropic glutamate receptor is a G-protein-coupled receptor that exists as different C-terminal splice variants. When expressed in mammalian cells, the mGluR1 splice variants exhibit diverse transduction mechanisms and also slightly differ in their apparent agonist affinities. In the present study, we used an affinity-purified antiserum, specifically reactive to the mGluRlb splice variant, in combination with a highly sensitive preembedding immunocytochemical method for light microscopy to investigate the distribution of this receptor in the rat hypothalamus. An intense immunoreactivity for mGluRlb was observed in distinct hypothalamic nuclei. Thus, neuronal cell bodies and dendrites were stained in the preoptic area, suprachiasmatic nucleus, dorsal hypothalamus, lateral hypothalamus, dorsomedial nucleus, tuberomammilary nucleus, and lateral mammilary body. The ventromedial nucleus exhibited neuropil immunostaining but neuronal cell bodies were not labeled. Strong mGluRlb immunoreactivity was observed in magnocellular neurons of the neuroendocrine supraoptic, paraventricular, and arcuate nuclei. Also, neuronal cell bodies were heavily labeled in the retrochiasmatic nucleus, anterior commissural nucleus, and periventricular nucleus. These immunocytochemical observations, together with previous studies, suggest that mGluRlb is coexpressed with other class I mGluRs in some nuclei throughout the hypothalamus. However, mGluRlb is so far the only receptor of this class strongly expressed in the supraoptic, paraventricular, and arcuate nuclei, which might have relevant implications in the physiological control of the neuroendocrine hypothalamic-pituitary system.
Abstract: We investigated the effects of oxygen (O2)/glucose deprivation on intracellular sodium concentration ([Na+]i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O2/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na+]i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (D-amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na+/Ca2+-exchanger, affected these responses to O2/ glucose. The present results demonstrate that, in cortical neurons, O2/glucose deprivation induces an early rise in [Na+]i which cannot be ascribed to the activity of voltage gated Na+-channels, glutamate receptors or of the Na+/Ca2+-exchanger.
Abstract: Alternative splicing has been shown to occur at the metabotropic glutamate receptor 1 (mGluR1) gene. Three main isoforms that differ in their carboxy-termini have been described so far and named mGluR1alpha, mGluR1beta and mGluR1c. These variants when expressed in recombinant systems all activate phospholipase C, although the [Ca2+] signals generated have different kinetics. Tissue distribution studies of specific mGluR1 splice variants are limited to the mGluR1alpha isoform. In the present work, we examined the localization of mGluR1beta in the adult rat and mouse forebrain by using a specific antipeptide antibody. Furthermore, the mGluR1beta immunostaining was compared with that obtained with antibodies specific for mGluR1alpha or with a pan-mGluR1 antibody which recognizes all isoforms. mGluR1beta-like immunoreactivity (LI) was found confined to the neuropil and neuronal perikarya and appeared discretely distributed in the rodent forebrain. Differential cellular distribution between mGluR1alpha and mGluR1beta was observed. In the hippocampus, mGluR1alpha-LI was restricted to non-principal neurons in all fields, whereas mGluR1beta-LI was strongest in principal cells of the CA3 field and dentate granule cells but absent in CA1. We have also shown that the vast majority of neurons in the striatum express mGluR1. The predominant form appeared to be mGluR1beta, with a distribution pattern reflecting the patch-matrix organization of the striatum. The specificity of the immunoreactivity described for mGluR1 splice variants was confirmed in mGluR1-deficient mice. The observation of a different cellular and regional distribution of mGluR1 splice variants, in particular in the hippocampus, suggests that they may mediate different roles in synaptic transmission.
Abstract: We investigated the hypoxia-induced disturbance of cytosolic sodium concentration ([Na+]i) and of cytosolic calcium concentration ([Ca2+]i) in dopamine neurons of the substantia nigra pars compacta in rat midbrain slices, by combining whole cell patch-clamp recordings and microfluorometry. Transient hypoxia (3-5 min) induced an outward current (118.7 +/- 15.1 pA, mean +/- SE; VH = -60 mV). The development of this outward current was associated with an elevation in [Na+]i and in [Ca2+]i. The hypoxia-induced outward current as well as the elevations in [Na+]i and [Ca2+]i were not affected by the ionotropic and metabotropic glutamate receptor antagonists -amino-phosphonovalerate (50 microM), 6nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (10 microM) and S-(alpha)-methyl-4-carboxyphenylglycine (500 microM). Tolbutamide, a blocker of ATP-dependent K+ channels, depressed the hypoxia-induced outward current but did not affect the increases in [Na+]i or [Ca2+]i. Increasing the concentration of ATP in the internal solution from 2 to 10 mM strongly reduced the hypoxia-induced outward current but did not reduce the rise in [Na+]i. Decreasing the concentration of extracellular Na+ to 19.2 mM depressed the hypoxia-induced outward current and resulted in a decrease in resting [Na+]i. Under this condition hypoxia still increased [Na+]i, albeit to levels not exceeding those of resting [Na+]i observed under control conditions. We conclude that 1) a major component of the hypoxia-induced outward current of these cells is caused by a depletion of intracellular ATP in combination with an increase in [Na+]i, 2) that the [Na+]i and [Ca2+]i responses are not mediated by glutamate receptors, 3) that the [Na+]i and [Ca2+]i responses are not depressed by activation of sulfonylurea receptors, and 4) that the rise in [Na+]i induced by short-lasting hypoxia is not due to a ATP depletion-induced failure of Na+ extrusion.
Abstract: The subcellular localization of the mGlu4a metabotropic glutamate receptor was investigated in rat cerebellum. At the light microscopical level, strong mGlu4a immunoreactivity was found in the molecular layer. A post-embedding immunogold method for electron microscopy revealed gold particles at the presynaptic sites of synapses made by parallel fiber terminals with dendritic spines of Purkinje cells. These observations support electrophysiological evidence indicating an autoreceptor function of mGlu4 receptors at these synapses.
Abstract: We investigated the expression and coupling to the phospholipase C signal transduction pathway of metabotropic glutamate receptor (mGluR) subtypes by Western blot analysis and agonist-stimulated inositol monophosphate formation in several brain regions of postnatal day 9 (P9) and adult rats. In the cerebral cortex, hippocampus, corpus striatum, olfactory bulb, cerebellum and hypothalamus, the expression level of mGluR5 was greater at P9 than in adulthood. The mGluR5 signal was very low or absent in the adult cerebellum and hypothalamus. The expression of mGluR1a was slightly greater at P9 in the hypothalamus, hippocampus and olfactory bulb, whereas it substantially increased with age in the cerebellum, and did not change in the cerebral cortex and corpus striatum. mGluR1b and -1c were nearly undetectable by Western blot analysis. The expression level of mGluR5, but not that of mGluR1a, was significantly correlated with the extent of phosphoinositide hydrolysis stimulated by mGluR agonists in slices prepared from these brain regions. The mGluR antagonist cyclopropan[b]chromen-1a-carboxylic acid ethylester (CPCCOEt), potently antagonized responses mediated by mGluR1, but much less potently those mediated by mGluR5a in recombinant cells. CPCCOEt, at a concentration which efficiently blocks mGluR1 responses, did not substantially affect the polyphosphoinositide response in hippocampal or cerebellar slices from newborn animals, and antagonized only a minor component of the polyphosphoinositide response in adult hippocampal slices. CPCCOEt, however, prevented the small stimulation of polyphosphoinositide hydrolysis by mGluR agonists in adult cerebellar slices. We conclude that (i) the efficient mGluR-mediated polyphosphoinositide hydrolysis in 9-day-old rats is mediated by mGluR5; (ii) the increased expression of mGluR1 in the adult cerebellum does not substitute for the decline of mGluR5 expression in the ability to mediate polyphosphoinositide hydrolysis; and therefore (iii) mGluR1a might couple less efficiently than mGluR5 to polyphosphoinositide hydrolysis.
Abstract: The group-II metabotropic glutamate (mGlu) receptor agonists (2S,1'R, 2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), S-4-carboxy-3-hydroxyphenylglycine (4C3HPG), and (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) protected mouse cortical neurons grown in mixed cultures against excitotoxic degeneration induced by a 10 min pulse with NMDA. Protection was observed not only when agonists were added in combination with NMDA but also when they were transiently applied to cultures 6-20 hr before the NMDA pulse. In both cases, neuroprotection was reduced by the group-II mGlu receptor antagonist (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV), as well as by the protein synthesis inhibitor cycloheximide (CHX). Both neurons and astrocytes in mixed cultures were immunostained with an antibody that recognized mGlu2 and mGlu3 receptors in recombinant cells. To determine whether astrocytes played any role in the neuroprotection mediated by group-II mGlu receptors, we exposed pure cultures of cortical astrocytes to DCG-IV, 4C3HPG, or L-CCG-I for 10 min. The astrocyte medium collected 2-20 hr after the exposure to any of these drugs was highly neuroprotective when transferred to mixed cultures treated with NMDA. This protective activity was reduced when CHX was applied to astrocyte cultures immediately after the transient exposure to group-II mGlu receptor agonists. We conclude that neuroprotection mediated by group-II mGlu receptors in cultured cortical cells requires new protein synthesis and involves an interaction between neurons and astrocytes.
Abstract: The mGluR-mediated EPSP at parallel fibre-Purkinje cell synapses in the cerebellum was blocked concentration-dependently and reversibly by antagonists acting selectively on group-I mGluRs but not by an inhibitor of group-III receptors. The results provide pharmacological evidence that the receptor type responsible for the mGluR-EPSP is mGluR1.
Abstract: By combining biochemical, molecular and immunohistochemical approaches, we have investigated the presence of metabotropic glutamate receptors (mGluRs) belonging to the subtype 5 in rat and human spinal cords and the developmental changes in their expression. A polyclonal antibody raised against the carboxy-terminal portion of mGluR5 was used to study the distribution of the receptor in rat foetal (Et15), neonatal (P8) and adult spinal cords and dorsal root ganglia (DRG). mGluR5 appeared to be predominantly expressed in regions containing the primary sensory afferents. Immunoblotting with anti-mGluR5 antibody revealed lower receptor protein levels in rat adult spinal cord when compared with P8 rat spinal cord. Reverse transcriptase-polymerase chain reaction showed both mGluR5a and mGluR5b mRNAs expression in rat spinal cord. The mGluR5a variant was found more abundant in young animals than in adults. The pattern of mGluR5 immunostaining was also studied in foetal (6-8, 10, 12 and 22 weeks of gestation) and adult human spinal cord. At all stages of human development, a strong mGluR5 immunoreactivity was observed in the dorsal roots and in the dorsal and dorsolateral funiculi with maximum levels of staining at week 12 of gestation. Foetal DRG neurons were heterogeneously labeled. mGluR5 was also diffusely detectable in the mantle layer. In adult human spinal cords, immunoreactivity was confined to laminae I and II of the dorsal horns. These results demonstrate for the first time the presence of mGluR5 in human spinal cord. The distribution of this receptor suggests a role in the development of somatosensory pathways and in the control of nociceptive neurotransmission.
Abstract: The periaqueductal grey matter (PAG) is known to adjust somatic and neurovegetative elements of the defence behaviour. We have used specific polyclonal antibodies to examine the distribution of the metabotropic glutamate receptor subtype 5 (mGluR5) in this region. Immunolabelling for mGluR5 displayed a net preference for dorsolateral areas at rostral and intermediate levels. Electronmicroscopic examination revealed that mGluR5 is expressed in neuronal perikarya and in dendrites receiving synaptic contacts of Gray I type. To investigate the possible relevance of mGluR5 to integration of somatosensory information, spinoannular (SA) neurones were peroxidase-labelled and their relationship with mGluR5-expressing PAG neurones was examined at the ultrastructural level. A number of synaptic terminals of the SA pathway established synaptic contact of asymmetric type onto mGluR5-immunoreactive dendrites. It is suggested that mGluR5 might be involved in the temporal integration of somatosensory inputs to the PAG.
Abstract: The metabotropic glutamate (mGlu) receptor agonists, quisqualate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), but not (RS)-3,5-dihydroxyphenylglycine or (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine, stimulated [3H]inositolmonophosphate ([3H]InsP) formation in primary cultures of rat hepatocytes. 1S,3R-ACPD-stimulated [3H]InsP formation was inhibited by alpha-methyl-4-carboxyphenylglycine, indicating that cultured hepatocytes express functional mGlu receptors coupled to polyphosphoinositide hydrolysis. The identity of these receptors is not similar to that of any of the known mGlu receptor subtypes characterized in heterologous expression systems.
Abstract: The cellular and subcellular localization of the mGluR5 metabotropic glutamate receptor subtype was studied in the rat cerebellar cortex, by using the preembedding immunoperoxidase and immunogold techniques. Light microscopic observations revealed an abundant, intense labeling of neurons in the granular layer as well as in the molecular layer. Lugaro and Golgi cells exhibited an intense mGluR5 immunoreactivity, while only a fraction of the neurons in the molecular layer were found to be mGluR5 immunopositive. In addition to a dense plexus of immunoreactive dendrites in the molecular layer of the cerebellar cortex, the mGluR5 immunopositive Golgi cell dendrites resembling axons at the light microscopic level were also labeled in the granular layer. At the ultrastructural level, mGluR5 immunoreactivity was present in neuronal elements postsynaptic to axon terminals of different morphology. By using a pre-embedding immunogold method, it was found that mGluR5 immunoreactivity is accumulated at the plasma membranes extrasynaptically as well as at the periphery of the postsynaptic specializations, mainly of the parallel fiber synaptic contacts. These findings provide morphological evidence that mGluR5 is expressed by a population of neurons in the cerebellar cortex and can synaptically be activated via the parallel fiber system.
Abstract: We investigated the neuroprotective efficacy of the P-type Ca2+ channel antagonist daurisoline against electroshock-induced convulsions in rats and mice, hypoxic/hypoglycemic-induced damage in rat hippocampal slices and brain damage induced by occlusion of the middle cerebral artery (MCA) in rats. Daurisoline applied intravenously (i.v.) (bolus of 1-60 mg/kg) reduced the spontaneous activity of rat cerebellar Purkinje cells in a dose-dependent manner, a result demonstrating activity in the brain with systemic administration of the compound. While this effect reversed rapidly in about 10-20 min following bolus-application of the drug at doses of up to 30 mg/kg, a dose of 60 mg/kg consistently induced a depression of respiration followed by death of the animals. Daurisoline administered at 10-30 mg/kg did not prevent electroshock-induced convulsions in mice or rats, nor did it reduce the neuronal damage in hippocampal slices induced by a hypoxic/hypoglycemic insult in vitro by MCA occlusion in vivo. These observations do not support the hypothesis that P-type Ca2+ channels are promising drug targets for the acute treatment of epileptic convulsions and/or ischemic stroke.
Abstract: Two splice variants of the human metabotropic glutamate receptor 7, named hmGluR7a and hmGluR7b, were isolated from a human brain cDNA library. The isoforms differ by an out-of-frame insertion of 92 nucleotides close to the C-terminus of the hmGluR7 coding region, hmGluR7a has a length of 915 amino acids and represents the human homolog of the recently cloned rat mGluR7. hmGluR7b is seven amino acids longer and exhibits a novel C-terminus of 23 amino acids in length. RT-PCR analysis demonstrated the existence of mGluR7b transcripts in wild-type mouse brain and its absence in mGluR7 knockout mice. Northern blot analysis indicate that mGluR7 expression is developmentally regulated. It is expressed at high levels in human fetal brain and at a lower level in many regions of adult human brain. Stimulation of hmGluR7b with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) or L-glutamate in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas (1S,3R)-1-aminocyclopentane-1,3,-dicarboxylic acid ((1S,3R)-ACPD) and quisqualate (both at 1mM) had no significant effects. As described for rat mGluR7, the rank order of agonist potencies is: L-SOP, L-AP4 > L-glutamate > (1S,3R)-ACPD, quisqualate.
Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (-1a, -1b, and -1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca2+ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca2+ concentration ([Ca2+]i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate >> (2S,1'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca2+]i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I >> (1S,3R)-ACPD approximately quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.
Abstract: (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG) and (S)-alpha-methyl-3-carboxyphenylalanine (M3CPA), two novel preferential antagonists of group III metabotropic glutamate (mGlu) receptors, antagonized the neuroprotective activity of L-2-amino-4-phosphono-butanoate (L-AP4) or L-serine-O-phosphate in mice cultured cortical cells exposed to a toxic pulse of N-methyl-D-aspartate. In contrast, MPPG did not influence the neuroprotective activity of the selective group II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxy-cyclopropyl) glycine (DCG-IV). These results indicate that activation of group III mGu receptors exerts neuroprotective activity against excitotoxic neuronal death. At least one of the two major group III mGlu receptor subtypes, i.e. mGlu4 receptor, is expressed by cultured cortical neurons, as shown by immunocytochemical analysis with specific polyclonal antibodies.
Abstract: We investigated whether the expression of human alpha-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca2+]i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM. Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K+. During this period, [Ca2+]i was monitored by video microfluorimetry using the Ca2+ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca2+]i. The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca2+]i than non-parvalbumin-expressing cells. Resting [Ca2+]i did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca2+]i in neuroblastoma cells can be suppressed by parvalbumin.
Abstract: Pre-embedding immunogold histochemistry was combined with Phaseolus vulgaris leucoagglutinin anterograde tract tracing in order to analyse the relationship between the subcellular localization of the GluR1a metabotropic glutamate receptors and the distribution of corticothalamic synapses in the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior nucleus (LP) of the rat. The injection of the tracer into area 17 labelled two types of corticothalamic terminals: (i) the small boutons constituting the majority of the labelled fibres which form asymmetrical synapses both in the dLGN and LP; and (ii) the giant terminals typically participating in glomerulus-like synaptic arrangements and found exclusively in the lateral posterior nucleus. The small corticothalamic terminals often established synapses with mGluR1a-immunopositive dendrites, with immunometal particles concentrated at the periphery of their postsynaptic membranes. In contrast, the synapses formed by giant boutons in the lateral posterior nucleus were always mGluR1a-immunonegative. We conclude that the corticothalamic fibres forming the small synaptic terminals are the most likely candidates for the postulated mGluR-mediated modulation of visual information flow by corticothalamic feedback mechanisms.
Abstract: Metabotropic glutamate (mGlu) receptors are a large, heterogeneous family of G-protein coupled receptors, which modulate excitatory synaptic transmission through various transduction pathways. Evidence is now accumulating that individual mGlu-receptor subtypes mediate distinct, facilitatory (group I subtypes) or inhibitory (group II and group III subtypes), actions on neurodegenerative processes. Drugs interacting with mGlu receptors are expected to influence both the induction and progression of neuronal degeneration without hampering the efficiency of fast excitatory synaptic transmission. For these reasons, mGlu receptors can be considered as promising drug targets in the experimental therapy of acute or chronic neurodegenerative diseases.
Abstract: Brief tetanic stimulation of parallel fibres can evoke a slow excitatory postsynaptic potential (EPSP) in cerebellar Purkinje cells that is mediated by metabotropic glutamate receptors (mGluRs). It is likely that the receptor subtype involved is mGluR1, which couples to the production of diacylglycerol and inositol-1,4,5-trisphosphate (IP3). We therefore examined whether the mGluR-EPSP is associated with an increase in cytosolic free calcium [Ca2+]i using simultaneous Ca2+ imaging and electrophysiological recordings. An mGluR-EPSP could be evoked in all nine Purkinje cells tested. In all but one this potential was not associated with measurable changes in [Ca2+]i whereas single calcium spikes produced large Ca2+ transients. In the one Purkinje cell where [Ca2+]i was elevated, the rise was estimated to be roughly 20-fold smaller than that produced by a single Ca2+ spike.
Abstract: It has been proposed that neurotransmitter signalling can occur between axons and glia in the mammalian optic nerve in the absence of synaptic specialisations, and that this may be glutamate mediated. Here, the cellular distribution of five metabotropic glutamate receptors (mGluR's 1a, 1b, 1c, 2/3 and 5) have been assessed in the rat optic pathway using specific antibodies. Positive immunoreactivity is found for mGluR2/3 and 5. Both are found in axons, although only mGluR5 is present in the majority of these. Strong immunoreactivity for mGluR2/3 is found in cells in the optic pathway and thalamus. The cellular morphology and distribution is consistent with their being astrocytes. Examination of brain sections stained for mGluR2/3 is consistent with this notion, with many cells having end-feet processes terminating on blood vessels or the pial surface. The axonal immunoreactivity could represent the presence of these receptors on axons, but it is more probable that the receptor protein synthesised in the ganglion cell soma is being transported to the cell terminal in sufficient concentration to be revealed by immunohistochemistry. The reason for the axon-astrocyte signalling is unclear, and may be associated with metabolic coupling. In development, communication between axons and glia mediates a range of functions including pathway selection and myelination. It is probable that in the adult this form of signalling underpins a range of functions that have yet to be described.
Abstract: We have characterized the expression pattern and pharmacological profile of activation of metabotropic glutamate receptors (mGluRs) in immortalized, gonadotropin releasing hormone (GnRH)-secreting GT1-7 cells, which represent a homogeneous cellular population of hypothalamic origin. These cells are known to respond to the mGluR agonist (1S,3R)-cyclopentanedicarboxylic acid (1S,3R-ACPD) with increased GnRH release. To establish which specific mGluR subtypes are expressed by GT1-7 cells, we used polyclonal antibodies raised against non-conserved regions of the carboxy-terminal domains of individual subtypes. The selectivity of these antibodies was tested in HEK 293 cells transiently transfected with each mGluR subtype. GT1-7 cells stained positively for the subtypes mGluR1a, -1b and -5 (belonging to group I mGluR2/3 (group II) and mGluR7 (group III). Agonists of group I mGluRs, including 1S,3R-ACPD, activated phosphoinositide hydrolysis in GT1-7 cells. This effect, however, was manifested only when cell density was low, and it disappeared when cells reached confluence. Stimulation of phosphoinositide hydrolysis could not therefore have been related to hormone secretion because 1S,3R-ACPD effectively released GnRH in confluent cultures. We then focused on group II and III mGluRs, which in transfected cells are negatively linked to adenylate cyclase activity. Unexpectedly, however, agonist which preferentially activate group II and III mGluRs increased both basal and forskolin-stimulated cAMP accumulation in GT1-7 cells. Stimulation of cAMP accumulation by mGluR agonists was not prevented by enzymatic depletion of endogenous adenosine, but was obliterated when cells were incubated with agonists of receptors positively coupled to adenylate cyclase, such as beta-adrenergic and prostaglandin E2 receptors. These results suggest that GT1-7 cells express a novel mGluR subtype positively coupled to adenylate cyclase, which shares the same transduction pathway of other classical receptors coupled with a Gs-type of GTP-binding protein.
Abstract: The metabotropic glutamate receptors (mGluRs) can be classified into three families based on amino acid sequence homology, signal transduction mechanisms and pharmacological properties. Generally, class I mGluRs mediate an excitation of neurons while activation of class II and III mGluRs results in a depression of synaptic transmission. In this study we have analyzed the expression pattern of mGluRs in human hippocampus using a panel of polyclonal antibodies specific for mGluR1b, mGluR2/3, mGluR4a, and mGluR5. Immunoreactivity for mGluR1b and mGluR5, i.e., the subtypes representing class I mGluRs, was found in all hippocampal neurons. The mGluR1b antiserum stained perikarya and proximal dendrites, whereas immunoreactivity for mGluR5 was also detectable in the distal dendritic compartments. Immunoreactivity for mGluR2/3, members of class II mGluRs, was present in all principle neurons in the dentate gyrus as well as in the CA4, CA3 and CA2 regions. Pyramidal cells of the CA1 region exhibited only weak labeling for mGluR2/3. Glial cells were also mGluR2/3-immunoreactive. The reaction obtained with an antiserum directed against mGluR4a, a member of class III mGluRs, was confined to the mossy fiber projection field in CA3 stratum lucidum. These data demonstrate differential expression of mGluR variants in the human hippocampus and may provide an important basis for future studies of mGluRs under various neuropathological conditions such as temporal lobe epilepsy, ischemia and neurodegenerative disorders.
Abstract: L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is mediated via ionotropic and metabotropic glutamate receptors (mGluR). Eight mGluRs have already been cloned. In the present study the hypothalamic distribution of mGluR1a has been investigated by immunocytochemistry using monoclonal antibodies recently produced by some of the present authors (T. J. G., R. K., T. K.). The observations have been compared with findings obtained with a polyclonal antibody. A widespread and heterogeneous distribution of mGluR1a was found with the monoclonal antibodies. Intense immunolabelling of perikarya and dendrites occurred in several hypothalamic cell groups including the suprachiasmatic, anterior periventricular, anterior hypothalamic (posterior part), paraventricular, supraoptic, arcuate, tuberal magnocellular, dorsomedial and mammillary nuclei (particularly in the medial). It was only the ventromedial nucleus in which several perikarya were stained by the polyclonal antibody but appeared to be negative by the monoclonal antibodies. The findings fit extremely well with the data on the hypothalamic distribution of mGluR1 mRNA with the exception of the ventromedial nucleus. It remains to be elucidated whether alternatively spliced variants of mGluR1 (mGluR1b and 1c) are expressed in this nucleus. Further, they confirm the results of former immunohistochemical studies. In addition, they indicate that a significant part of the neuroendocrine region of the hypothalamus (including the paraventricular, supraoptic and arcuate nuclei) also contains mGluR1 suggesting that this receptor may play a role also in neuroendocrine regulation.
Abstract: Metabotropic glutamate receptors (mGluR) share no sequence homology with any other G-protein-coupled receptors (GPCRs). The characterization of their G-protein coupling domains will therefore help define the general rules for receptor-G-protein interaction. To this end, the intracellular domains of mGluR3 and mGluR1, receptors coupled negatively to adenylyl cyclase and positively to phospholipase C, respectively, were systematically exchanged. The ability of these chimeric receptors to induce Ca2+ signals were examined in Xenopus oocytes and HER 293 cells. The chimeric receptors that still possessed the second intracellular loop (i2) of these proteins were targeted correctly to the plasma membrane. Consistent Ca2+ signals could be recorded only with chimeric mGluR3 receptors that contains i2 and at least one other intracellular domains of mGluR3 have to be replaced by their mGluR1 equivalent to produce optimal coupling to G protein. These observations indicate that i2 of mGluR1 is a critical element in determining the transduction mechanism of this receptor. These results suggest that i2 of mGluRs may play a role similar to i3 of most other GPCRs in the specificity of coupling to the G-proteins. Moreover, as in many other GPCRs, our data revealed cooperation between the different mGluR intracellular domains to control efficient coupling to G-proteins.
Abstract: We have tested the two enantiomers of trans-azetidine-2,4-dicarboxylic acid, (2S,4S)-azetidine-2,4-dicarboxylic acid ((2S,4S)-ADA) and (2R,4R)-azetidine-2,4-dicarboxylic acid ((2R,4R)-ADA) for activity at the human metabotropic glutamate receptors mGlu1b, mGlu2, mGlu4a and mGlu5a expressed in mammalian cells. In Chinese hamster ovary (CHO) cells expressing human mGlu2 receptors, 500 microM (2S,4S)-ADA inhibited forskolin-stimulated cAMP accumulation by 33 +/- 3% while 100 microM (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid induced an inhibition by 66 +/- 5%. The (2R,4R)-ADA enantiomer was inactive at human mGlu2 receptors. In CHO cells expressing human mGlu4a receptors, 10 microM L-AP4 inhibited forskolin-stimulated cAMP levels by 37 +/- 4% whereas both ADA enantiomers of trans-azetidine-2,4-dicarboxylic acid (500 microM) had no such effect. In CHO cells expressing human mGlu1b receptors and L cells expressing human mGlu5a receptors, both enantiomers, applied at 500 microM or 1 mM, were ineffective in stimulating inositolmonophosphate accumulation and did not affect quisqualate-stimulated inositolmonophosphate accumulation. We conclude that (2S,4S)-azetidine-2,4-dicarboxylic acid is a weak human mGlu2 receptor agonist and that (2R,4R)-azetidine-2,4-dicarboxylic acid is inactive at human mGlu2 receptors. Trans-azetidine-2,4-dicarboxylic acid has no significant agonistic effect on human mGlu4a receptors and neither agonistic nor antagonistic effects on human mGlu1b and mGlu5a receptors.
Abstract: A cDNA encoding the human metabotropic glutamate receptor type 2 (hmGluR2) was isolated from human brain cDNA libraries by cross-hybridization with rat mGluR2 probes. The deduced amino acid sequence of the human mGluR2 receptor consists of 872 residues and shows a sequence identity of 97% to the amino acid sequence of rat mGluR2. Northern blot analyses showed that hmGluR2 is widely expressed in different regions of the adult brain as well as in fetal human brain. Genomic Southern blotting localized the mGluR2 gene to human chromosome 3. Chinese hamster ovary (CHO) cells stably transfected with the cloned hmGluR2 cDNA exhibit agonist induced depression of forskolin-stimulated cAMP accumulation. A direct comparison of CHO cells stably expressing human and rat mGluR2 with five agonists revealed the same rank order of potency [(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine >> (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid = L-glutamate >> quisqualate = L-2-amino-4-phosphonobutyric acid] and similar EC50 values for both homologous receptors. (R,S)-alpha-methyl-4-carboxyphenylglycine, a reported antagonist at some mGluR subtypes, reduced the depression of forskolin-induced cAMP accumulation by (1S,3R)-ACPD in both human and rat mGluR2.
Abstract: A cDNA encoding the human metabotropic glutamate receptor type 4 (hmGluR4) was isolated from human brain cDNA libraries by cross-hybridization with rat mGluR4 probes. The deduced amino acid sequence of human mGluR4 consists of 912 residues and shows a sequence identity of 96% to the amino acid sequence of rat mGluR4. Northern blot analyses indicate that hmGluR4 is strongly expressed in the cerebellum of the adult human brain but also at low levels in hippocampus, hypothalamus and thalamus. Stimulation of hmGluR4 with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP), L-glutamate or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas quisqualate (0.5 mM) was ineffective. The rank order of agonist potencies is: L-AP4 > L-SOP > L-glutamate > (1S,3R)-ACPD >> quisqualate. (R,S)-alpha-methyl-4-carboxyphenylglycine (1 mM), a reported antagonist at some mGluR subtypes, did not reduce the depression of forskolin-induced cAMP accumulation by L-AP4.
Abstract: We have studied the influence of class I metabotropic glutamate receptors (mGluRs) on excitotoxic neuronal degeneration in cultured murine cortical neurons grown on a monolayer of astrocytes. These cultures expressed high levels of mGluR5 mRNA, which were comparable to those found in RNA extracts from cerebral cortex. Cortical neurons in mixed cultures were heavily stained with antibodies raised against mGluR5 and were also stained--albeit to a much lower extent--with mGluR1a but not with mGluR1b or c antibodies. Preferential agonists of class I mGluRs, such as quisqualate, 3,5-dihydroxyphenylglycine (DHPG), and trans-azetidine-2,4-dicarboxylic acid (t-ADA), as well as the mixed mGluR agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) all stimulated PPI hydrolysis in cultured cortical cells. The potency of N-methyl-D-aspartate (NMDA) in inducing neuronal degeneration was substantially enhanced when the drug was coincubated with quisqualate, DHPG or t-ADA during a 10-min pulse (paradigm of "fast" toxicity). None of the mGluR agonists influenced neuronal viability by itself. The amplification of NMDA toxicity by quisqualate or DHPG was attenuated by a series of protein kinase C (PKC) inhibitors, suggesting that class I mGluRs operate, at least in part, through activation of PKC. Quisqualate and, in particular, DHPG enhanced excitoxic neuronal degeneration even when applied after the toxic pulse with NMDA. This action is likely to occur early in the maturation of excitotoxic damage, because the functional activity of class I mGluRs was substantially reduced at 2 or 3 hr after the NMDA pulse. These results suggest that activation of class I mGluRs enhances NMDA-receptor mediated neuronal toxicity and encourage the search for selective antagonists for the experimental therapy of acute or chronic neurodegenerative diseases.
Abstract: The two reported metabotropic glutamate receptor (mGluR) antagonists, alpha-methyl-cyclopropyl glycine (MCCG) and alpha-methyl-aminophosphonobutyrate (MAP4) were tested on the mGluR1b, mGluR2 and mGluR4a subtypes of human mGluRs. Neither MCCG (500 microM) nor MAP4 (500 microM) antagonized the activation of mGluR1b by 10 microM quisqualate. MCCG was found to potently antagonize the action of 30 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] at mGluR2 (IC50 = 87.5 microM; apparent Kd = 25 microM) but did not block the action of 1 microM S-2-amino-4-phosphonobutyric acid at mGluR4a (IC50 >> 1 mM). MAP4 was found to be a weak antagonist or partial agonist at mGluR4a (IC50 > 500 microM) and, less potently, also antagonized the action of 30 microM (1S,3R)-ACPD) at mGluR2 (IC50 approximately 2 mM).
Abstract: To test the hypothesis that the determinants for agonist selectivity of class III metabotropic glutamate receptors (mGluRs) are localized in the N-terminal extracellular domain, a chimaeric cDNA was constructed where 519 amino acids of the N-terminal extracellular domain of human mGluR1b were exchanged with the corresponding region of human mGluR4. The pharmacological profile of the chimaera, designated hmGlu(R4)1-519/1b, was analysed by recordings of intracellular calcium concentration ([Ca2+]i) in transiently transfected HEK 293 cells and compared with that of human mGluR1b and human mGluR4a stably expressed in Chinese hamster ovary cells. Application of 100 microM L-2-amino-4-phosphonobutyrate (L-AP4), a class III mGluR-specific agonist, induced a rise in [Ca2+]i in hmGlu(R4)1-519/1b but not in hmGluR1b expressing cells. In contrast, application of quisqualate (100 microM) induced a rise in [Ca2+]i at hmGluR1b but not at hmGlu(R4)1-519/1b. Dose-response analysis with L-AP4 and L-glutamate at hmGlu(R4)1-519/1b revealed a half-maximal effect (EC50) of 16.0 microM and 196 microM, respectively. The EC50 values for quisqualate, glutamate and (1S,3R)-ACPD at hmGluR1b were 10.25 microM, 225 microM and 3060 microM, respectively. The rank order of agonist potency of hmGlu(R4)1-519/1b corresponds to that of hmGluR4 (L-AP4 > L-glutamate > (1S,3R)-ACPD > quisqualate) but is different from that of hmGluR1b (quisqualate > glutamate >> (1S,3R)-ACPD).
Abstract: 1. We have developed compartmental models of guinea-pig medial vestibular nuclei neurons (MVNns). The structure and the parameters of the model cells were chosen to reproduce the responses of type A and type B MVNns as described in electrophysiological recordings. 2. Dynamics of membrane potentials were modeled in 46 and 61 branched electrical compartments for Type A and Type B MVNns, respectively. Each compartment was allowed to contain up to nine active ionic conductances: a fast inactivating sodium conductance, gNa, a persistent sodium conductance, gNap, a low-voltage activated calcium conductance, gCa(LVA), a high-voltage activated calcium conductance, gCa(HVA), a fast-voltage activated potassium conductance, gK(fast), a slowly relaxing voltage activated potassium conductance, gK(slow), a fast transient potassium channel, gK(A), a slowly relaxing mixed sodium-potassium conductance activating at hyperpolarized membrane potentials, gH, and a calcium-activated potassium conductance gK(AHP). The kinetics of these conductances were derived from voltage-clamp studies in a variety of preparations. Kinetic parameters as well as distribution and density of ion channels were adjusted to yield the reported electrophysiological behavior of medial vestibular neurons. 3. Dynamics of intracellular free [Ca2]i were modeled by inclusion of a Ca(2+)-pump and a Na(+)-Ca2+ exchanger for extrusion of calcium. Diffusion of calcium between submembraneous sites and the center of an electrical compartment was modeled by 25 and 6 shell-like chemical compartments for the cell body and the proximal dendrites, respectively. These compartments also contained binding sites for calcium. 4. The dynamics of active conductances were the same in both models except for gK(fast). This was necessary to achieve the different shape of spikes and of spike afterhyperpolarization in type A and type B MVNns. An intermediate depolarizing component of the spike afterhyperpolarization of type B neurons in part depended on their dendritic cable structure. 5. Variation of the low threshold calcium conductance, gCa(LVA), shows that the ability to generate low-threshold spike bursts critically depends on the density of this conductance. Sodium plateaus were generated when increasing the density of gNap. 6. The type B model cell generated rhythmic bursts of spiking activity under simulation of two distinct experimental conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: Antibodies were raised against C-terminal peptides of the splice variants a, b and c of the rat mGluR1 metabotropic glutamate receptor. Affinity purified antibodies each specifically reacting with mGluR1a, mGluR1b and mGluR1c were used to study the cellular localization of these receptors in rat cerebellum. The mGluR1a antibody strongly labelled Purkinje cells at their cells bodies, portions of their dendritic trees and numerous small punctate elements reminiscent of dendritic spines. Also labelled were some stellate, basket, Golgi and Lugaro cells. Granule cells were devoid of staining. The mGluR1b antibody strongly labelled Purkinje cell bodies and their dendrites at levels which varied within the same lobule of the vermis or the hemispheres. No significant labelling was observed at stellate, basket, Golgi and granule cells, while occasionally a fraction of basket cells and cerebellar glomeruli was moderately immunoreactive. The mGluR1c antibody strongly labelled cell bodies and thick principal dendrites of Purkinje cells but not dendritic spines. Immunonegative Purkinje cells were intermingled with strongly labelled ones in lobules 4-10, while in lobules 1, 2 and 3, no stained Purkinje cells were detected. The mGluR1c antibody also labelled stellate, basket, some Golgi and some Lugaro cells as well as granule cells.
Abstract: We investigated the synaptic transmission in the parallel fiber-Purkinje cell system at high spatio-temporal resolution by using voltage-sensitive dyes and an imaging system. In rat cerebellar slices, cut in the frontal plane or in a plane of the cerebellar surface, local electrical stimulation induced volleys of action potentials in the parallel fibers; subsequent postsynaptic responses from Purkinje cells were observed along the volleys' entire trajectories. Furthermore, the formation of an ordered spatial gradient in parallel fiber conduction velocity across the depth of the molecular layer during postnatal development was observed. In preparations of adult, but not of immature rats, the conduction velocity of parallel fibers in the deep molecular layer was faster than in its more superficial regions. Our observations demonstrate that parallel fibers can mediate Purkinje cell excitation effectively and over considerable distances in a well-organized spatio-temporal manner, thus supporting the classical view of the physiological role assigned to the parallel fibers.
Abstract: 1. We have developed a compartmental model of a turtle cerebellar granule cell consisting of 13 compartments that represent the soma and 4 dendrites. We used this model to investigate the synaptic integration of mossy fiber inputs in granule cells. 2. The somatic compartment contained six active ionic conductances: a sodium conductance with fast activation and inactivation kinetics, gNa; a high-voltage-activated calcium conductance, gCa(HVA); a delayed potassium conductance, gK(DR); a transient potassium conductance, gK(A); a slowly relaxing mixed Na+/K+ conductance activating at hyperpolarized membrane potentials, gH, and a calcium- and voltage-dependent potassium conductance, gK(Ca). The kinetics of these conductances was derived from electrophysiological studies in a variety of preparations, including turtle and rat granule cells. 3. In the soma, dynamics of intracellular free Ca2+ was modeled by incorporation of a Na+/Ca2+ exchanger, radial diffusion, and binding sites for Ca2+. 4. The model of the turtle granule cell exhibited depolarization-induced action potential firing with properties closely resembling those seen with intracellular recordings in turtle granule cells in vitro. 5. In the most distal compartments of the dendrites, mossy fiber activity induced synaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type of glutamate receptors. The strength of synaptic inputs chosen was such that the synaptic potential induced by synchronous activation of two mossy fiber synapses reached threshold for induction of a single action potential. 6. The slow time course of the NMDA synaptic current together with the slow relaxation kinetics of gH significantly affected the temporal summation of excitatory synaptic potentials. A priming action potential evoked by mossy fiber stimulation increased the maximal time interval between two synaptic potentials capable to reach again threshold for a subsequent action potential. This time interval then decreased in parallel with the decay of the NMDA synaptic current, reached a minimum after 200 ms, and slowly recovered with reactivation of gH. 7. Repetitive, steady activation of synaptic conductances by a single mossy fiber at different frequencies induced action potential firing with a sharp threshold at 12 Hz. Activity of a single or of several mossy fibers induced firing of the granule cell at a frequency close to that induced when the average synaptic current was directly injected into the cell. The mossy fiber activity-granule cell firing frequency curve was close to linear with a slope of about one-half for input frequencies < or = 400 Hz.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: The cellular and subcellular distribution of the mGluR5a metabotropic glutamate receptor was studied in the spinal cord of the rat using an antibody raised against a mGluR5a-specific carboxy-terminal peptide. Strong mGluR5a-immunoreactivity (mGluR5a-ir) was found in the laminae I-II of the dorsal horn, which gradually decreased towards the deeper layers. At the electron microscopical level, mGluR5a-ir was present exclusively in neuronal somata and dendrites. Immunometal labelling revealed that mGluR5a-ir is concentrated at the periphery of postsynaptic densities of asymmetrical synapses or localized extrasynaptically at dendritic and somatic membranes. The mGluR5a-immunoreactive dendritic profiles were often targeted by synaptic boutons with the morphological characteristics of C-fibre terminals. These observations provide evidence for mGluR5a being involved in the nociceptive transmission at the dorsal horn.
Abstract: We investigated the spread of membrane voltage changes from the soma into the dendrites of cerebellar Purkinje cells by using voltage-imaging techniques in combination with intracellular recordings and by performing computer simulations using a detailed compartmental model of a cerebellar Purkinje cell. Fluorescence signals from single Purkinje cells in cerebellar cultures stained with the styryl dye di-4-ANEPPS were detected with a 10 x 10 photodiode array and a charge coupled device (CCD). Fluorescence intensity decreased and increased with membrane depolarization and hyperpolarization, respectively. The relation between fractional fluorescence change (delta F/F) and membrane potential could be described by a linear function with a slope of up to -3%/100 mV. Hyperpolarizing and depolarizing voltage jumps applied to Purkinje cells voltage-clamped with an intrasomatic recording electrode induced dendritic dye signals, demonstrating that these voltage transients invaded the dendrites. Dye signals induced by depolarizing somatic voltage jumps were weaker in the dendrites, when compared with those induced by hyperpolarizing voltage jumps. Dendritic responses to hyperpolarizing voltage steps applied at the soma were attenuated when membrane conductance was increased by muscimol, an agonist for GABA(A) receptors. Corresponding experimental protocols were applied to a previously developed detailed compartmental model of a Purkinje cell. In the model, as in the electrophysiological recordings, voltage attenuation from soma to dendrites increased under conditions where membrane conductance is increased by depolarization or by activation of GABA(A) receptors, respectively. We discuss how these results affect voltage clamp studies of synaptic currents and synaptic integration in Purkinje cells.
Abstract: IN looking for a structurally defined non-peptide P-channel blocker we have tested the alkaloid daurisoline which has been isolated from traditional Chinese medicinal herb (Menispermum dauricum) used for the treatment of epilepsy, hypertension and asthma. We have found that daurisoline is an inhibitor of omega-Aga-IVA sensitive barium currents in cerebellar Purkinje cells and of excitatory postsynaptic potentials evoked in Purkinje cells by stimulating parallel fibres in acutely prepared cerebellar slices. Daurisoline did not significantly affect omega-Aga-IVA-insensitive barium currents recorded from granule cells freshly isolated from rat cerebellum. Daurisoline passes the blood-brain barrier and will, therefore, facilitate the functional characterization of brain calcium channels as well as the exploration of P-type calcium channels as possible drug targets.
Abstract: 1. Depolarization-induced changes in the cytosolic free calcium concentration ([Ca2+]i) were examined in slice-cultured neurons of the deep cerebellar nuclei by combined intracellular and multisite fura-2 recording techniques. 2. Firing of tetrodotoxin (TTX)-sensitive action potentials induced by depolarizing current pulses caused large elevations in somatic as well as proximal dendritic [Ca2+]i. In the dendrites, rise and decay times of [Ca2+]i were faster than in the soma. [Ca2+]i changes associated with depolarizations to < or = -40 mV in the presence of TTX were small compared with changes induced by Na+ spike firing, suggesting that Ca2+ influx through high voltage-activated Ca2+ channels is a major cause for Na+ spike-associated [Ca2+]i increases. 3. During sustained Na+ spike firing at a constant frequency (> 20 Hz), [Ca2+]i approached a constant level, after approximately 1 s in the dendrites and 2 s in the soma, respectively. The amplitude of the attained level was positively correlated with the firing frequency. We suggest that during tonic activity [Ca2+]i reaches a steady state determined by Ca2+ influx and extrusion. 4. TTX-resistant plateau potentials caused substantially greater [Ca2+]i increases in the dendrites than in the soma. In the dendrites, plateau-associated Ca2+ transients were comparable in amplitude to Ca2+ transients triggered by short (50 ms) Na+ spike trains, in the soma, they were considerably smaller. 5. Low-threshold spikes (LTSs) in association with a burst of Na+ spikes induced a sharp increase in [Ca2+]i both in the soma and in dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The responses of slice-cultured Purkinje cells to trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) were examined by intracellular recording techniques and fura-2 microfluorometry. Bath-application of t-ACPD (100 microM, 30 s), a selective agonist of metabotropic glutamate receptors (mGluRs), to Purkinje cells voltage-clamped near their resting potential -65 to -60 mV) consistently induced a transient inward current, followed by a slower outward current (Iout). This outward current was characterized by a linear current-voltage relationship in the range from -130 to -60 mV and accompanied by a significant decrease in membrane conductance. The extrapolated reversal potential of Iout was positive to 0 mV. When t-ACPD was applied for 60 s or more it became apparent that Iout emerged in parallel to the wash-out of t-ACPD. Microfluorometric fura-2 measurements in combination with electrophysiological recordings were used to assess the relation between Iout and intracellular free calcium concentration ([Ca2+]i). In contrast to the inward current that was associated with a transient elevation in [Ca2+]i. Iout was not correlated with an elevated [Ca2+]i. When t-ACPD was applied in the presence of caffeine (5 mM), Iout was reversibly enhanced in amplitude. Caffeine affected neither the t-ACPD-induced calcium signal nor the resting [Ca2+]i. While longer applications of caffeine alone induced outward currents with a current-voltage relationship similar to that of Iout, short applications (30 s) of caffeine had no detectable effect per se but still were effective in enhancing Iout when applied in conjunction with t-ACPD. 3-Isobutyl-1-methylxanthine (IBMX, 0.5 mM), a more selective and potent phosphodiesterase inhibitor than caffeine, exhibited caffeine-like effects at a 10-fold lower concentration. We propose that Iout is generated by a transient inhibition of an inward current that is tonically active at rest and largely voltage-independent in the range tested. Our observations provide evidence for an involvement of cyclic nucleotide second messenger systems in the regulation of this current.
Abstract: The effects of iontophoretically applied (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), an agonist of metabotropic glutamate receptors, were examined in rat cerebellar Purkinje cells in vivo. Multibarrel electrodes were used for extracellular recordings of spontaneous single unit discharges and iontophoretic ejection of 1S,3R-ACPD. The effect of 1S,3R-ACPD depended on both the strength and the duration of the iontophoretic current. Application of the agonist with ejection currents at or slightly above the response threshold for up to 60 s resulted in an increased rate of action potential firing. With larger ejection currents of the same duration the initial increase in activity was followed by a depression and eventually a cessation of activity. In the transition phase between low frequency firing and firing arrest, Purkinje cells generated almost exclusively complex spikes. When the drug application was continued for longer durations (1-10 min) the initial response was followed by a characteristic cyclic firing pattern. These cycles consisted of alternating phases of mainly simple spike activity, predominantly complex spike activity and silent intervals. At the end of drug applications using large ejection currents, a prolonged period (on average 66 s) with almost no spiking activity was observed. This period ended with an abrupt onset of simple spike firing. These findings point to an important function of cerebellar metabotropic glutamate receptors in the regulation of Purkinje cell activity.
Abstract: A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca2+ concentration ([Ca2+]i). Action potentials elicited by intracellular injection of short-lasting (50-100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50-100 ms) depolarizing voltage jumps from -60 mV to between -30 and 0 mV induced a slowly decaying outward aftercurrent (IAHP) which was depressed by bath application of muscarine (0.5 microM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (IADP) in voltage-clamp mode. This current was also induced in the presence of trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 5 microM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 microM), while both of these agonists depressed IAHP. IADP was depressed by reducing the external Ca2+ concentration from 3.8 to 0.5 mM, by external Co2+ (1 mM) and by external Cd2+ (10-100 microM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca2+]i using the Ca2+ indicator fura-2 revealed that the amplitude of IADP was correlated with the amplitude of depolarization-induced Ca2+ influx. IADP was absent at membrane potentials < -90 mV, and reached maximal amplitudes at approximately -55 mV. Raising the extracellular K+ concentration from 2.7 to 13.5 mM increased the amplitude of IADP and resulted in a positively directed shift of the apparent reversal potential of IADP. When the external Na+ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external CI- concentration from 159 to 20 mM did not affect IADP. We conclude that IADP most likely represents a Ca(2+)-activated cation current, rather than a Ca2+ tail current, or an electrogenic Ca2+ extrusion current.
Abstract: The interactions of the phenylglycine derivatives (S)-4-carboxyphenylglycine (S-4CPG) and (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) with responses of rat cerebellar Purkinje cells to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) were examined by intracellular recordings in acute cerebellar slices and extracellular recordings in vivo, using multibarrel electrodes. In vitro, both S-4CPG (100 microM to 1 mM) and MCPG (250 microM to 1 mM) reversibly and dose-dependently reduced an inward current induced by bath-applied 1S,3R-ACPD, an agonist at metabotropic glutamate receptors (mGluRs), in Purkinje cells voltage-clamped at -60 to -65 mV. S-4CPG applied at a concentration of 1 mM reduced the 1S,3R-ACPD induced current to 17% of control values but when applied alone also produced an inward current amounting to 26.8% of that induced by 1S,3R-ACPD. MCPG bath-applied at 250 microM, 500 microM, or 1 mM reduced the 1S,3R-ACPD-induced current to 85%, 56% or 3% of control values, respectively, and did not cause any current when applied alone even at a concentration of 1 mM. In vivo, iontophoretic application of 1S,3R-ACPD induced a transient increase followed by a decrease in the firing rate of Purkinje cells. The excitatory response of Purkinje cells to 1S,3R-ACPD was suppressed during ejection of either one of the phenylglycine derivatives, while the mechanism resulting in the decreased firing rate was not affected. Our observations demonstrate that both S-4CPG and MCPG antagonized the excitatory response of cerebellar Purkinje cells to 1S,3R-ACPD.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The possible role of protein kinase C activation in the inhibitory action of cholinergic transmitters on the slow Ca-dependent afterhyperpolarizing current (IAHP) in hippocampal CA3 pyramidal neurons was investigated using hippocampal slice cultures. IAHP was inhibited reversibly by methacholine (100 - 600 nM) and irreversibly by the protein kinase C activator, phorbol-12,13-dibutyrate (PDBu, 10 nM to 1 microM). The inhibitory action of PDBu was antagonized by prior (15 - 60 min) exposure to staurosporin (1 microM). In contrast, the inhibitory effect of methacholine on IAHP was not reduced after up to 3 h of exposure to this compound. In addition, methacholine produced a reversible inward current at the holding potential, which was augmented by staurosporin. However, prior exposure to PDBu reduced the effect of methacholine on IAHP and occluded the methacholine-induced inward current. This effect of PDBu was also observed in the presence of staurosporin, suggesting that it might be exerted through a protein kinase C-independent pathway. Noradrenalin (2 - 5 microM) and 8-bromo cyclic adenosine 3',5'monophosphate (8-Br-cAMP, 1 mM) also produced a reversible block of IAHP. Their action was antagonized by staurosporin, probably via its effect on protein kinase A. Thus the present experiments suggest that the action of muscarinic agonists on IAHP cannot be explained by an effect on protein kinase C, but support a role for protein kinase A in mediating the action of noradrenalin.
Abstract: 1. Depolarization-induced elevations of intracellular calcium concentration ([Ca2+]i) were examined in slice-cultured hippocampal pyramidal and nonpyramidal cells of the CA3 region by combined intracellular and multisite fura-2 recording techniques. 2. In pyramidal cells, spiking activity induced by depolarizing current pulses (200-800 ms) induced transient elevations of somatic as well as of proximal dendritic [Ca2+]i. The calcium signals from the proximal dendrites were larger in amplitude and decayed much faster than those from the soma. Depolarization of presumed interneurons induced comparable somatic and dendritic calcium transients, which decayed faster than those observed in pyramidal cell somata. 3. The calcium transients of pyramidal cells, but not those of nonpyramidal cells, were associated with a slow afterhyperpolarization (sAHP), whose time course was correlated with that of the somatic calcium signal. We conclude that the lack of a sAHP in non-pyramidal cells cannot be explained by the absence of an efficient rise in [Ca2+]i but rather by the absence of the potassium conductance underlying the sAHP in pyramidal cells.
Abstract: Excitatory postsynaptic potentials evoked in neurons of the deep cerebellar nuclei, either by electrical stimulation within the nuclei in cerebellar slice cultures or by electrical stimulation of olivary explants in olivo-cerebellar co-cultures, were investigated in the rat by means of intracellular recordings. In neurons of the deep cerebellar nuclei, stimulation of the nuclear tissue, as well as stimulation of the olivary tissue, induced a fast rising excitatory postsynaptic potential, followed by an inhibitory postsynaptic potential and a long-lasting excitation. The fast rising excitatory postsynaptic potential and the following inhibitory postsynaptic potential were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The remaining depolarization was abolished by D-(-)-2-amino-5-phosphonovalerate, suggesting that this potential was mediated by N-methyl-D-aspartate receptors. With only D-(-)-2-amino-5-phosphonovalerate added to the bath, the slow excitation was depressed, whereas the fast excitatory and inhibitory postsynaptic potentials were not affected. In the presence of bicuculline, the 6-cyano-7-nitroquinoxaline-2,3-dione- and the D-(-)-2-amino-5-phosphonovalerate-sensitive excitatory postsynaptic potentials elicited by stimulation of the olivary tissue had the same latency, and were both graded with stimulation strength. The time-to-peak and the duration of the D-(-)-2-amino-5-phosphonovalerate-sensitive excitatory postsynaptic potentials were considerably longer than those of the 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive excitatory postsynaptic potentials.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Amino acid levels were measured in perfusates from biplanar slices of rat cerebellum installed in a Krebs-filled three-compartment chamber. The two lateral compartments housed the white matter and a section containing parallel fibres respectively. The central compartment housed cortical structures, including the Purkinje cell and granule cell bodies. This arrangement allows selective electrical stimulation of the parallel fibre or mossy fibre pathways, recording of the evoked responses to such stimulation and collection of the perfusion medium passing through the central chamber for amino acid analysis using high-pressure liquid chromatography (HPLC). Both, 2-Hz and 5-Hz stimulation of white matter caused a delayed increase in arginine levels in the perfusate. Since L-arginine is the physiological precursor of nitric oxide, a neuronal messenger in the brain, the data suggest that physiological stimuli can result in the release of this precursor, possibly to supply the nitric oxide synthase.
Abstract: The responses to activation of metabotropic glutamate receptors (mGluRs) of Purkinje cells in rat cerebellar slice cultures were investigated using intracellular recordings in single-electrode voltage-clamp mode combined with microfluorometric measurements of cytosolic free calcium using fura-2. Purkinje cells were perfused with saline containing 0.5 microM tetrodotoxin and 10 microM bicuculline and voltage-clamped at -60 mV. Bath-applied trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 50 - 100 microM), a selective agonist of mGluRs, induced a transient inward current that was followed by an outward current. The response induced by t-ACPD was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, up to 40 microM). In contrast, inward currents caused by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1 - 2 microM) were completely abolished, while inward currents caused by quisqualate (0.25 microM) were only partially depressed by CNQX (5 - 40 microM). The inward current induced by t-ACPD was unaffected by external Ba2+ (1 mM), tetraethylammonium (10 mM) and Cs+ (1 mM), and was associated with an increase in apparent input conductance of the cell membrane. The extrapolated reversal potential of inward currents induced by t-ACPD was +18 mV while Cl- currents induced by muscimol reversed at -66 mV. Inward currents induced by t-ACPD, but not those induced by AMPA, were associated with a rise in cytosolic Ca2+ concentration and suppressed by intracellular injection of a calcium chelator. Replacement of external Na+ by choline or Li+ depressed the inward current and resulted in a slower decay of the Ca2+ signal.
Abstract: We describe techniques for measurements of cytosolic calcium dynamics in single current- or voltage-clamped nerve cells. The calculations of calcium dynamics are based on continuous recordings of fura-2 fluorescence intensity at one excitation wavelength after an initial reference measurement at two excitation wavelengths. We show that such single wavelength recordings are not only sufficient for the calculation of Ca2+ concentrations, but also lead to a superior signal-to-noise ratio at a high temporal resolution. Moreover, this strategy diminishes requirements for the experimental setup, such as a device necessary to switch quickly between excitation filters. We have applied this approach on measurements of cytosolic free Ca2+ in single-electrode voltage-clamped CA3 pyramidal cells in hippocampal slice cultures.
Abstract: A combination of intracellular recording and fluorometric measurements of cytosolic calcium [( Ca2+]i) was used to locate changes in [Ca2+]i induced by the specific metabotropic glutamate receptor (mGluR) agonist trans-D,L-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), in Purkinje cells of rat cerebellar slices. Under voltage-clamp conditions, application of t-ACPD (100 microM) induced an inward current accompanied by a large increase in [Ca2+]i located primarily in the soma but also, to a lesser degree, in restricted parts of the dendrites. In contrast, elevations of [Ca2+]i associated with calcium spikes were confined to the dendrites and inward currents of a similar amplitude induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an agonist of ionotropic glutamate receptors, did not raise [Ca2+]i.
Abstract: Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca(2+)-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.
Abstract: The generation of climbing fibre responses in cerebellar Purkinje cells has been analysed in co-cultured slices of rat cerebellum and inferior olive. Complex spikes were evoked in Purkinje cells by climbing fibre activation or by intrasomatic injection of depolarizing current pulses. Microfluorometric measurements of cytosolic free calcium ([Ca2+]i) by means of intracellularly injected fura-2 combined with intracellular recordings revealed that both types of complex spikes were accompanied by a transient rise in [Ca2+]i, which was most prominent at dendritic locations. Synaptically induced Ca2+ transients were completely and reversibly abolished by 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX, 5 microM), an antagonist of the ionotropic action mediated by non-N-methyl-d-aspartate excitatory amino acid receptors. Ca2+ transients evoked by injections of depolarizing current pulses were not affected by CNQX. These observations indicate that Ca2+ transients induced by climbing fibre activity are generated by voltage-gated Ca2+ channels, which are activated by a CNQX-sensitive synaptic depolarization.
Abstract: The dynamics of cytosolic free Ca2+ ([Ca2+]i) of single voltage-clamped CA3 pyramidal cells in hippocampal slice cultures is reviewed. [Ca2+]i amounts to about 30 nM at resting membrane potential and increases slowly when the membrane potential is clamped at more positive values (up to 500 nM at -30 mV). Short lasting depolarizations (40-100 ms) induce a transient rise in [Ca2+]i which activates a slow aftercurrent (IAHP). The muscarinic or beta-adrenergic depression of IAHP is not accompanied by any change in the dynamics of Ca2+ and appears, therefore, to result primarily from an inhibition of the K(+)-current itself or of the ability of Ca2+ to activate the current. At higher concentrations than those required to inhibit IAHP, muscarine produces a pronounced inward current and this is accompanied by a rise in resting [Ca2+]i concentration.
Abstract: Cerebellar slices prepared from newborn rats were co-cultured with slices derived from the inferior olive of 4-day-old rats. After several weeks in vitro olivary fibres projecting into the cerebellar tissue could be assessed by anterograde labelling with the fluorescent dye 1,1-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (Dil). Following electrical field stimulation of the olivary tissue, all-or-nothing complex spikes were generated in Purkinje cells, which closely resembled climbing fibre responses as seen in situ. These responses were completely and reversibly abolished by 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX, 5 microM), an antagonist of non-N-methyl-d-aspartate excitatory amino acid receptors. Wash in of smaller concentrations of CNQX (0.5 - 2 microM) resulted in a graded dose-dependent depression of the climbing fibre-induced postsynaptic potentials and in a consecutive failure of distinct active components of the complex spikes. With climbing fibre synaptic transmission blocked by CNQX, complex spike-like potentials could, however, still be evoked by intrasomatic injection of depolarizing current pulses. Increasing the concentration of Mg2+ in the bathing solution from 0.5 to up to 8 mM depressed regenerative complex-spike components. Olivary stimulation elicited only monophasic postsynaptic potentials in Purkinje cells under these conditions. These observations indicate that voltage-gated conductances which are substantially involved in the generation of the complex spike, are gated by the climbing fibre synaptic depolarization rather than directly by the climbing fibre transmitter.
Abstract: In co-cultures of rat septum and hippocampus, cholinergic neurons, identified by immunocytochemical techniques using antibodies against choline acetyltransferase, were found to be exclusively located in septal tissue. The presence of nerve growth factor during the entire growth period of four weeks increased the activities of acetylcholinesterase and choline acetyltransferase about 10-fold and strongly increased the number of acetylcholinesterase-positive neurons. Application of nerve growth factor yielded different effects depending on the age of the cultures. During the first two weeks in vitro, nerve growth factor enhanced the number of acetylcholinesterase-positive neurons, an effect which was no longer observed following later applications of nerve growth factor. Nerve growth factor increased the activities of cholinergic enzymes during all phases of in vitro development, but the effects of one-week applications were always considerably smaller than those observed following continuous application of nerve growth factor. The results of different application schedules suggest that the continuous presence of nerve growth factor is needed for maximal increases in cholinergic enzyme activities and maintenance of cholinergic neurons in septohippocampal co-cultures.
Abstract: Combined intracellular and microfluorometric recording techniques were used to evaluate whether the inhibition by cholinergic or adrenergic transmitters of the Ca2(+)-activated potassium current (IAHP) in hippocampal CA3 pyramidal cells was mediated by an alteration of depolarization-induced change in cytosolic free Ca2+ concentration [(Ca2+]i). Low concentrations of isoproterenol (1-10 microM) and muscarine (0.25-1 microM) reversibly abolished IAHP without affecting concomitant Ca2+ transients or the steady-state [Ca2+]i. Only after application of higher concentrations of muscarine, [Ca2+]i increased; in the presence of potassium channel blockers, muscarine depressed Ca2+ currents and concomitant Ca2+ transients. These observations provide direct evidence that the inhibition of IAHP by isoproterenol and muscarine are not mediated by an alteration of Ca2+ dynamics.
Abstract: Glucose deprivation (GD) results in a hyperpolarization by turning on a potassium conductance (gK,GD) in hippocampal CA3 pyramidal cells. We used combined intracellular and microfluorometric recording techniques to evaluate whether gK,GD is activated by a rise in the concentration of intracellular calcium ([Ca2+]i). We found that the activation of gK,GD is only followed, but not preceded by a rise in [Ca2+]i. Furthermore, gK,GD is not blocked by the sulfonylurea glibenclamide, a blocker of ATP-regulated potassium conductance. We conclude that activation of gK,GD does not simply reflect breakdown of the calcium of ATP homeostasis, but on the contrary might represent an active restoring mechanism which delays the pathological consequences of sustained glucose deficiency.
Abstract: 1. The actions of the endogenous excitatory amino acids (EAAS) glutamate (Glu), aspartate (Asp) and homocysteate (HCA) on Purkinje cells and neurones of the deep nuclei in cerebellar slice cultures were investigated using intracellular recordings in the single-electrode voltage-clamp mode and the whole-cell configuration of the patch-clamp technique. 2. Purkinje cells and neurones of deep cerebellar nuclei were identified according to their localization in the living cultures, their morphology as revealed by intracellular injections of Lucifer Yellow and their immunoreactivity to antibodies to the 28 kDa Ca2(+)-binding protein. 3. When Purkinje cells were voltage-clamped near their resting membrane potential in a TTX-containing salt solution, Glu, Asp and HCA induced inward currents which were abolished by 6-cyano-7-nitroxaline-2,3-dione (CNQX), a selective antagonist of the non-N-methyl-D-aspartate (NMDA) subtype of EAA receptors. The selective antagonist of NMDA receptors, D-(-)-2-amino-5-phosphonovaleric acid (D-APV), was ineffective in blocking the responses induced by these three amino acids. NMDA, even at high concentrations and in magnesium-free bathing solution, had no detectable effect on membrane properties of Purkinje cells grown in culture during 11-34 days. 4. In magnesium-containing saline, the amplitude of the responses induced by Glu, Asp and HCA was a linear function of the membrane potential. 5. In contrast, neurones of the deep cerebellar nuclei were responsive to NMDA and the inward currents induced by Glu, Asp and HCA were partially blocked both by CNQX and by D-APV. 6. In magnesium-containing saline, the amplitude of the currents induced by NMDA as well as by the three endogenous EAAs decreased at hyperpolarizing holding potentials whereas the current-voltage relation of the responses induced by quisqualate (QA) was strictly linear. 7. It is concluded that Purkinje cells in cerebellar slice cultures do not express NMDA receptors and that excitation of these neurones by the endogenous amino acids Glu, Asp and HCA is mediated exclusively through the activation of non-NMDA receptors. In the same preparation, neurones of the deep cerebellar nuclei possess NMDA and non-NMDA receptors which can be both activated by the three endogenous excitatory amino acids.
Abstract: Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.
Abstract: Slices from the brainstem at the level of the locus coeruleus and from the hippocampus of 5 - 7 day old rats were co-cultured using the roller tube technique. After 2 - 6 weeks in vitro the co-cultures were examined with antibodies raised against tyrosine-hydroxylase (TH). The cultures derived from the brainstem consistently contained a bilateral cluster of TH-positive neurons with 3 - 5 long slender dendrites. These neurons typically gave rise to several fine varicose fibres reminiscent of catecholaminergic axons. A morphologically distinct group of TH-positive neurons was detected in the hippocampal slices. The vast majority of them were located in the subicular region and a smaller number in the CA1/CA3 region of the hippocampal explant. TH-positive neurons were also present in mono-cultures of hippocampus or brainstem. In the vast majority of co-cultures, a variable number of TH-immunoreactive fibres of neurons derived from the locus coeruleus grew over considerable distances to terminate finally within the co-cultured hippocampus where they branched to form a diffuse innervation plexus with club-like endings. Even after several weeks in vitro, TH-positive fibres could still be seen exploring sites which were not related to their target, including the cell-free areas surrounding the cultures. Fibres in these outgrowth areas formed whirl-like endings. TH-positive fibres arising from neurons located in the hippocampus, on the other hand, did not branch extensively and never projected over long distances. Nerve growth factor had no apparent trophic effect on TH-positive cells in the hippocampus, the locus coeruleus, or in the co-cultures.
Abstract: Slices were prepared from septal and hippocampal tissue and co-cultured for periods up to one month. The presence of cholinergic neurons within the septal slices was demonstrated by histochemical staining techniques for acetylcholinesterase or by Golgi-like immunoperoxidase techniques with antibodies raised against the enzyme choline acetyltransferase. Cholinergic fibers originating in the septal explants started to grow radially in all directions. By day 7, the first fibers were seen to reach their target, but maximal hippocampal ingrowth occurred between day 8 and 14 in vitro. Only those fibers reaching the target were maintained, whereas cholinergic fibers growing in other directions degenerated. Electrophysiological studies showed that cholinergic fibers established functional cholinergic connections with hippocampal pyramidal cells. As a result of septal stimulation, two different potassium currents were inhibited in pyramidal cells: a calcium-independent current, IM, and a calcium-dependent current, IAHP, underlying spike afterhyperpolarization. Application of nerve growth factor (NGF) strongly increased the number of cholinergic fibers which invaded the hippocampal slices and raised the activities of the cholinergic enzymes choline acetyltransferase and acetylcholinesterase, effects which were completely blocked by anti-NGF antibodies. The response of septohippocampal co-cultures to NGF depended on the time of application. During the first two weeks in vitro, NGF elicited sustained increases in enzyme activities, whereas later administration of NGF produced effects which were only maintained for several days.
Abstract: The synaptic excitation of central vestibular neurons in the isolated superfused brainstem of chronic hemilabyrinthectomized (HL) frogs and of controls was studied electrophysiologically and pharmacologically. Central vestibular neurons were excited either through vestibular afferent fibers or through the vestibular commissural pathway by means of electrical stimulation of the ipsilateral or the contralateral VIIIth nerve. In chronic HL frogs, commissural field potential amplitudes were on the average larger than those of intact frogs and the shape parameters of intracellularly recorded commissural EPSPs of chronic animals were on the average shifted towards those of vestibular afferent EPSPs. In control frogs, vestibular afferent EPSPs were generated independently from N-methyl-D-aspartate (NMDA) receptors, whereas commissural EPSPs exhibited a delayed NMDA receptor mediated component. Commissural EPSPs of HL frogs exhibited a NMDA receptor mediated component as well. The size of this EPSP component was larger when the time to peak of the EPSP was longer. EPSPs with similar rise times exhibited NMDA mediated components of similar size, irrespective of whether they originated from chronic animals or controls. The tendency of these EPSPs towards shorter rise times in chronic animals was paralleled by a similar decrease of the relative size of their NMDA receptor mediated component. It is concluded that the increased synaptic efficacy of commissural fibers observed in chronic HL frogs does not result from an increased NMDA receptor component.
Abstract: The effect of (+/-)-beta-parachlorophenylglutamate (CP) on depolarizations induced by iontophoretically applied L-glutamate, L-aspartate, L-homocysteate (L-HCA) and D-HCA was investigated in neurons of the rat neocortex in vitro. CP, a reported blocker of amino acid uptake, strongly enhanced L-HCA responses whereas responses to the other amino acids remained little affected. This action was observed irrespective of whether CP was administered iontophoretically or pneumatically from micropipettes. CP (5 mM) administered alone had no effect on membrane potential. These findings suggest the existence of a specific uptake system for L-HCA providing further evidence in favour of a possible function of L-HCA as an endogenous ligand for the N-methyl-D-aspartate receptor in the rat neocortex.
Abstract: The effects of ionophoretically applied L-homocysteate (L-HCA), L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were compared in rat neocortical neurons recorded intracellularly in vitro. The firing pattern and the time course of membrane depolarization induced by L-HCA resembled those of NMDA responses. Action potentials evoked by NMDA and L-HCA were superimposed upon slow depolarizations in a burst-like pattern, while L-Glu elicited single spike discharges. Ionophoretically applied D-2-amino-5-phosphonovalerate (2-APV) at doses sufficient to abolish NMDA responses, markedly reduced the L-HCA induced depolarizations but had no detectable effect on the L-Glu responses. The present findings are consistent with a possible role of L-HCA as an NMDA receptor preferring neurotransmitter in the rat frontal cortex.
Abstract: We have investigated the role of N-methyl-D-aspartate (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg2+ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-applied D-2-amino-5-phosphonovaleric acid (D-APV, 25-50 microM), a selective NMDA antagonist. In a low Mg2+ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as in their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and has a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of hte latter was reduced by D-APV (25-50 microM) to about 65% of the control value in the presence of 1 mM Mg2+. During bath application of NMDA (100 microM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg2+ concentration. Bath application of NMDA (0.1-1 microM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were simulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these simulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.
Abstract: Compensatory torsional and vertical eye movements were recorded in the frog during sinusoidal linear acceleration along the longitudinal and transverse body axes, respectively. Stimulus frequencies ranged between 0.1 and 1.0 Hz and peak accelerations from 0.01 g to 0.1 g corresponding to body tilts ranging from 0.57 to 5.7 degrees. In addition, static compensatory eye movements were studied during fore-and-aft and lateral body tilt over ranges of +/- 10 degrees. The evoked eye movements were generally quite small (+/- 0.5 degree). Dynamic gain (rotation of the eye/apparent rotation of gravity direction) was 0.10-0.20 at 0.1 Hz and decreased to about 0.05 at 1.0 Hz. The gain of vertical eye movements was somewhat higher than that of torsional eye movements. Phase lag relative to peak accelerations increased from about 10 degrees to about 45 degrees over the same frequency range. Static compensatory eye movements evoked by nose-up and ipsilateral side-up tilt were larger in amplitude than those evoked by nose-down and ipsilateral side-down tilt. Static gain (rotation of the eye/tilt of the whole body) was about 0.10 for vertical and about 0.06 for torsional eye movements. No consistent eye movements could be evoked by vertical sinusoidal accelerations (maximal modulation amplitudes +/- 0.025 g). The results indicate that, as in other vertebrates, maculo-ocular reflexes contribute to gaze stabilization in the frog mainly during low frequency and static head and body tilts.
