Increased GABAergic function in mouse models of Huntington's disease: reversal by BDNF

Huntington's disease (HD) is characterized by loss of striatal gamma-aminobutyric acid (GABA)ergic medium-sized spiny projection neurons (MSSNs), whereas some classes of striatal interneurons are relatively spared. Striatal interneurons provide most of the inhibitory synaptic input to MSSNs and use GABA as their neurotransmitter. We reported previously alterations in glutamatergic synaptic activity in the R6/2 and R6/1 mouse models of HD. In the present study, we used whole-cell voltage clamp recordings to examine GABAergic synaptic currents in MSSNs from striatal slices in these two mouse models compared to those in age-matched control littermates. The frequency of spontaneous GABAergic synaptic currents was increased significantly in MSSNs from R6/2 transgenics starting around 5-7 weeks (when the overt behavioral phenotype begins) and continuing in 9-14-week-old mice. A similar increase was observed in 12-15-month-old R6/1 transgenics. Bath application of brain-derived neurotrophic factor, which is downregulated in HD, significantly reduced the frequency of spontaneous GABAergic synaptic currents in MSSNs from R6/2 but not control mice at 9-14 weeks. Increased GABA current densities also occurred in acutely isolated MSSNs from R6/2 animals. Immunofluorescence demonstrated increased expression of the ubiquitous alpha1 subunit of GABA(A) receptors in MSSNs from R6/2 animals. These results indicate that increases in spontaneous GABAergic synaptic currents and postsynaptic receptor function occur in parallel to progressive decreases in glutamatergic inputs to MSSNs. In conjunction, both changes will severely alter striatal outputs to target areas involved in the control of movement.     

Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease

Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.     

Endogenous cannabinoids mediate the effect of BDNF at CA1 inhibitory synapses in the hippocampus

Brain‐derived neurotrophic factor (BDNF), traditionally known for promoting neuronal growth and development, is also a modulator of synaptic transmission. In addition to the well‐characterized effects at excitatory synapses, BDNF has been shown to acutely suppress inhibitory neurotransmission; however, the underlying mechanisms are unclear. We have previously shown that at inhibitory synapses in layer 2/3 of the somatosensory cortex, BDNF induces the mobilization of endogenous cannabinoids (eCBs) that act retrogradely to suppress GABA release. Here, we hypothesized that in the hippocampus, BDNF acts similarly via eCB signaling to suppress GABAergic transmission. We found that the acute application of BDNF reduced the spontaneous inhibitory postsynaptic currents (sIPSCs) via postsynaptic TrkB receptor activation. The suppressive effects of BDNF required eCB signaling, as this effect on sIPSCs was prevented by a CB1 receptor antagonist. Further, blocking the postsynaptic eCB release prevented the effect of BDNF, whereas eCB reuptake inhibition enhanced the effect of BDNF. These results suggest that BDNF triggers the postsynaptic release of eCBs. To identify the specific eCB release by BDNF, we tested the effects of disrupting the synthesis or degradation of 2‐arachidonoylcglycerol (2‐AG). Blocking 2‐AG synthesis prevented the effect of BDNF and blocking 2‐AG degradation enhanced the effect of BDNF. However, there was no change in the effect of BDNF when anandamide degradation was blocked. Collectively, these results suggest that in the hippocampus, BDNF‐TrkB signaling induces the postsynaptic release of the endogenous cannabinoid 2‐AG, which acts retrogradely on the presynaptic CB1 receptors to suppress GABA release.     

Chronic cocaine sensitizes striatal GABAergic synapses to the stimulation of cannabinoid CB1 receptors

Behavioural studies indicate that cannabinoid receptors are implicated in cocaine addiction. The synaptic underpinning of cocaine-cannabinoid receptor interaction is however, obscure. We have studied electrophysiologically the sensitivity of cannabinoid receptors modulating synaptic transmission in the striatum of rats exposed to cocaine. One-day treatment with cocaine did not modify the synaptic response to HU210, a cannabinoid CB1 receptor agonist. Seven days cocaine-treatment, conversely, caused conditioned place preference, and sensitized striatal GABAergic synapses to the presynaptic effect of cannabinoid CB1 receptor stimulation. The cannabinoid receptor-induced modulation of glutamate transmission was unaltered by cocaine. Furthermore, the effects of chronic cocaine on cannabinoid-mediated regulation of striatal GABA synapses were attenuated one week after the discontinuation of cocaine, and absent two weeks later, indicating the progressive reversibility of the adaptations of cannabinoid system during abstinence of drug consumption. Our data support the concept that modulation of cannabinoid receptors might be useful against drug abuse.     

Impaired striatal GABA transmission in experimental autoimmune encephalomyelitis

Synaptic dysfunction triggers neuronal damage in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). While excessive glutamate signaling has been reported in the striatum of EAE, it is still uncertain whether GABA synapses are altered. Electrophysiological recordings showed a reduction of spontaneous GABAergic synaptic currents (sIPSCs) recorded from striatal projection neurons of mice with MOG_(35−55)-induced EAE. GABAergic sIPSC deficits started in the acute phase of the disease (20-25 days post immunization, dpi), and were exacerbated at later time-points (35, 50, 70 and 90 dpi). Of note, in slices they were independent of microglial activation and of release of TNF-α. Indeed, sIPSC inhibition likely involved synaptic inputs arising from GABAergic interneurons, because EAE preferentially reduced sIPSCs of high amplitude, and was associated with a selective loss of striatal parvalbumin (PV)-positive GABAergic interneurons, which contact striatal projection neurons in their somatic region, giving rise to more efficient synaptic inhibition. Furthermore, we found also that the chronic persistence of pro-inflammatory cytokines were able, per se, to produce profound alterations of electrophysiological network properties, that were reverted by GABA administration.The results of the present investigation indicate defective GABA transmission in MS models depending from alteration of PV cells number and, in part, deriving from the effects of a chronic inflammation, and suggest that pharmacological agents potentiating GABA signaling might be considered to limit neuronal damage in MS patients.     

CCL2/CCR2 Contributes to the Altered Excitatory-inhibitory Synaptic Balance in the Nucleus Accumbens Shell Following Peripheral Nerve Injury-induced Neuropathic Pain

The medium spiny neurons (MSNs) in the nucleus accumbens (NAc) integrate excitatory and inhibitory synaptic inputs and gate motivational and emotional behavior output. Here we report that the relative intensity of excitatory and inhibitory synaptic inputs to MSNs of the NAc shell was decreased in mice with neuropathic pain induced by spinal nerve ligation (SNL). SNL increased the frequency, but not the amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs), and decreased both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in the MSNs. SNL also decreased the paired-pulse ratio (PPR) of evoked IPSCs but increased the PPR of evoked EPSCs. Moreover, acute bath application of C–C motif chemokine ligand 2 (CCL2) increased the frequency and amplitude of sIPSCs and sEPSCs in the MSNs, and especially strengthened the amplitude of N-methyl-D-aspartate receptor (NMDAR)-mediated miniature EPSCs. Further Ccl2 overexpression in the NAc in vivo decreased the peak amplitude of the sEPSC/sIPSC ratio. Finally, Ccr2 knock-down improved the impaired induction of NMDAR-dependent long-term depression (LTD) in the NAc after SNL. These results suggest that CCL2/CCR2 signaling plays a role in the integration of excitatory/inhibitory synaptic transmission and leads to an increase of the LTD induction threshold at the synapses of MSNs during neuropathic pain.     

Altered function of glutamatergic cortico-striatal synapses causes output pathway abnormalities in a chronic model of parkinsonism

ObjectiveIn Parkinson's disease, chronic striatal dopamine depletion results in over-activity and under-activity of the indirect and direct striatal output pathways respectively. In this study, we investigated changes in the function of glutamatergic cortico-striatal synapses that contribute to abnormalities in striatal efferents.MethodsWhole-cell recordings were performed in striatal slices prepared from adult bacterial artificial chromosome mice, chronically lesioned with 6-hydroxydopamine (6-OHDA). Paired pulse facilitation, spontaneous synaptic activity, the ratio of AMPAR to NMDAR-mediated components of excitatory postsynaptic currents, AMPAR and NMDAR kinetics, current–voltage relationship and intrinsic membrane properties were assessed in indirect and direct pathway medium spiny neurons (MSNs), which were identified on the basis of expression of GFP, driven by the promoters of A2A or D1 receptor expression. The trajectory of striatal efferents, with respect to selective targeting of the globus pallidus and substantia nigra was also compared in sham-operated versus 6-OHDA-lesioned mice.ResultsDopamine depletion did not affect the number of pathway specific output neurons or the trajectory of striatal outputs. In sham-operated animals, cortico-striatal synapses of both striatal efferent populations exhibited paired pulse facilitation and similar ratios of AMPAR to NMDAR-mediated components of excitatory postsynaptic currents. Following striatal dopamine depletion, indirect pathway neurons exhibited decreased levels of paired pulse facilitation, enhanced sensitivity to presynaptic stimulation and an increase in the relative contribution of NMDAR to the EPSC but no change in spontaneous synaptic activity. In sham-operated mice, neurons of the direct pathway exhibited lower firing frequency compared to the indirect pathway following current injection. However, in 6-OHDA-lesioned mice, in the direct pathway, firing threshold was reduced, spike frequency adaptation developed and the frequency of spontaneous activity was also reduced. In addition, changes in the properties of NMDAR kinetics suggest that these receptors were desensitised.DiscussionIncreased synchronicity between pre and postsynaptic neurons, as indicated by decreased paired pulse facilitation, and increased sensitivity to extracellular stimulation, combined with an increase in the contribution of NMDARs to the EPSC at cortico-striatal synapses, may contribute to the over-activity of indirect pathway neurons in the parkinsonian striatum. In contrast, a decrease in spontaneous activity, postsynaptic desensitisation to excitatory stimuli and spike frequency adaptation of cortico-striatal synapses may underlie under-activity of the direct pathway.     

Intermittent morphine treatment induces a long-lasting increase in cholinergic modulation of GABAergic synapses in nucleus accumbens of adult rats

Repeated exposure to drugs of abuse causes persistent behavioral sensitization and associated adaptations of striatal neurotransmission, which is thought to play an important role in certain aspects of drug addiction. Microdialysis and neurochemical studies suggest that intermittent morphine treatment may lead to a long-term increase in both ACh and dopaminergic neurotransmission in the nucleus accumbens (NAc). This implies that both cholinergic modulation of GABA synapses and their sensitivity to dopaminergic transmission might be changed, ultimately leading to a modified NAc output. Here we investigate to what extent cholinergic modulation and sensitivity to amphetamine, causing endogenous dopamine efflux, of GABAergic transmission in the nucleus accumbens are affected 3 weeks after a period of daily morphine injections in adult rats. To this end, we recorded medium spiny neurons using whole cell voltage clamp and monitored the frequency and amplitude of spontaneous GABAergic synaptic currents. We observed that the effect of nicotine on the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was suppressed in rats pretreated with morphine, whereas the effects of mecamylamine and tetrodotoxin (TTX) were increased. These results indicate that the probability of GABA release was increased and that this effect resulted from an upregulation of the endogenous activation of presynaptic nicotinic receptors. In addition, we observed an increased sensitivity to in vitro application of amphetamine. This suggests that the long-term increase in dopaminergic transmission caused by the morphine treatment affects GABA synapses in the NAc. Hence, there may be two parallel synaptic mechanisms by which drugs of abuse may affect processing and integration of NAc inputs.     

Endocannabinoid-dependent plasticity at GABAergic and glutamatergic synapses in the striatum is regulated by synaptic activity

Long-term depression (LTD) at striatal synapses is mediated by postsynaptic endocannabinoid (eCB) release and presynaptic cannabinoid 1 receptor (CB₁R) activation. Previous studies have indicated that eCB mobilization at excitatory synapses might be regulated by afferent activation. To further address the role of neuronal activity in synaptic plasticity we examined changes in synaptic strength induced by the L-type calcium channel activator 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176, FPL) at glutamatergic and γ-aminobutyric acid (GABA)ergic synapses in the striatum. We found that the basic mechanisms for FPL-mediated eCB signaling are the same at glutamatergic and GABAergic synapses. FPL-induced LTD (FPL-LTD) was blocked in slices treated with the CB₁R antagonist AM251 (2 μ m ), but established depression was not reversed by AM251. FPL-LTD was temperature dependent, blocked by protein translation inhibitors and prevented by intracellular loading of the anandamide transporter inhibitor VDM11 (10 μ m ) at both glutamatergic and GABAergic synapses. FPL-LTD at glutamatergic synapses required paired-pulse afferent stimulation, while FPL-LTD at GABAergic synapses could be induced even in the absence of explicit afferent activation. By evaluating tetrodotoxin-insensitive spontaneous inhibitory postsynaptic currents we found that neuronal firing is vital for eCB release and LTD induction at GABAergic synapses, but not for short-term depression induced by CB₁R agonist. The data presented here suggest that the level of neuronal firing regulates eCB signaling by modulating release from the postsynaptic cell, as well as interacting with presynaptic mechanisms to induce LTD at both glutamatergic and GABAergic synapses in the striatum.     

Two distinct classes of muscarinic action on hippocampal inhibitory synapses: M2-mediated direct suppression and M1/M3-mediated indirect suppression through endocannabinoid signalling

The cholinergic system in the CNS plays important roles in higher brain functions, primarily through muscarinic acetylcholine receptors. At cellular levels, muscarinic activation produces various effects including modulation of synaptic transmission. Here we report that muscarinic activation suppresses hippocampal inhibitory transmission through two distinct mechanisms, namely a cannabinoid-dependent and cannabinoid-independent mechanism. We made paired whole-cell recordings from cultured hippocampal neurons of rats and mice, and monitored inhibitory postsynaptic currents (IPSCs). When cannabinoid receptor type 1 (CB1) was blocked, oxotremorine M (oxo-M), a muscarinic agonist, suppressed IPSCs in a subset of neuron pairs. This suppression was associated with an increase in paired-pulse ratio, blocked by the M(2)-preferring antagonist gallamine, and was totally absent in neuron pairs from M(2)-knockout mice. When CB1 receptors were not blocked, oxo-M suppressed IPSCs in a gallamine-resistant manner in cannabinoid-sensitive pairs. This suppression was associated with an increase in paired-pulse ratio, blocked by the CB1 antagonist AM281, and was completely eliminated in neuron pairs from M(1)/M(3)-compound-knockout mice. Our immunohistochemical examination showed that M(2) and CB1 receptors were present at inhibitory presynaptic terminals of mostly different origins. These results indicate that two distinct mechanisms mediate the muscarinic suppression. In a subset of synapses, activation of M(2) receptors at presynaptic terminals suppresses GABA release directly. In contrast, in a different subset of synapses, activation of M(1)/M(3) receptors causes endocannabinoid production and subsequent suppression of GABA release by activating presynaptic CB1 receptors. Thus, the muscarinic system can influence hippocampal functions by controlling different subsets of inhibitory synapses through the two distinct mechanisms.     

Depolarizing GABAergic synaptic input triggers endocannabinoid‐mediated retrograde synaptic signaling

Endocannabinoids released by postsynaptic neurons inhibit neurotransmitter release from presynaptic axon terminals. One typical stimulus of endocannabinoid production is an increase of calcium concentration in postsynaptic neurons. The aim of the present study was to clarify whether depolarizing GABAergic synaptic input, by increasing calcium concentration in postsynaptic neurons, can trigger endocannabinoid production. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in Purkinje cells in mouse cerebellar slices with patch‐clamp pipettes containing 151 mM chloride (a usual recording mode). sIPSCs were depolarizing inward currents under this condition. Combined electrophysiological and fluorometric calcium imaging experiments indicated that sIPSCs frequently triggered calcium spikes. After the calcium spikes, a short‐term suppression of sIPSCs occurred. This suppression was prevented by the CB₁ cannabinoid receptor antagonist rimonabant and the diacylglycerol lipase inhibitor orlistat, but not changed by URB597, an inhibitor of anandamide degradation. It is, therefore, likely that CB₁ receptors and 2‐arachidonoylglycerol were involved. For testing the physiological significance of the above observation, we carried out experiments on brains of 3‐ to 5‐day‐old mice. The gramicidin‐induced perforated patch‐clamp mode was used for preserving the physiological intracellular chloride concentration of the neurons. Depolarizing GABAergic sIPSCs occurred under this condition, but at a very low rate. Rimonabant did not change the frequency of these sIPSCs, arguing against the persistence of an endocannabinoid tone. The results point to a new kind of trigger of endocannabinoid production: depolarizing GABAergic synaptic input can elicit endocannabinoid production in postsynaptic neurons by activating calcium channels. The produced endocannabinoid suppresses GABA release from presynaptic axon terminals. Synapse 63:643–652, 2009. © 2009 Wiley‐Liss, Inc.     

Exogenous and endogenous cannabinoids suppress inhibitory neurotransmission in the human neocortex

Activation of CB(1) receptors on axon terminals by exogenous cannabinoids (eg, Δ(9)-tetrahydrocannabinol) and by endogenous cannabinoids (endocannabinoids) released by postsynaptic neurons leads to presynaptic inhibition of neurotransmission. The aim of this study was to characterize the effect of cannabinoids on GABAergic synaptic transmission in the human neocortex. Brain slices were prepared from neocortical tissues surgically removed to eliminate epileptogenic foci. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in putative pyramidal neurons using patch-clamp techniques. To enhance the activity of cannabinoid-sensitive presynaptic axons, muscarinic receptors were continuously stimulated by carbachol. The synthetic cannabinoid receptor agonist WIN55212-2 decreased the cumulative amplitude of sIPSCs. The CB(1) antagonist rimonabant prevented this effect, verifying the involvement of CB(1) receptors. WIN55212-2 decreased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, but did not change their amplitude, indicating that the neurotransmission was inhibited presynaptically. Depolarization of postsynaptic pyramidal neurons induced a suppression of sIPSCs. As rimonabant prevented this suppression, it is very likely that it was due to endocannabinods acting on CB(1) receptors. This is the first demonstration that an exogenous cannabinoid inhibits synaptic transmission in the human neocortex and that endocannabinoids released by postsynaptic neurons suppress synaptic transmission in the human brain. Interferences of cannabinoid agonists and antagonists with synaptic transmission in the cortex may explain the cognitive and memory deficits elicited by these drugs.     

Endocannabinoids limit metabotropic glutamate 5 receptor-mediated synaptic inhibition of striatal principal neurons

Synaptic transmission in the striatum is regulated by metabotropic glutamate (mGlu) receptors through pre- and postsynaptic mechanisms. We investigated the involvement of mGlu 1 and 5 receptors in the control of both excitatory and inhibitory transmission in the striatum. The mGlu 1 and 5 receptor agonist 3,5-DHPG failed to affect glutamate transmission, while it caused a biphasic effect on GABA transmission, characterized by early increase and late decrease in the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from striatal principal neurons. Both mGlu 1 and 5 receptors were involved in the early response to 3,5-DHPG, through membrane depolarization of striatal GABAergic interneurons and action potential generation. The 3,5-DHPG-mediated late depression of inhibitory inputs to striatal principal neurons was conversely secondary to mGlu 5 receptor activation and subsequent endocannabinoid release. In conclusion, we have identified an mGlu-dependent mechanism of GABA transmission regulation of potential relevance for physiological neuronal activity.     

Functional mapping of GABA A receptor subtypes in the amygdala

The physiological significance of the large diversity of GABA A receptors is poorly understood. Using mice, which carry a point mutation that renders specific subtypes of GABA A receptors diazepam insensitive, it was recently discovered that particular types of GABA A receptors are involved in specific, behaviorally relevant signaling pathways. We have used these mice to study inhibitory synaptic transmission in the amygdala. GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) per se were not affected by the point mutations. Their modulation by diazepam, however, was altered depending on the genotype of the mice studied. Based on the different responses to diazepam, we found that IPSCs in the lateral/basolateral amygdala were mediated by both alpha2- and alpha1-subunit-containing GABA A receptors whereas those in the central amygdala were mediated only by alpha2-subunit-containing GABA A receptors. Immunohistochemical staining corroborated these findings at a morphological level. To investigate a possible link between interneuron and receptor diversity, we selectively depressed release from the subset of GABAergic terminals carrying type 1 cannabinoid receptors. These receptors are known to modulate amygdala-mediated behavior. Application of a type 1 cannabinoid receptor agonist resulted in a selective reduction of inhibitory current mediated by alpha1-subunit-containing GABA A receptors. Mice with specific diazepam-insensitive GABA A receptor subtypes therefore provide a novel tool to investigate GABA A receptor distribution and the organization of inhibitory circuits at a functional level. The crucial role of the amygdala for the mediation of anxiety is in agreement with the part that alpha2-subunit-containing GABA A receptors play in anxiolysis and their important function in this area of the brain.     

Plasticity of synaptic inhibition in mouse spinal cord lamina II neurons during early postnatal development and after inactivation of the glycine receptor α3 subunit gene

Synaptic inhibition mediated by GABA_A receptors and glycine receptors (GlyRs) in the outer laminae of the spinal cord dorsal horn efficiently filters ascending nociceptive messages, controlling pathological pain symptoms. However, although many studies have utilized transgenic models to study spinal nociceptive processing, very little is known about the development of functional inhibitory synapses onto these interneurons in mice. Here we report that most interneurons in lamina II are placed under phasic control by both GABAergic and glycinergic synapses, a number of which exhibit dual GABA/glycine co‐release. A developmental switch is also apparent: a subpopulation of lamina II interneurons controlled exclusively by either GABAergic or glycinergic synapses becomes detectable only after postnatal days 15 and 21, respectively. Using mice older than postnatal day 21, we also characterized the plastic changes in glycinergic transmission resulting from the inactivation of the GlyR α3 subunit gene, a key player in inflammatory pain pathways. This allowed us to demonstrate that synapses containing GlyR α3 contribute in large part to synaptic inhibition in lamina II. In Glra3 knockout mice, we found that synaptic currents at the remaining glycinergic synapses, containing GlyR α1, showed faster decay kinetics with unchanged amplitudes but increased frequency. These findings explain the absence of any basal nociceptive hypersensitivity in Glra3 knockout mice, as GlyR α1 is still available for mediating synaptic inhibition at lamina II synapses, but cannot be modulated by the prostaglandin–E‐prostanoid type 2 (EP2) receptor–protein kinase A signalling cascade. Our results clearly demonstrate that presynaptic GABA/glycine release properties are influenced by the nature and complexity of postsynaptic inhibitory receptor subtypes.     

Development of Action Potential-dependent and Independent Spontaneous GABAA Receptor-mediated Currents in Granule Cells of Postnatal Rat Cerebellum

The postnatal development of spontaneous GABAergic transmission between cerebellar Golgi cells and granule cells was investigated with voltage-clamp recording from rat cerebellar slices, in symmetrical Cl^-conditions. Between postnatal days 7 and 14 (P7–14), bicuculline-and TTX (tetrodotoxin)-sensitive spontaneous inhibitory postsynaptic currents (sIPSCs), occurred at high frequency in 56% of granule cells. Between P10 and P14, sIPSCs were superimposed on tonic current of-12 ± 1.8 pA at -70 mV, that was accompanied by noise with variance of 17 ± 3 pA². Both the current and noise were inhibited by bicuculline. TTX blocked the bicuculline-sensitive current and noise by?60%. Between P18 and P25, sIPSCs were less frequent; all cells showed tonic, bicuculline-sensitive currents, but these were partially inhibited by TTX (?35%). Between P40 and P53, slPSCs were rare; tonic, bicuculline-sensitive currents and noise were greater in amplitude, with mean values of-17 pA and 22 pA² at-70 mV, they were present in all cells but they were not inhibited by TTX. Glycine receptor channels that were expressed in immature, but not adult cells, did not mediate spontaneous currents. Our results indicate that spontaneous transmission onto cerebellar granule cells in immature animals consists primarily of action potential-dependent, phasic release of vesicular GABA. This generates GABA_A receptor-mediated slPSCs. The effects of GABA transporter blockers suggest that it also produces the TTX-sensitive current-noise, as GABA spills out of synapses to activate extrasynaptic receptors or receptors in neighbouring synapses. In older animals, action potential-independent release of transmitter is predominant and results in tonic activation of GABA_A receptors. This does not appear to be spontaneous vesicular release of GABA. Neither does it appear to be reversed uptake of GABA, although further work is required to rule out these possibilities.     

Gephyrin clustering is required for the stability of GABAergic synapses

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.     

Pre- and postsynaptic contribution of GABAC receptors to GABAergic synaptic transmission in rat collicular slices and cultures

The mammalian superior colliculus (SC) is reported to contain gamma-aminobutyric acid (GABA)C receptors (GABACRs) at high concentration. However, their role in GABAergic synaptic transmission is not yet known. The aim of the present study was: (i) to clarify whether GABACRs are activated by endogenous GABA; and (ii), to determine whether GABACRs play a role in inhibitory synaptic transmission. Experiments were performed on acute horizontal slices from the postnatal rat SC or on collicular neurons in dissociated cell culture. In both preparations, bicuculline-resistant current responses to exogenous GABA and currents elicited by cis-4-aminocrotonic acid (CACA) were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA), a GABACR antagonist. The CACA-induced currents exhibited a linear current-voltage relationship and reversed at the Cl- equilibrium potential. These results indicate that functional GABACRs are present in the somato-dendritic membrane of collicular neurons. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the whole-cell patch clamp technique. TPMPA significantly decreased mIPSC amplitudes in slices, but not in cultured neurons. As TPMPA decreased also the coefficient of variation of mIPSCs, we suggest that somatodendritic GABACRs are located extrasynaptically but can be involved in the generation of IPSCs if GABA diffusion is constrained. In cultures, individual connections were activated by focal electrical stimulation of single neurons, and evoked inhibitory postsynaptic currents (eIPSCs) were recorded. Paired-pulse stimulation revealed that TPMPA significantly decreased the paired-pulse ratio at short (50 ms) interstimulus intervals, and this effect was inversely dependent on the amplitude of the first eIPSC. We conclude that presynaptic GABACRs are activated by endogenous GABA and can alleviate the short-term depression resulting from a preceding episode of GABA release. Thus, in GABAergic synapses of the SC GABACRs are involved in pre- and postsynaptic functions and may therefore contribute to the activity-dependent adjustment of GABAergic inhibition.     

Cannabinoids modulate synaptic activity in the rat supraoptic nucleus

In the present study, we investigated the effects of the cannabinoid receptor agonist CP55,940 on excitatory and inhibitory synaptic transmission in the rat supraoptic nucleus. Whole-cell patch clamp recordings were performed on supraoptic neurones in in vitro brain slice preparations. CP55,940 significantly reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents in a concentration-dependent manner. These changes were potently reversed by the CB1 receptor antagonist AM251. The results indicate that cannabinoids modulate the activity of magnocellular neurosecretory neurones by presynaptic inhibition of both excitatory and inhibitory synaptic transmission.     

Distinct localization of GABA(B) receptors relative to synaptic sites in the rat cerebellum and ventrobasal thalamus

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) are involved in modulation of synaptic transmission and activity of cerebellar and thalamic neurons. We used subtype-specific antibodies in pre- and postembedding immunohistochemistry combined with three-dimensional reconstruction of labelled profiles and quantification of immunoparticles to reveal the subcellular distribution of pre- and postsynaptic GABA(B)R1a/b and GABA(B)R2 in the rat cerebellum and ventrobasal thalamus. GABA(B)R1a/b and R2 were extensively colocalized in most brain regions including the cerebellum and thalamus. In the cerebellum, immunoreactivity for both subtypes was prevalent in the molecular layer. The most intense immunoreactivity was found in Purkinje cell spines with a high density of immunoparticles at extrasynaptic sites peaking at around 240 nm from glutamatergic synapses between spines and parallel fibre varicosities. This is in contrast to dendrites at sites around GABAergic synapses where sparse and random distribution was found for both subtypes. In addition, more than one-tenth of the synaptic membrane specialization of spine-parallel fibre synapses were labelled at pre- or postsynaptic sites. Weak immunolabelling for both subtypes was also seen in parallel fibres but only rarely in GABAergic axons. In the ventrobasal thalamus, immunolabelling for both receptor subtypes was intense over the dendritic field of thalamocortical cells. Electron microscopy demonstrated an extrasynaptic localization of GABA(B)R1a/b and R2 exclusively in postsynaptic elements. Quantitative analysis further revealed the density of GABA(B)R1a/b around GABAergic synapses was higher than glutamatergic synapses on thalamocortical cell dendrites. The distinct localization of GABA(B)Rs relative to synaptic sites in the cerebellum and ventrobasal thalamus suggests that GABA(B)Rs differentially regulate activity of different neuronal populations.