INSERM UMR 1072 Unité de Neurobiologie des canaux Ioniques et de la Synapse (UNIS) Faculté de Médecine - Secteur Nord Université d'Aix-Marseille CS80011 Bd Pierre Dramard 13344 Marseille Cedex 15 - FRANCE
Abstract: Frequency and timing of action potential discharge are key elements for coding and transfer of information between neurons. The nature and location of the synaptic contacts, the biophysical parameters of the receptor-operated channels and their kinetics of activation are major determinants of the firing behaviour of each individual neuron. Ultimately the intrinsic excitability of each neuron determines the input-output function. Here we evaluate the influence of spontaneous GABAergic synaptic activity on the timing of action potentials in Layer 2/3 pyramidal neurones in acute brain slices from the somatosensory cortex of young rats. Somatic dynamic current injection to mimic synaptic input events was employed, together with a simple computational model that reproduce subthreshold membrane properties. Besides the well-documented control of neuronal excitability, spontaneous background GABAergic activity has a major detrimental effect on spike timing. In fact, GABA(A) receptors tune the relationship between the excitability and fidelity of pyramidal neurons via a postsynaptic (the reversal potential for GABA(A) activity) and a presynaptic (the frequency of spontaneous activity) mechanism. GABAergic activity can decrease or increase the excitability of pyramidal neurones, depending on the difference between the reversal potential for GABA(A) receptors and the threshold for action potential. In contrast, spike time jitter can only be increased proportionally to the difference between these two membrane potentials. Changes in excitability by background GABAergic activity can therefore only be associated with deterioration of the reliability of spike timing.
Abstract: Analysis of synaptic transmission, synaptic plasticity, axonal processing, synaptic timing or electrical coupling requires the simultaneous recording of both the pre- and postsynaptic compartments. Paired-recording technique of monosynaptically connected neurons is also an appropriate technique to probe the function of small molecules (calcium buffers, peptides or small proteins) at presynaptic terminals that are too small to allow direct whole-cell patch-clamp recording. We describe here a protocol for obtaining, in acute and cultured slices, synaptically connected pairs of cortical and hippocampal neurons, with a reasonably high probability. The protocol includes four main stages (acute/cultured slice preparation, visualization, recording and analysis) and can be completed in approximately 4 h.
Abstract: In the cortex, synaptic latencies display small variations ( approximately 1-2 ms) that are generally considered to be negligible. We show here that the synaptic latency at monosynaptically connected pairs of L5 and CA3 pyramidal neurons is determined by the presynaptic release probability (Pr): synaptic latency being inversely correlated with the amplitude of the postsynaptic current and sensitive to manipulations of Pr. Changes in synaptic latency were also observed when Pr was physiologically regulated in short- and long-term synaptic plasticity. Paired-pulse depression and facilitation were respectively associated with increased and decreased synaptic latencies. Similarly, latencies were prolonged following induction of presynaptic LTD and reduced after LTP induction. We show using the dynamic-clamp technique that the observed covariation in latency and synaptic strength is a synergistic combination that significantly affects postsynaptic spiking. In conclusion, amplitude-related variation in latency represents a putative code for short- and long-term synaptic dynamics in cortical networks.
Abstract: Dendritic spines are morphing structures believed to provide a cellular substrate for synaptic plasticity. It has been suggested that the actin cytoskeleton is the target of molecular mechanisms regulating spine morphology. Here we hypothesized that acidic calponin, an actin-binding protein, is one of the key regulators of actin filaments during spine plasticity. Our data showed that the overexpression of acidic calponin-GFP (green fluorescent protein) in primary cultures of rat hippocampal neurons causes an elongation of spines and an increase of their density as compared with those of GFP-expressing neurons. These effects required the actin-binding domains of acidic calponin. The close apposition of the presynatic marker synaptophysin to these long spines and the presence of specific postsynaptic markers actin, PSD-95, NR1, and GluR1 suggested the existence of functional excitatory synaptic contacts. Indeed, electrophysiological data showed that the postsynaptic overexpression of acidic calponin enhanced the frequency of miniature excitatory postsynaptic currents as compared with that of GFP-expressing neurons, but did not affect their properties such as amplitude, rise time, and half width. Studies in heterologous cells revealed that acidic calponin reorganized the actin filaments and stabilized them. Taken together, these findings show that acidic calponin regulates dendritic spine morphology and density, likely via regulation of the actin cytoskeleton reorganization and dynamic. Furthermore, the acidic calponin-induced spines are able to establish functional glutamatergic synapses. Such data suggest that acidic calponin is a key factor in the regulation of spine plasticity and synaptic activity.
Abstract: The consequences of sustained activation of GABA(B) receptors on GABA(B)-mediated inhibition and network activity were investigated in the neonatal rat hippocampus using whole-cell and extracellular field recordings. GABA(B)-mediated presynaptic control of gamma-aminobutyric acid (GABA) release progressively diminished with time in spite of the continued presence of the agonist (100 microM baclofen, 15 min), indicating acute desensitization of presynaptic GABA(B)-mediated inhibition on GABAergic terminals. By contrast, neither GABA(B)-mediated inhibition of glutamate release nor postsynaptic GABA(B)-mediated inhibition seemed to produce this desensitization. Efficacy of presynaptic GABA(B) receptors was still reduced by 49% 30 min after baclofen washout, suggesting a long timeframe for recovery from desensitization. The 15-min baclofen application was followed by a dramatic modification of the spontaneous network activity, with the occurrence of epileptiform events called ictal-like discharges (ILDs). Extracellular field recordings confirmed the epileptic nature of the discharges that could be recorded up to 4 h after baclofen washout. ILDs did not occur when the GABA(B) receptor antagonist CGP35348 was coapplied with baclofen. This indicates that ILD induction is a consequence of the sustained activation of GABA(B) receptors and the correlated changes in GABA(B)-mediated inhibition. Furthermore, ILDs were also induced when blocking with CGP35348 an amount of GABA(B) receptors that exactly mimicked the loss of inhibition obtained with desensitization. These results show that presynaptic GABA(B)-mediated inhibition of GABA release acutely and specifically desensitizes following a sustained application of the GABA(B) receptor agonist baclofen. Conditions that induce desensitization of the GABA(B)-mediated responses also trigger persistent epileptiform discharges in the neonatal rat hippocampus.
Abstract: Here we examine the role of the spontaneous synaptic activity generated by the developing rat hippocampus in the formation of functional gamma-aminobutyric acid (GABA) synapses. Intact hippocampal formations (IHFs) were dissected at birth and incubated for 1 day in control or tetrodotoxin (TTX)-supplemented medium at 25 degrees C. After the incubation, miniature GABA(A)-mediated postsynaptic currents (mGABA(A)-PSCs) were recorded in whole-cell voltage-clamped CA3 pyramidal neurones from IHF-derived slices. After 1 day in vitro in control medium, the frequency of mGABA(A)-PSCs was similar to that recorded in acute slices obtained 1 day after birth, but significantly higher than the frequency recorded from acute slices just after birth. These results suggest that the factors required in vivo for the formation of functional GABAergic synapses are preserved in the IHFs in vitro. The frequency increase was prevented when IHFs were incubated for 1 day with TTX. TTX treatment affected neither the morphology of CA3 pyramidal neurones nor cell viability. The TTX effects were reproduced when IHFs were incubated in the presence of glutamatergic or GABAergic ionotropic receptor antagonists or in high divalent cationic medium. The present results indicate that the spontaneous synaptic activity generated by the developing hippocampus is a key player in the formation of functional GABAergic synapses, possibly via network events requiring both glutamatergic and GABAergic receptors.
Abstract: Activity-dependent long-term changes in synaptic efficacy are thought to be important in learning, memory formation, neuronal development and pathological states of neuronal excitability in the CNS. For the past two decades, numerous studies have investigated long-term changes in synaptic efficacy at excitatory glutamatergic synapses. Although inhibitory synapses are essential for proper functioning of the neuronal network, attention has focused only recently on describing and characterizing plasticity at these types of synapse. Not surprisingly, different forms of plasticity at GABAergic, and the closely related glycinergic, synapses have been reported in several regions of the brain. Here we review these different forms of plasticity and focus on their possible roles in developing and adult neuronal networks.
Abstract: Morphological studies suggest that the primate hippocampus develops extensively before birth, but little is known about its functional development. Patch-clamp recordings of hippocampal neurons and reconstruction of biocytin-filled pyramidal cells were performed in slices of macaque cynomolgus fetuses delivered by cesarean section. We found that during the second half of gestation, axons and dendrites of pyramidal cells grow intensively by hundreds of micrometers per day to attain a high level of maturity near term. Synaptic currents appear around midgestation and are correlated with the level of morphological differentiation of pyramidal cells: the first synapses are GABAergic, and their emergence correlates with the growth of apical dendrite into stratum radiatum. A later occurrence of glutamatergic synaptic currents correlates with a further differentiation of the axodendritic tree and the appearance of spines. Relying on the number of dendritic spines, we estimated that hundreds of new glutamatergic synapses are established every day on a pyramidal neuron during the last third of gestation. Most of the synaptic activity is synchronized in spontaneous slow ( approximately 0.1 Hz) network oscillations reminiscent of the giant depolarizing potentials in neonatal rodents. Epileptiform discharges can be evoked by the GABA(A) receptor antagonist bicuculline by the last third of gestation, and postsynaptic GABA(B) receptors contribute to the termination of epileptiform discharges. Comparing the results obtained in primates and rodents, we conclude that the template of early hippocampal network development is conserved across the mammalian evolution but that it is shifted toward fetal life in primate.
Abstract: GABAergic (GABA = gamma-aminobutyric acid) neurons from different brain regions contain high levels of parvalbumin, both in their soma and in their neurites. Parvalbumin is a slow Ca(2+) buffer that may affect the amplitude and time course of intracellular Ca(2+) transients in terminals after an action potential, and hence may regulate short-term synaptic plasticity. To test this possibility, we have applied paired-pulse stimulations (with 30- to 300-ms intervals) at GABAergic synapses between interneurons and Purkinje cells, both in wild-type (PV+/+) mice and in parvalbumin knockout (PV-/-) mice. We observed paired-pulse depression in PV+/+ mice, but paired-pulse facilitation in PV-/- mice. In paired recordings of connected interneuron-Purkinje cells, dialysis of the presynaptic interneuron with the slow Ca(2+) buffer EGTA (1 mM) rescues paired-pulse depression in PV-/- mice. These data show that parvalbumin potently modulates short-term synaptic plasticity.
Abstract: The role of internal calcium stores in the induction of long-term depression at GABAergic synapses was investigated in the neonatal rat hippocampus. Whole-cell recordings of CA3 pyramidal neurons were performed on hippocampal slices from neonatal (2-4 d old) rats. In control conditions, tetanic stimulation (TS) evoked an NMDA-dependent long-term depression of GABA(A) receptor-mediated postsynaptic responses (LTD(GABA-A)). LTD(GABA-A) was prevented when the cells were loaded with ruthenium red, a blocker of Ca2+-induced Ca2+ release (CICR) stores, whereas loading the cells with heparin, a blocker of IP3-induced Ca2+ release stores, had no effect. The effects of ryanodine, another compound that interferes with CICR stores, were also investigated. Intracellular injection of ryanodine prevented the induction of LTD(GABA-A) only when the TS was preceded by depolarizing pulses that increase intracellular Ca2+ concentration. When applied in the bath, ryanodine prevented the induction of LTD(GABA-A). Altogether, these results suggest that ryanodine acts as a Ca2+-dependent blocker of CICR stores and that the induction of LTD(GABA-A) required the activation of both presynaptic and postsynaptic CICR stores.
Abstract: Synaptic plasticity at excitatory glutamatergic synapses is believed to be instrumental in the maturation of neuronal networks. Using whole-cell patch-clamp recordings, we have studied the mechanisms of induction and expression of long-term depression at excitatory GABAergic synapses in the neonatal rat hippocampus (LTD(GABA-A)). We report that the induction of LTD(GABA-A) requires a GABA(A) receptor-mediated membrane depolarization, which is necessary to remove the Mg(2+) block from postsynaptic NMDA receptors. LTD(GABA-A) is associated with an increase in the coefficient of variation of evoked GABA(A) receptor-mediated synaptic currents and a decrease in the frequency, but not amplitude, of Sr(2+)-induced asynchronous GABA(A) quantal events. We conclude that LTD(GABA-A) induction requires the activation of both GABA(A) and NMDA postsynaptic receptors and that its expression is likely presynaptic.
Abstract: 1. The plasticity of GABAergic synapses was investigated in neonatal rat hippocampal slices obtained between postnatal days 3 and 6 using intracellular recording techniques. Ionotropic glutamate receptor antagonists were present throughout the experiments to isolate GABAA receptor-mediated postsynaptic potentials (GABAA PSPs) or currents (GABAA PSCs). 2. Repetitive depolarizing pulses (20 pulses, 0.5 s duration, at 0.1 Hz, each pulse generating 4-6 action potentials) induced a long-term potentiation in the slope and amplitude of the evoked GABAA PSPs and GABAA PSCs. 3. Long-term potentiation was prevented by intracellular injection of the calcium chelator BAPTA (50 mM), or when the voltage-dependent calcium channels blockers Ni2+ (50 microM) and nimodipine (10 microM) were bath applied. 4. Repetitive depolarizing pulses induced a persistent (over 1 h) increase in the frequency of spontaneous GABAA PSCs. 5. Repetitive depolarizing pulses induced a long-lasting increase in the frequency of miniature GABAA PSCs, without altering their amplitude or decay-time constant. 6. It is concluded that the postsynaptic activation of voltage-dependent calcium channels leads to a long-term potentiation of GABAergic synaptic transmission in neonatal rat hippocampus. This form of plasticity is expressed as an increase in the probability of GABA release or in the number of functional synapses, rather than as an upregulation of postsynaptic GABAA receptor numbers or conductance at functional synapses.
Abstract: gamma-aminobutyric acid (GABA) is the principal neurotransmitter of inhibition in the adult mammalian brain. However, at early stages of development, including the embryonic period and first week of postnatal life, GABA plays the role of main neurotransmitter of excitation. The paradoxical excitatory effect of GABA is caused by an inverted chloride gradient and, therefore, a depolarizing direction of GABA type A (GABAA) receptor mediated responses. In addition, another type of GABAergic inhibition mediated by postsynaptic GABA type B (GABAB) receptors is not functional at early stage of life. In the neonatal rat hippocampus, GABA, acting via GABAA receptors, activates voltage-gated sodium and calcium channels and potentiates the activity of N-methyl-D-aspartate (NMDA) receptors by reducing their voltage-dependent Mg2+ block. The temporal window when GABA exerts excitatory actions coincides with a particular pattern of activity of hippocampal neuronal network that is characterized by periodical giant depolarizing potentials (GDPs) reminiscent of interictal-like epileptiform discharges. Recent studies have shown that GDPs result from the synchronous discharge of GABAergic interneurons and principal glutamatergic pyramidal cells, and they are mediated by the synergistic excitatory actions of GABAA and glutamate receptors. GDPs provide synchronous intracellular Ca2+ oscillations and may, therefore, be implicated in hebbian modulation of developing synapses and activity-dependent formation of the hippocampal network.
Abstract: gamma-Aminobutyric acid-B(GABAB) receptor-dependent and -independent components of paired-pulse depression (PPD) were investigated in the rat CA3 hippocampal region. Intracellular and whole cell recordings of CA3 pyramidal neurons were performed on hippocampal slices obtained from neonatal (5-7 day old) and adult (27-34 day old) rats. Electrical stimulation in the hilus evoked monosynaptic GABAA postsynaptic currents (eIPSCs) isolated in the presence of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)2-amino-5-phosphovaleric acid (-AP5, 50 microM) with 2(triethylamino)-N-(2,6-dimethylphenyl) acetamine (QX314) filled electrodes. In adult CA3 pyramidal neurons, when a pair of identical stimuli was applied at interstimulus intervals (ISIs) ranging from 50 to 1,500 ms the amplitude of the second eIPSC was depressed when compared with the first eIPSC. This paired-pulse depression (PPD) was partially blocked by P-3-aminoprophyl -P-diethoxymethylphosphoric acid (CGP35348, 0.5 mM), a selective GABAB receptor antagonist. In neonates, PPD was restricted to ISIs shorter than 200 ms and was not affected by CGP35348. The GABAB receptor agonist baclofen reduced the amplitude of eIPSCs in a dose-dependent manner with the same efficiency in both adults and neonates. Increasing the probability of transmitter release with high Ca2+ (4 mM)/low Mg2+ (0.3 mM) external solution revealed PPD in neonatal CA3 pyramidal neurons that was 1) partially prevented by CGP35348, 2) independent of the membrane holding potential of the recorded cell, and 3) not resulting from a change in the reversal potential of GABAA eIPSCs. In adults the GABA uptake blocker tiagabine (20 microM) increased the duration of eIPSCs and the magnitude of GABAB receptor-dependent PPD. In neonates, tiagabine also increased duration of eIPSCs but to a lesser extent than in adult and did not reveal a GABAB receptor-dependent PPD. These results demonstrate that although GABAB receptor-dependent and -independent mechanisms of presynaptic inhibition are present onGABAergic terminals and functional, they do not operate at the level of monosynaptic GABAergic synaptic transmission at early stages of development. Absence of presynaptic autoinhibition of GABA release seems to be due to the small amount of transmitter that can access presynaptic regulatory sites.
Abstract: 1. We investigated the effects of the selective gamma-aminobutyric acid-B (GABAB) receptor antagonist, P-3 aminopropyl-P-diethoxymethyl phosphoric acid (CGP 35348), on spontaneous and evoked postsynaptic potentials (PSPs) and currents (PSCs) in CA3 pyramidal cells and interneurons of hippocampal slices obtained between postnatal day 3 and 7 with the use of intracellular and whole cell recording techniques. The intracellular pipette solution contained either 2 M CsCl or 50 mM 2(triethylamino)-N-(2,6-dimethylphenyl) acetamine (QX314) dissolved in 2 M KMeSO4. Cesium and QX314 block postsynaptic responses mediated by GABAB receptors. 2. Under control conditions, bath application of CGP 35348 (0.5-1 mM) progressively increased the duration of spontaneous and evoked polysynaptic giant GABAergic PSPs leading to the appearance of ictal-like discharges. The effects of CGP 35348 were dose dependent and voltage independent. 3. In CA3 pyramidal neurons, CGP 35348 (0.5 mM) had no effect on monosynaptic GABAergic inhibitory PSPs (IPSPs) that were isolated in the presence of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)2-amino-5-phosphovaleric acid (D-APV, 50 microM). Similarly, CGP 35348 (0.5 mM) had no effect on monosynaptic glutamatergic excitatory PSPs (EPSPs) that were isolated in the presence of bicuculline (10 microM) and high divalent cation artificial cerebrospinal fluid (ACSF; 6 mM Mg2+/4 mM Ca2+). 4. In CA3 pyramidal neurons exposed to CNQX (20 microM) and D-APV (50 microM), application of the potassium channel blocker 4-aminopyridine (4-AP, 50 microM) generated synchronous giant GABAergic PSPS that were blocked in the presence of high divalent cation ACSF (6 mM Mg2+/4 mM Ca2+) or bicuculline (10 microM). The duration of these synchronous GABAergic PSPs was prolonged in the presence of CGP 35348 (0.5 mM) but did not lead to the appearance of ictal-like discharges. 5. In the presence of bicuculline, interictal giant glutamatergic potentials were observed in simultaneously recorded CA3 pyramidal cells and interneurons. CGP 35348 (0.5 mM) progressively increased the duration of these bicuculline-induced glutamatergic bursts leading to the simultaneous appearance of ictal discharges in both pyramidal cells and interneurons. 6. These results suggest that in the neonatal CA3 hippocampal region, when synchronous giant polysynaptic GABAergic PSPs are present (i.e., under basal, control conditions), spontaneously released GABA reaches a critical level and activates GABAB receptors on both pyramidal cells and interneurons thus regulating the level of glutamatergic and GABAergic activity in the CA3 neuronal network.
Abstract: 1. Activity-dependent plasticity of GABAergic synaptic transmission was investigated in neonatal rat hippocampal slices obtained between postnatal day (P) 2-10 using intracellular recording techniques. In all experiments, AMPA receptors were blocked by continual application of CNQX (10 microM). 2. Between P2 and P4, tetanic stimulation (TS) evoked NMDA receptor-dependent long-term depression of monosynaptic GABAA EPSPS (LTDGABAA). In contrast, when NMDA receptors were blocked by D-AP5 (50 microM), the same TS evoke long-term potentiation of GABAA EPSPS (LTPGABAA). 3. Between P6 and P10, TS failed to produce either LTP or LTD or hyperpolarizing monosynaptic GABAA IPSPS under the same recording conditions. However, when GABAergic potentials were rendered depolarizing (KCl-filled electrode) Ts induced either LTPGABAA or LTDGABAA in the presence or absence of D-AP5, respectively. 4. Both LTPGABAA and LTDGABAA were specific to the conditioned pathway and could be sequentially expressed at the same synapses. Potentiation of GABAergic synaptic efficacy was induced more easily following previous induction of LTDGABAA than in naive slices. 5. In conclusion, early in development, bidirectional synaptic plasticity is expressed by GABAA receptors and the activation (or not) of NMDA receptors determines the induction of either LTPGABAA or LTDGABAA.
