Abstract: Ipsilateral and contralateral hippocampal CA3-CA1 and CA2-CA1 projections were investigated in adult male Long-Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior-posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3-CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2-CA1 stratum oriens and CA2-CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.
Abstract: Calcium-dependent activator protein for secretion 2 (CAPS2) is a dense-core vesicle-associated protein that is involved in the secretion of BDNF. BDNF has a pivotal role in neuronal survival and development, including the development of inhibitory neurons and their circuits. However, how CAPS2 affects BDNF secretion and its biological significance in inhibitory neurons are largely unknown. Here we reveal the role of CAPS2 in the regulated secretion of BDNF and show the effect of CAPS2 on the development of hippocampal GABAergic systems. We show that CAPS2 is colocalized with BDNF, both synaptically and extrasynaptically in axons of hippocampal neurons. Overexpression of exogenous CAPS2 in hippocampal neurons of CAPS2-KO mice enhanced depolarization-induced BDNF exocytosis events in terms of kinetics, frequency, and amplitude. We also show that in the CAPS2-KO hippocampus, BDNF secretion is reduced, and GABAergic systems are impaired, including a decreased number of GABAergic neurons and their synapses, a decreased number of synaptic vesicles in inhibitory synapses, and a reduced frequency and amplitude of miniature inhibitory postsynaptic currents. Conversely, excitatory neurons in the CAPS2-KO hippocampus were largely unaffected with respect to field excitatory postsynaptic potentials, miniature excitatory postsynaptic currents, and synapse number and morphology. Moreover, CAPS2-KO mice exhibited several GABA system-associated deficits, including reduced late-phase long-term potentiation at CA3-CA1 synapses, decreased hippocampal theta oscillation frequency, and increased anxiety-like behavior. Collectively, these results suggest that CAPS2 promotes activity-dependent BDNF secretion during the postnatal period that is critical for the development of hippocampal GABAergic networks.
Abstract: Global brain state dynamics regulate plasticity in local cortical circuits, but the underlying cellular and molecular mechanisms are unclear. Here, we demonstrate that astrocyte Ca(2+) signaling provides a critical bridge between cholinergic activation, associated with attention and vigilance states, and somatosensory plasticity in mouse barrel cortex in vivo. We investigated first whether a combined stimulation of mouse whiskers and the nucleus basalis of Meynert (NBM), the principal source of cholinergic innervation to the cortex, leads to enhanced whisker-evoked local field potential. This plasticity is dependent on muscarinic acetylcholine receptors (mAChR) and N-methyl-d-aspartic acid receptors (NMDARs). During the induction of this synaptic plasticity, we find that astrocytic [Ca(2+)](i) is pronouncedly elevated, which is blocked by mAChR antagonists. The elevation of astrocytic [Ca(2+)](i) is crucial in this type of synaptic plasticity, as the plasticity could not be induced in inositol-1,4,5-trisphosphate receptor type 2 knock-out (IP(3)R2-KO) mice, in which astrocytic [Ca(2+)](i) surges are diminished. Moreover, NBM stimulation led to a significant increase in the extracellular concentration of the NMDAR coagonist d-serine in wild-type mice when compared to IP(3)R2-KO mice. Finally, plasticity in IP(3)R2-KO mice could be rescued by externally supplying d-serine. Our data present coherent lines of in vivo evidence for astrocytic involvement in cortical plasticity. These findings suggest an unexpected role of astrocytes as a gate for cholinergic plasticity in the cortex.
Abstract: Dendrite arborization patterns are critical determinants of neuronal connectivity and integration. Planar and highly branched dendrites of the cerebellar Purkinje cell receive specific topographical projections from two major afferent pathways; a single climbing fiber axon from the inferior olive that extend along Purkinje dendrites, and parallel fiber axons of granule cells that contact vertically to the plane of dendrites. It has been believed that murine Purkinje cell dendrites extend in a single parasagittal plane in the molecular layer after the cell polarity is determined during the early postnatal development. By three-dimensional confocal analysis of growing Purkinje cells, we observed that mouse Purkinje cells underwent dynamic dendritic remodeling during circuit maturation in the third postnatal week. After dendrites were polarized and flattened in the early second postnatal week, dendritic arbors gradually expanded in multiple sagittal planes in the molecular layer by intensive growth and branching by the third postnatal week. Dendrites then became confined to a single plane in the fourth postnatal week. Multiplanar Purkinje cells in the third week were often associated by ectopic climbing fibers innervating nearby Purkinje cells in distinct sagittal planes. The mature monoplanar arborization was disrupted in mutant mice with abnormal Purkinje cell connectivity and motor discoordination. The dendrite remodeling was also impaired by pharmacological disruption of normal afferent activity during the second or third postnatal week. Our results suggest that the monoplanar arborization of Purkinje cells is coupled with functional development of the cerebellar circuitry.
Abstract: We describe a low-cost, small, remotely triggerable LED device for wireless control of transcranial optical stimulation of cortical neurons, for use in freely moving mice. The device is easily mountable on the head of a mouse with a high-polymer block. Using the Thy1-ChR2-YFP transgenic mice, we demonstrate that the device is capable of remotely triggering muscle twitches upon activation of the primary motor cortex in freely moving conditions.
Abstract: MicroRNAs (miRNAs) have been demonstrated to be potent post-trascriptional modulators of protein expression. miRNA expression was profiled in the left and right dorsal hippocampal CA3 of mature rats by high-throughput deep sequencing. Among the sequenced and cross-mapped small RNAs, 88% belonged to the miRNAs annotated in the miRBase 15 database. Nearly half of the small RNAs belonged to the let-7 family miRNA. Seven percent of the sequenced small RNAs were not annotated in miRBase 15. Bioinformatic analysis of the unannotated small RNA sequences suggested seventeen novel miRNA candidates with relatively high expression levels (>100 tags per million). The left:right expression ratios were similar for all highly expressed miRNAs with less than 10% differences. These results provide a basic idea of the relative expression strengths of known and unknown miRNAs in the dorsal hippocampal CA3.
Abstract: Previous anatomical and in vitro electrophysiology studies suggest that astrocytes are heterogeneous in physiology, morphology, and biochemical content. However, the extent to which this diversity applies to in vivo conditions is largely unknown. To characterize and classify the physiological and morphological properties of cerebral cortical and hippocampal astrocytes in the intact brain, we performed in vivo intracellular recordings from single astrocytes using anesthetized mature rats. Astrocytes were classified based on their glial fibrillary acidic protein (GFAP) immunoreactivity and cell body locations. We analyzed morphometric measures such as the occupied volume and polarity, as well as physiological characteristics such as the mean membrane potential. These measurements did not show obvious segregation into subpopulations, suggesting that gray matter astrocytes in the cortex and hippocampus are composed of a homogeneous population in mature animals. The membrane potential of astrocytes in both cortex and hippocampus fluctuated within a few millivolts in the presence of spontaneous network activity. These membrane potential fluctuations of an astrocyte showed a significant variability that depended on the local field potential state and cell body location. We attribute the variability of the membrane potential fluctuations to local potassium concentration changes due to neuronal activity.
Abstract: The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors. In diabetes and Alzheimer's disease, pathological progression is accelerated by activation of RAGE. However, how RAGE influences gross behavioral activity patterns in basal condition has not been addressed to date. In search for a functional role of RAGE in normal mice, a series of standard behavioral tests were performed on adult RAGE knockout (KO) mice. We observed a solid increase of home cage activity in RAGE KO. In addition, auditory startle response assessment resulted in a higher sensitivity to auditory signal and increased prepulse inhibition in KO mice. There were no significant differences between KO and wild types in behavioral tests for spatial memory and anxiety, as tested by Morris water maze, classical fear conditioning, and elevated plus maze. Our results raise a possibility that systemic therapeutic treatments to occlude RAGE activation may have adverse effects on general activity levels or sensitivity to auditory stimuli.
Abstract: Synaptic plasticity is considered to be the main mechanism for learning and memory. Excitatory synapses in the cerebral cortex and hippocampus undergo plastic changes during development and in response to electric stimulation. It is widely accepted that this process is mediated by insertion and elimination of various glutamate receptors. In a series of recent investigations on left-right asymmetry of hippocampal CA3-CA1 synapses, glutamate receptor subunits have been found to have distinctive expression patterns that depend on the postsynaptic density (PSD) area. Particularly notable are the GluR1 AMPA receptor subunit and NR2B NMDA receptor subunit, where receptor density has either a supralinear (GluR1 AMPA) or inverse (NR2B NMDAR) relationship to the PSD area. We review current understanding of structural and physiological synaptic plasticity and propose a scheme to classify receptor subtypes by their expression pattern with respect to PSD area.
Abstract: Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.
Abstract: S100B is the principal calcium-binding protein of astrocytes and known to be secreted to extracellular space. Although secreted S100B has been reported to promote neurite extension and cell survival via its receptor [receptor for advanced glycation end products (RAGE)], effects of extracellular S100B on neural activity have been mostly unexplored. Here, we demonstrate that secreted S100B enhances kainate-induced gamma oscillations. Local infusion of S100B in S100B(-/-) mice enhanced hippocampal kainate-induced gamma oscillations in vivo. In a complementary set of experiments, local application of anti-S100B antibody in wild-type mice attenuated the gamma oscillations. Both results indicate that the presence of extracellular S100B enhances the kainate-induced gamma oscillations. In acutely isolated hippocampal slices, kainate application increased S100B secretion in a neural-activity-dependent manner. Further pharmacological experiments revealed that S100B secretion was critically dependent on presynaptic release of neurotransmitter and activation of metabotropic glutamate receptor 3. Moreover, the kainate-induced gamma oscillations were attenuated by the genetic deletion or antibody blockade of RAGE in vivo. These results suggest RAGE activation by S100B enhances the gamma oscillations. Together, we propose a novel pathway of neuron-glia communications--astrocytic release of S100B modulates neural network activity through RAGE activation.
Abstract: Cumulative evidence supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement of neuronal information processing in the brain. Cytosolic calcium (Ca(2+)) concentration is important for astrocytes as Ca(2+) surges co-occur with gliotransmission and neurotransmitter reception. Cerebral cortex is organized in layers which are characterized by distinct cytoarchitecture. We asked if astrocyte-dominant layer 1 (L1) of the somatosensory cortex was different from layer 2/3 (L2/3) in spontaneous astrocytic Ca(2+) activity and if it was influenced by background neural activity. Using a two-photon laser scanning microscope, we compared spontaneous Ca(2+) activity of astrocytic somata and processes in L1 and L2/3 of anesthetized mature rat somatosensory cortex. We also assessed the contribution of background neural activity to the spontaneous astrocytic Ca(2+) dynamics by investigating two distinct EEG states ("synchronized" vs. "de-synchronized" states). We found that astrocytes in L1 had nearly twice higher Ca(2+) activity than L2/3. Furthermore, Ca(2+) fluctuations of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. Pharmacological blockades of metabotropic receptors for glutamate, ATP, and acetylcholine, as well as suppression of action potentials did not have a significant effect on the spontaneous somatic Ca(2+) activity. These results suggest that spontaneous astrocytic Ca(2+) surges occurred in large part intrinsically, rather than neural activity-driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous astrocytic activity.
Abstract: S100B is a calcium-binding protein predominantly expressed in astrocytes. Previous studies using gene-manipulated animals have suggested that the protein has a role in synaptic plasticity and learning. In order to assess the physiological roles of the protein in active neural circuitry, we recorded spontaneous neural activities from various layers of the neocortex and hippocampus in urethane-anesthetized S100B knockout (KO) and wildtype (WT) control mice. Typical local field oscillation patterns including the slow (0.5-2 Hz) oscillations in the neocortex, theta (3-8 Hz) and sharp wave-associated ripple (120-180 Hz) oscillations in the hippocampus were observed in both genotypes. Comparisons of the frequency, power and peak amplitude have shown that these oscillatory patterns were virtually indistinguishable between WT and KO. When seizure was induced by intraperitoneal injection of kainic acid, a difference between WT and KO appeared in the CA1 radiatum local field potential pattern, where seizure events were characterized by prominent appearance of hyper-synchronous gamma band (30-80 Hz) activity. Although both genotypes developed seizures within 40 min, the gamma amplitude was significantly smaller during the development of seizures in KO mice. Our results suggest that deficiency of S100B does not have a profound impact on spontaneous neural activity in normal conditions. However, when neural activity was sufficiently raised, activation of S100B-related pathways may take effect, resulting in modulation of neural activities.
Abstract: Glial cells have traditionally been considered to play supportive roles in the central nervous system. As recent experimental evidence suggests glial cells' participation in neural information processing, there has been a need to monitor the physiology of glial cells in vivo in the matured brain. Concurrently, identification and classification of the recorded glial cells is essential as there are at least several different kinds of glial cells. Past studies have achieved in vivo intracellular electrophysiological recording of glial cells using sharp glass microelectrodes, however, morphological recovery and identification of the recorded cells have hardly been done, due to technical difficulties. We demonstrate that use of large fragment biotinylated dextran amine (BDA) is an effective way to label a single glial cell recorded with a sharp microelectrode in vivo. Furthermore, the tracer signal amplification was achieved by a combination of avidin biotinylated horseradish peroxidase macromolecular complex (ABC) and tyramide-based methods, making multiple immunohistochemistry feasible. Using the method described in this study, we have successfully recorded and labeled cortical glial cells including astrocytes, oligodendrocytes, and microglia.
Abstract: Evidence for astrocytic participation of neuronal signal processing has cumulated in recent years. In particular, large and long-lasting cytosolic calcium surges in astrocytes, which may result in neurotransmitter release from the astrocytes, have been described in in vivo preparations. While the mechanisms for astrocytic calcium events have been extensively studied in vivo, their existence and functions in the intact brain (i.e. in vivo condition) have just started to be addressed. With the recent progress in genetics and molecular biology, it is now possible to generate gene-manipulated animals that are targeted to assess neuron-glia interaction questions. As we gain molecular tools to study neuron-glia interaction, there is an increasing need to verify in vivo experiments' findings in in vivo experimental preparations. Three physiological methods that can assess contribution of glial cells in vivo are discussed: (1) intracellular recording from a single astrocyte to record the membrane potential fluctuation; (2) multi-channel extracellular recordings to monitor mass activity of neuronal dynamics in gene-manipulated animals; (3) optical imaging using two-photon microscopy to monitor calcium dynamics in astrocytes. Combination of these methods will hopefully lead to a new development in the field of neuron-glia biology.
Abstract: Cytochrome c (CC) immunoreactivity was quantified in functionally distinct rat hippocampal inhibitory neuron populations using double immunocytochemistry and laser scanning confocal microscopy to measure the CC expression level as well as the amount of mitochondria within the cells, which is a sign of neuronal activity. The CC signal showed a similar distribution to cytochrome c oxidase histochemical staining. Strongly stained somata, dendrites and axon terminal clouds were dispersed over the low intensity neuropil staining. The staining was granular and electron microscopic investigation confirmed that the signal was localized in mitochondria. Intensively labeled neurons, showing the morphological features of inhibitory cells, were most frequently found in the principal cell layers, stratum oriens of the CA1-3 areas, stratum lucidum and hilus. These neurons contained not only a higher number of mitochondria than the principal cells but the intensity of the mitochondrial staining was evidently stronger. Among the examined interneuronal populations, parvalbumin-immunoreactive neurons were intensively labeled for CC. Calbindin D28k- (CB), somatostatin- and cholecystokinin-labeled cells showed heterogeneous CC levels, whereas calretinin-immunoreactive cells never showed a strong CC signal. CB cells in stratum oriens and alveus layers, lucidum and the hilus were strongly labeled for CC. CB cells in such regions are known to project to the medial septum and contain somatostatin. We have demonstrated that the CA1 interneurons that project to the medial septum (hippocampo-septal neurons) express a high level of CC. Thus, similar to the parvalbumin-containing basket and axo-axonic cells, the hippocampo-septal neurons potentially have a high average activity level.
Abstract: The cellular components of the brain are neurons, glia and vascular cells. These three entities form a metabolic network to sustain brain activity. Interactions among these cell types have been studied extensively in vitro, where the cells are easily accessible to physiological and pharmacological manipulations. With the advent of optical tools, it has become possible to investigate the cerebral metabolic network in vitro at the cellular and subcellular levels. However, the metabolic and homeostatic nature of neuronal-glial-vascular interactions must eventually be examined in vivo, and multi-photon imaging now provides a means to monitor neurovascular units in living experimental animals.
Abstract: Pericytes in the central nervous system (CNS) are hypothesized to be involved in important circulatory functions, including local blood flow regulation, angiogenesis, immune reaction, and regulation of blood-brain barrier. Despite these putative functions, functional correlates of pericytes in vivo are scarce. We have labeled CNS pericytes using the dextran-conjugated fluorescent calcium indicator Calcium Green I and imaged them in somatosensory cortex of the mouse in vivo. Intracellular calcium concentration in pericytes showed spontaneous surges lasting for several seconds. Furthermore, population bursts of neuronal activity were associated with increased Ca(2+) signal in a portion of the pericytes. Selective in vivo labeling of pericytes with functional markers may help reveal their physiological function in neuronal activity-associated regulation of local cerebral blood flow.
Abstract: Most neuronal interactions in the cortex occur within local circuits. Because principal cells and GABAergic interneurons contribute differently to cortical operations, their experimental identification and separation is of utmost important. We used 64-site two-dimensional silicon probes for high-density recording of local neurons in layer 5 of the somatosensory and prefrontal cortices of the rat. Multiple-site monitoring of units allowed for the determination of their two-dimensional spatial position in the brain. Of the approximately 60,000 cell pairs recorded, 0.2% showed robust short-term interactions. Units with significant, short-latency (<3 ms) peaks following their action potentials in their cross-correlograms were characterized as putative excitatory (pyramidal) cells. Units with significant suppression of spiking of their partners were regarded as putative GABAergic interneurons. A portion of the putative interneurons was reciprocally connected with pyramidal cells. Neurons physiologically identified as inhibitory and excitatory cells were used as templates for classification of all recorded neurons. Of the several parameters tested, the duration of the unfiltered (1 Hz to 5 kHz) spike provided the most reliable clustering of the population. High-density parallel recordings of neuronal activity, determination of their physical location and their classification into pyramidal and interneuron classes provide the necessary tools for local circuit analysis.
Abstract: Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca(2+)](i) events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca(2+)](i) events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca(2+)](i) activity in individual cells and a robust coordination of [Ca(2+)](i) signals in neighboring astrocytes. These findings indicate potential neuron-glia communication in the intact brain.
Abstract: Local hemodynamics of the cerebral cortex is the basis of modern functional imaging techniques, such as fMRIand PET. Despite the importance of local regulation of the blood flow, capillary level quantification of cerebral blood flow has been limited by the spatial resolution of functional imaging techniques and the depth penetration of conventional optical microscopy. Two-photon laser scanning microscopic imaging technique has the necessary spatial resolution and can image capillaries in the depth of the cortex. We have loaded the serum with fluorescein isothiocyanate dextran and quantified the flow of red blood cells (RBCs) in capillaries in layers 2/3 of the mouse somatosensory cortex in vivo. Basal capillary flux was quantified as approximately 28.9+/-13.6 RBCs/s (n=50, mean+/-S.D.) under ketamine-xylazine anesthesia and 26.7+/-16.0 RBCs/s (n=31) under urethane anesthesia. Focal interictal (epileptiform) activity was induced by local infusion of bicuculline methochloride in the cortex. We have observed that capillary blood flow increased as the cortical local field events developed into epileptiform in the vicinity of GABA receptor blockade (<300 microm from the administration site). Local blood flow in the interictal focus increased significantly (42.5+/-18.5RBCs/s, n=52) relative to the control conditions or to blood flow measured in capillaries at distant (>1mm from the focus) sites from the epileptic focus (27.8+/-12.9 RBCs/s, n=30). These results show that hyper-synchronized neural activity is associated with increased capillary perfusion in a localized cortical area. This volume is significantly smaller than the currently available resolution of the fMRI signal.
Abstract: Neurons can produce action potentials with high temporal precision. A fundamental issue is whether, and how, this capability is used in information processing. According to the 'cell assembly' hypothesis, transient synchrony of anatomically distributed groups of neurons underlies processing of both external sensory input and internal cognitive mechanisms. Accordingly, neuron populations should be arranged into groups whose synchrony exceeds that predicted by common modulation by sensory input. Here we find that the spike times of hippocampal pyramidal cells can be predicted more accurately by using the spike times of simultaneously recorded neurons in addition to the animals location in space. This improvement remained when the spatial prediction was refined with a spatially dependent theta phase modulation. The time window in which spike times are best predicted from simultaneous peer activity is 10-30 ms, suggesting that cell assemblies are synchronized at this timescale. Because this temporal window matches the membrane time constant of pyramidal neurons, the period of the hippocampal gamma oscillation and the time window for synaptic plasticity, we propose that cooperative activity at this timescale is optimal for information transmission and storage in cortical circuits.
Abstract: Information in neuronal networks is thought to be represented by the rate of discharge and the temporal relationship between the discharging neurons. The discharge frequency of neurons is affected by their afferents and intrinsic properties, and shows great individual variability. The temporal coordination of neurons is greatly facilitated by network oscillations. In the hippocampus, population synchrony fluctuates during theta and gamma oscillations (10-100 ms scale) and can increase almost 10-fold during sharp wave bursts. Despite these large changes in excitability in the sub-second scale, longer-term (minute-scale) firing rates of individual neurons are relatively constant in an unchanging environment. As a result, mean hippocampal output remains stable over time. To understand the mechanisms responsible for this homeostasis, we address the following issues: (i) Can firing rates of single cells be modified? (ii) Once modified, what mechanism(s) can maintain the changes? We show that firing rates of hippocampal pyramidal cells can be altered in a novel environment and by Hebbian pairing of physiological input patterns with postsynaptic burst discharge. We also illustrate a competition between single spikes and the occurrence of spike bursts. Since spike-inducing (suprathreshold) inputs decrease the ability of strong ('teaching') inputs to induce a burst discharge, we propose that the single spike versus burst competition presents a homeostatic regulatory mechanism to maintain synaptic strength and, consequently, firing rate in pyramidal cells.
Abstract: We pulsed the activation of neurons using a femtosecond laser. Pyramidal neurons are depolarized and fire action potentials when high intensity mode-locked infrared light irradiates somatic membranes and axon initial segments. This depolarization is reversible, does not occur with CW laser light, and appears to be due to multiphoton excitation. We describe two regimes of multiphoton optical stimulation. Low intensity, long duration laser irradiation produces a sustained depolarization, insensitive to sodium channel blockers yet sensitive to antioxidants. On the other hand, high intensity, short duration irradiation can induce fast depolarizations, which appear due to different mechanism. The combination of multiphoton stimulation and optical probing could enable systematic analysis of circuits.
Abstract: The behavior of immature cortical networks in vivo remains largely unknown. Using multisite extracellular and patch-clamp recordings, we observed recurrent bursts of synchronized neuronal activity lasting 0.5 to 3 seconds that occurred spontaneously in the hippocampus of freely moving and anesthetized rat pups. The influence of slow rhythms (0.33 and 0.1 hertz) and the contribution of both gamma-aminobutyric acid A-mediated and glutamate receptor-mediated synaptic signals in the generation of hippocampal bursts was reminiscent of giant depolarizing potentials observed in vitro. This earliest pattern, which diversifies during the second postnatal week, could provide correlated activity for immature neurons and may underlie activity-dependent maturation of the hippocampal network.
Abstract: According to the temporal coding hypothesis, neurons encode information by the exact timing of spikes. An example of temporal coding is the hippocampal phase precession phenomenon, in which the timing of pyramidal cell spikes relative to the theta rhythm shows a unidirectional forward precession during spatial behaviour. Here we show that phase precession occurs in both spatial and non-spatial behaviours. We found that spike phase correlated with instantaneous discharge rate, and processed unidirectionally at high rates, regardless of behaviour. The spatial phase precession phenomenon is therefore a manifestation of a more fundamental principle governing the timing of pyramidal cell discharge. We suggest that intrinsic properties of pyramidal cells have a key role in determining spike times, and that the interplay between the magnitude of dendritic excitation and rhythmic inhibition of the somatic region is responsible for the phase assignment of spikes.
Abstract: The interplay between principal cells and interneurons plays an important role in timing the activity of individual cells. We investigated the influence of single hippocampal CA1 pyramidal cells on putative interneurons. The activity of CA1 pyramidal cells was controlled intracellularly by current injection, and the activity of neighboring interneurons was recorded extracellularly in the urethane-anesthetized rat. Spike transmission probability between monosynaptically connected pyramidal cell-interneuron pairs was frequency dependent and highest between 5 and 25 Hz. In the awake animal, interneurons were found that had place-modulated firing rates, with place maps similar to their presynaptic pyramidal neuron. Thus, single pyramidal neurons can effectively determine the firing patterns of their interneuron targets.
Abstract: Cortical pyramidal cells fire single spikes and complex spike bursts. However, neither the conditions necessary for triggering complex spikes, nor their computational function are well understood. CA1 pyramidal cell burst activity was examined in behaving rats. The fraction of bursts was not reliably higher in place field centers, but rather in places where discharge frequency was 6-7 Hz. Burst probability was lower and bursts were shorter after recent spiking activity than after prolonged periods of silence (100 ms-1 s). Burst initiation probability and burst length were correlated with extracellular spike amplitude and with intracellular action potential rising slope. We suggest that bursts may function as "conditional synchrony detectors," signaling strong afferent synchrony after neuronal silence, and that single spikes triggered by a weak input may suppress bursts evoked by a subsequent strong input.
Abstract: A modular multichannel microdrive ('hyperdrive') is described. The microdrive uses printed circuit board technology and flexible fused silica capillaries. The modular design allows for the fabrication of 4-32 independently movable electrodes or 'tetrodes'. The drives are re-usable and re-loading the drive with electrodes is simple.
Abstract: Local versus distant coherence of hippocampal CA1 pyramidal cells was investigated in the behaving rat. Temporal cross-correlation of pyramidal cells revealed a significantly stronger relationship among local (<140 microm) pyramidal neurons compared with distant (>300 microm) neurons during non-theta-associated immobility and sleep but not during theta-associated running and walking. In contrast, cross-correlation between local pyramidal cell-interneuron pairs was significantly stronger than between distant pairs during theta oscillations but were similar during non-theta-associated behaviors. We suggest that network state-dependent functional clustering of neuronal activity emerges because of the differential contribution of the main excitatory inputs, the perforant path, and Schaffer collaterals during theta and non-theta behaviors.
Abstract: What determines the firing rate of cortical neurons in the absence of external sensory input or motor behavior, such as during sleep? Here we report that, in a familiar environment, the discharge frequency of simultaneously recorded individual CA1 pyramidal neurons and the coactivation of cell pairs remain highly correlated across sleep-wake-sleep sequences. However, both measures were affected when new sets of neurons were activated in a novel environment. Nevertheless, the grand mean firing rate of the whole pyramidal cell population remained constant across behavioral states and testing conditions. The findings suggest that long-term firing patterns of single cells can be modified by experience. We hypothesize that increased firing rates of recently used neurons are associated with a concomitant decrease in the discharge activity of the remaining population, leaving the mean excitability of the hippocampal network unaltered.
Abstract: Transfer of neuronal patterns from the CA3 to CA1 region was studied by simultaneous recording of neuronal ensembles in the behaving rat. A nonlinear interaction among pyramidal neurons was observed during sharp wave (SPW)-related population bursts, with stronger synchrony associated with more widespread spatial coherence. SPW bursts emerged in the CA3a-b subregions and spread to CA3c before invading the CA1 area. Synchronous discharge of >10% of the CA3 within a 100 ms window was required to exert a detectable influence on CA1 pyramidal cells. Activity of some CA3 pyramidal neurons differentially predicted the ripple-related discharge of circumscribed groups of CA1 pyramidal cells. We suggest that, in SPW behavioral state, the coherent discharge of a small group of CA3 cells is the primary cause of spiking activity in CA1 pyramidal neurons.
Abstract: Simultaneous recording from large numbers of neurons is a prerequisite for understanding their cooperative behavior. Various recording techniques and spike separation methods are being used toward this goal. However, the error rates involved in spike separation have not yet been quantified. We studied the separation reliability of "tetrode" (4-wire electrode)-recorded spikes by monitoring simultaneously from the same cell intracellularly with a glass pipette and extracellularly with a tetrode. With manual spike sorting, we found a trade-off between Type I and Type II errors, with errors typically ranging from 0 to 30% depending on the amplitude and firing pattern of the cell, the similarity of the waveshapes of neighboring neurons, and the experience of the operator. Performance using only a single wire was markedly lower, indicating the advantages of multiple-site monitoring techniques over single-wire recordings. For tetrode recordings, error rates were increased by burst activity and during periods of cellular synchrony. The lowest possible separation error rates were estimated by a search for the best ellipsoidal cluster shape. Human operator performance was significantly below the estimated optimum. Investigation of error distributions indicated that suboptimal performance was caused by inability of the operators to mark cluster boundaries accurately in a high-dimensional feature space. We therefore hypothesized that automatic spike-sorting algorithms have the potential to significantly lower error rates. Implementation of a semi-automatic classification system confirms this suggestion, reducing errors close to the estimated optimum, in the range 0-8%.
Abstract: We examined whether excitation and inhibition are balanced in hippocampal cortical networks. Extracellular field and single-unit activity were recorded by multiple tetrodes and multisite silicon probes to reveal the timing of the activity of hippocampal CA1 pyramidal cells and classes of interneurons during theta waves and sharp wave burst (SPW)-associated field ripples. The somatic and dendritic inhibition of pyramidal cells was deduced from the activity of interneurons in the pyramidal layer [int(p)] and in the alveus and st. oriens [int(a/o)], respectively. Int(p) and int(a/o) discharged an average of 60 and 20 degrees before the population discharge of pyramidal cells during the theta cycle, respectively. SPW ripples were associated with a 2.5-fold net increase of excitation. The discharge frequency of int(a/o) increased, decreased ("anti-SPW" cells), or did not change ("SPW-independent" cells) during SPW, suggesting that not all interneurons are innervated by pyramidal cells. Int(p) either fired together with (unimodal cells) or both before and after (bimodal cells) the pyramidal cell burst. During fast-ripple oscillation, the activity of interneurons in both the int(p) and int(a/o) groups lagged the maximum discharge probability of pyramidal neurons by 1-2 msec. Network state changes, as reflected by field activity, covaried with changes in the spike train dynamics of single cells and their interactions. Summed activity of parallel-recorded interneurons, but not of pyramidal cells, reliably predicted theta cycles, whereas the reverse was true for the ripple cycles of SPWs. We suggest that network-driven excitability changes provide temporal windows of opportunity for single pyramidal cells to suppress, enable, or facilitate selective synaptic inputs.
Abstract: In contrast to sensory cortical areas of the brain, the relevant physiological inputs to the hippocampus, leading to selective activation of pyramidal cells, are largely unknown. Pyramidal cells are thought to be phasically activated by spatial cues and a variety of sensory and motor stimuli. Here, we used a behavioural 'space clamp' method, which involved the confinement of the actively running animal in a defined position in space (running wheel) and kept sensory inputs constant. Twelve percent of the recorded CA1 pyramidal cells were selectively active while the rat was running in the wheel. Cell firing was specific to the direction of running and disappeared after rotating the recording apparatus. The discharge frequency of pyramidal cells and interneurons was sustained as long as the rat ran continuously in the wheel. Furthermore, the discharge frequency of pyramidal cells and interneurons increased with increasing running velocity, even though the frequency of hippocampal theta waves remained constant. The discharge frequency of some 'wheel-related' pyramidal cells could increase more than 10-fold between 10 and 100 cm/s, whereas the firing rate of 'non-wheel' cells remained constantly low. We hypothesize that: (i) a necessary condition for place-specific discharge of hippocampal pyramidal cells is the presence of theta oscillation; and (ii) relevant stimuli can tonically and selectively activate hippocampal pyramidal cells as long as theta activity is present.
Abstract: This study examined intermittent, high-frequency (100-200 Hz) oscillatory patterns in the CA1 region of the hippocampus in the absence of theta activity, i.e., during and in between sharp wave (SPW) bursts. Pyramidal and interneuronal activity was phase-locked not only to large amplitude (>7 SD from baseline) oscillatory events, which are present mainly during SPWs, but to smaller amplitude (<4 SD) patterns, as well. Large-amplitude events were in the 140-200 Hz, "ripple" frequency range. Lower-amplitude events, however, contained slower, 100-130 Hz ("slow") oscillatory patterns. Fast ripple waves reversed just below the CA1 pyramidal layer, whereas slow oscillatory potentials reversed in the stratum radiatum and/or in the stratum oriens. Parallel CA1-CA3 recordings revealed correlated CA3 field and unit activity to the slow CA1 waves but not to fast ripple waves. These findings suggest that fast ripples emerge in the CA1 region, whereas slow (100-130 Hz) oscillatory patterns are generated in the CA3 region and transferred to the CA1 field.
Abstract: In the hippocampus, spatial representation of the environment has been suggested to be coded by either the firing rate of pyramidal cell assemblies or the relative timing of the action potentials during the theta EEG cycle. Here, we used a behavioural 'space clamp' method, which involved the confinement of the actively running animal in a defined position in space (running wheel) to examine how 'spatial' and other inputs affect firing rate and timing of hippocampal CA1 pyramidal cells and interneurons. Nineteen per cent of the recorded CA1 pyramidal cells were selectively active while the rat was running in the wheel in a given direction ('wheel' cells). Spatial rotation of the apparatus showed that selective discharge of pyramidal cells in the wheel was under the combined influence of distal and apparatus cues. During steady running, both discharge rate and theta phase were constant. Rotation of the wheel apparatus resulted in a shift of both firing rate and preferred theta phase. The discharge frequency of 'wheel' cells increased threefold (on average) with increasing running velocity. In contrast, change in running speed had relatively little effect on the theta phase-related discharge of 'wheel' cells. Our findings indicate that mechanisms that regulate rate and phase of spikes are overlapping but not necessarily identical.
Abstract: Information in neuronal networks may be represented by the spatiotemporal patterns of spikes. Here we examined the temporal coordination of pyramidal cell spikes in the rat hippocampus during slow-wave sleep. In addition, rats were trained to run in a defined position in space (running wheel) to activate a selected group of pyramidal cells. A template-matching method and a joint probability map method were used for sequence search. Repeating spike sequences in excess of chance occurrence were examined by comparing the number of repeating sequences in the original spike trains and in surrogate trains after Monte Carlo shuffling of the spikes. Four different shuffling procedures were used to control for the population dynamics of hippocampal neurons. Repeating spike sequences in the recorded cell assemblies were present in both the awake and sleeping animal in excess of what might be predicted by random variations. Spike sequences observed during wheel running were "replayed" at a faster timescale during single sharp-wave bursts of slow-wave sleep. We hypothesize that the endogenously expressed spike sequences during sleep reflect reactivation of the circuitry modified by previous experience. Reactivation of acquired sequences may serve to consolidate information.
Abstract: Spike transmission probability between pyramidal cells and interneurons in the CA1 pyramidal layer was investigated in the behaving rat by the simultaneous recording of neuronal ensembles. Population synchrony was strongest during sharp wave (SPW) bursts. However, the increase was three times larger for pyramidal cells than for interneurons. The contribution of single pyramidal cells to the discharge of interneurons was often large (up to 0.6 probability), as assessed by the presence of significant (<3 ms) peaks in the cross-correlogram. Complex-spike bursts were more effective than single spikes. Single cell contribution was higher between SPW bursts than during SPWs or theta activity. Hence, single pyramidal cells can reliably discharge interneurons, and the probability of spike transmission is behavior dependent.
Abstract: In learning matrix associative memory networks, the choice of threshold value is one of the most significant factors for determining the recall performance. Choice of threshold is especially important for multi-step recall, as each network state is dependent on the prior states. Recently, Gibson and Robinson used a statistical approximation to formalize the dynamics of partially connected recurrent networks. Using this formalism, we evaluate all of the possible thresholding sequences and find the sequence that results in the highest storage capacity. The resulting optimal strategy can be closely approximated by a threshold that is proportional to the activation of the network plus an offset. The performance of a simulated associative memory is shown to match well with the predictions of the theory. This strategy corresponds to one of the simplest putative roles of interneurons which provide a linear output proportional to the total unweighted input from the principal cells.
