Role of presynaptic L-type Ca2+ channels in GABAergic synaptic transmission in cultured hippocampal neurons

Using dual whole cell patch-clamp recordings of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons, we have previously demonstrated posttetanic potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic neuron leads to accumulation of Ca2+ in the presynaptic terminals. This enhances the probability of GABA-vesicle release for up to 1 min, which underlies PTP. In the present study, we have examined the effect of altering the probability of release on PTP of IPSCs. Baclofen (10 microM), which depresses presynaptic Ca2+ entry through N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs), caused a threefold greater enhancement of PTP than did reducing [Ca2+]o to 1.2 mM, which causes a nonspecific reduction in Ca2+ entry. This finding prompted us to investigate whether presynaptic L-type VDCCs contribute to the Ca2+ accumulation in the boutons during spike activity. The L-type VDCC antagonist, nifedipine (10 microM), had no effect on single IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 microM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also depressed IPSCs during the stimulation (i.e., they increased tetanic depression). The L-type VDCC "agonist" (-)BayK 8644 (4 microM) had no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train of 1 s by 91%. In conclusion, the results indicate that L-type VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation during high-frequency activity. This helps maintain vesicle release during tetanic stimulation and also enhances the probability of transmitter release during the posttetanic period, which is manifest as PTP. Involvement of L-type channels in these processes represents a novel presynaptic regulatory mechanism at fast CNS synapses.     

GABA(B) receptors are the first target of released GABA at lamina I inhibitory synapses in the adult rat spinal cord

We have previously provided functional evidence that glycine and GABA are contained in the same synaptic vesicles and coreleased at the same synapses in lamina I of the rat spinal dorsal horn. However, whereas both glycine receptors (GlyRs) and GABA(A) receptors (GABA(A)Rs) are expressed on the postsynaptic target, under certain conditions inhibitory events appeared to be mediated by GlyRs only. We therefore wanted to test whether GABA(B) receptors could be activated in conditions where GABA released was insufficient to activate GABA(A)Rs. Focal stimulation in the vicinity of visually identified lamina I neurons elicited monosynaptic IPSCs in the presence of ionotropic glutamate receptor antagonists. Pairs of stimuli were given at different interstimulus intervals (ISI), ranging from 25 ms to 1 s to study the depression of the second of evoked IPSCs (paired pulse depression; PPD). Maximal PPD of IPSCs was 60 +/- 14% (SE) (of the conditioning pulse amplitude), at ISI between 150 and 200 ms. PPD was observed with IPSCs evoked at stimulus intensities where they had no GABA(A)R component. PPD of small evoked IPSCs was not affected by the GABA(A)R antagonist bicuculline but significantly attenuated by 10-30 microM CGP52432, a specific GABA(B) receptor antagonist. These data indicate that, under conditions where GABA released is insufficient to affect postsynaptic GABA(A)Rs at lamina I inhibitory synapses, significant activation of presynaptic GABA(B) receptors can occur.     

Calcium from internal stores triggers GABA release from retinal amacrine cells

The Ca(2+) that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca(2+)channels. Using electrophysiology and Ca(2+) imaging, we show that, in amacrine cell dendrites, at least some of the Ca(2+) that triggers transmitter release comes from endoplasmic reticulum Ca(2+) stores. We show that both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca(2+)] during the brief depolarization of a dendrite. Only the Ca(2+) released through IP(3)Rs, however, seems to promote the release of transmitter. Antagonists for the IP(3)R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca(2+) from internal stores, enhanced both spontaneous and evoked transmitter release.     

Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies

Serotonin 5-hydroxytryptamine type 3 receptors (5HT3R) are Ca2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT3Rs, as it was mimicked by the selective 5HT3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT3R-mediated increase in GABA release was blocked by 100 microM cadmium or by omitting Ca2+ in external solutions, indicating the Ca2+-dependence of the effect. The high voltage-activated Ca2+ channel blockers omega-conotoxin GVIA and omega-conotoxin MVIIC and 10 microM cadmium had no significant effect on the 5-HT3R-mediated enhancement of GABA release, indicating that Ca2+ influx through the 5-HT3R facilitates GABA release. Taken together, these data provide direct evidence that Ca2+ entry via presynaptic 5HT3Rs facilitates the release of GABA from hippocampal interneurons.     

Distinct mechanisms of presynaptic inhibition at GABAergic synapses of the rat substantia nigra pars compacta

We investigated the mechanisms of presynaptic inhibition of GABAergic neurotransmission by group III metabotropic glutamate receptors (mGluRs) and GABA(B) receptors, in dopamine (DA) neurons of the substantia nigra pars compacta (SNc). Both the group III mGluRs agonist L-(+)-2-amino-4-phosphonobutyric acid (AP4, 100 microM) and the GABA(B) receptor agonist baclofen (10 microM) reversibly depressed the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) to 48.5 +/- 2.7 and 79.3 +/- 1.6% (means +/- SE) of control, respectively. On the contrary, the frequency of action potential-independent miniature IPSCs (mIPSCs), recorded in tetrodotoxin (TTX, 1 microM) and cadmium (100 microM) were insensitive to AP4 but were reduced by baclofen to 49.7 +/- 8.6% of control. When the contribution of voltage-dependent calcium channels (VDCCs) to synaptic transmission was boosted with external barium (1 mM), AP4 became effective in reducing TTX-resistant mIPSCs to 65.4 +/- 3.9% of control, thus confirming a mechanism of presynaptic inhibition involving modulation of VDCCs. Differently from AP4, baclofen inhibited to 58.5 +/- 6.7% of control the frequency mIPSCs recorded in TTX and the calcium ionophore ionomycin (2 microM), which promotes Ca2+-dependent, but VDCC-independent, transmitter release. Moreover, in the presence of alpha-latrotoxin (0.3 nM), to promote a Ca2+-independent vesicular release of GABA, baclofen reduced mIPSC frequency to 48.1 +/- 3.2% of control, while AP4 was ineffective. These results indicate that group III mGluRs depress GABA release to DA neurons of the SNc through inhibition of presynaptic VDCCs, while presynaptic GABA(B) receptors directly impair transmitter exocytosis.     

Activity-dependent depression of GABAergic IPSCs in cultured hippocampal neurons.

Short-term depression of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) evoked between pairs of cultured rat hippocampal neurons was investigated using dual whole cell patch-clamp recordings. Paired stimuli applied to the GABAergic neuron resulted in paired-pulse depression (PPD) of the second IPSC (IPSC2) at interpulse intervals from 25 to 2,000 ms. CGP 55845A, but not CGP 35348, reduced PPD marginally. Brief paired-pulse applications of exogenous GABA indicated that postsynaptic factors made only minimal contribution to PPD of IPSCs. IPSC1 and PPD was reduced on lowering [Ca2+]o and enhanced on increasing [Ca2+]o. The potassium-channel blocker 4-aminopyridine (4-AP), which increases presynaptic Ca2+ influx, enhanced IPSC1 and PPD. Chelation of residual Ca2+ in the GABAergic boutons with EGTA-AM enhanced PPD. Stimulation of the presynaptic neuron at frequencies (f) ranging from 2.5 to 80 Hz resulted in tetanic depression of IPSCs, which declined rapidly and reached a plateau depending on f and [Ca2+]o. CGP 55845A decreased tetanic depression in the first part of the train, but this could be overcome with continued stimulation. We show that GABAergic IPSCs are robustly depressed by paired-pulse stimulation in cultured hippocampal neurons. The depression of IPSCs is mainly independent of presynaptic GABAB receptors and could be caused by depletion of releasable vesicles. Depleted synapses recover with a slow time course, depending on factors that regulate [Ca2+]i in the GABAergic boutons.     

Endocannabinoid signaling depends on the spatial pattern of synapse activation

The brain's endocannabinoid retrograde messenger system decreases presynaptic transmitter release, but its physiological function is uncertain. We show that endocannabinoid signaling is absent when spatially dispersed synapses are activated on rodent cerebellar Purkinje cells but that it reduces presynaptic glutamate release when nearby synapses are active. This switching of signaling according to the spatial pattern of activity is controlled by postsynaptic type I metabotropic glutamate receptors, which are activated disproportionately when glutamate spillover between synapses produces synaptic crosstalk. When spatially distributed synapses are activated, endocannabinoid inhibition of transmitter release can be rescued by inhibiting glutamate uptake to increase glutamate spillover. Endocannabinoid signaling initiated by type I metabotropic glutamate receptors is a homeostatic mechanism that detects synaptic crosstalk and downregulates glutamate release in order to promote synaptic independence.     

Kinetics of GABAB autoreceptor-mediated suppression of GABA release in rat insular cortex

Release of GABA is controlled by presynaptic GABA receptor type B (GABA(B)) autoreceptors at GABAergic terminals. However, there is no direct evidence that GABA(B) autoreceptors are activated by GABA release from their own terminals, and precise profiles of GABA(B) autoreceptor-mediated suppression of GABA release remain unknown. To explore these issues, we performed multiple whole-cell, patch-clamp recordings from layer V rat insular cortex. Both unitary inhibitory and excitatory postsynaptic currents (uIPSCs and uEPSCs, respectively) were recorded by applying a five-train depolarizing pulse injection at 20 Hz. In connections from both fast-spiking (FS) and non-FS interneurons to pyramidal cells, the GABA(B) receptor antagonist CGP 52432 had little effect on the initial uIPSC amplitude. However, uIPSCs, responding to later pulses, were effectively facilitated. This CGP 52432-induced facilitation was prominent in the fourth uIPSCs, which were evoked 150 ms after the first uIPSC. The facilitation of uIPSCs was accompanied by an increase in the paired-pulse ratio. In addition, analysis of the coefficient of variation suggests the involvement of presynaptic mechanisms in CGP 52432-induced uIPSC facilitation. Paired-pulse stimulation (interstimulus interval = 150 ms) of presynaptic FS cells revealed that the second uIPSC was also facilitated by CGP 52432, which had little effect on the amplitude and interevent interval of miniature IPSCs. In contrast, uEPSCs, responding to all five stimulations of a presynaptic pyramidal cell, were less affected by CGP 52432. These results suggest that a single presynaptic action potential is sufficient to activate GABA(B) autoreceptors and to suppress GABA release in the cerebral cortex.     

Synaptically released GABA activates both pre- and postsynaptic GABA(B) receptors in the rat globus pallidus

The globus pallidus (GP) contains abundant GABAergic synapses and GABA(B) receptors. To investigate whether synaptically released GABA can activate pre- and postsynaptic GABA(B) receptors in the GP, physiological recordings were performed using rat brain slice preparations. Cell-attached recordings from GABA(A) antagonist-treated preparations revealed that repetitive local stimulation induced a GABA(B) antagonist-sensitive pause in spontaneous firings of GP neurons. Whole cell recordings revealed that the repetitive stimulation evoked fast excitatory postsynaptic potentials followed by a slow inhibitory postsynaptic potential (IPSP) in GP neurons. The slow IPSP was insensitive to a GABA(A) receptor antagonist, increased in amplitude with the application of ionotropic glutamate receptor antagonists, and was suppressed by the GABA(B) antagonist CGP55845. The reversal potential of the slow IPSP was close to the potassium equilibrium potential. These results suggest that synaptically released GABA activated postsynaptic GABA(B) receptors and induced the pause and the slow IPSP. On the other hand, in the neurons that were treated to block postsynaptic GABA(B) responses, CGP55845 increased the amplitudes of repetitive local stimulation-induced GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) but not the ionotropic glutamate-mediated excitatory postsynaptic currents. Moreover, the GABA(B) receptor specific agonist baclofen reduced the frequency of miniature IPSCs without altering their amplitude distributions. These results suggest that synaptically released GABA also activated presynaptic GABA(B) autoreceptors, resulting in decreased GABA release in the GP. Together, we infer that both pre- and postsynaptic GABA(B) receptors may play crucial roles in the control of GP neuronal activity.     

The effect of internal GTPγS on GABA-release in cultured hippocampal neurons

The effects of presynaptic guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in cultured hippocampal neurons using whole-cell recordings. Inclusion of GTPgammaS (0.5-1 mM) in the presynaptic electrode reduced both the amplitude and paired-pulse depression of IPSCs, indicating that the probability of GABA-release had been reduced. Presynaptic GTPgammaS increased the depression of IPSCs by the GABA(B)-receptor-agonist baclofen (10 microM), and the effect of baclofen was poorly reversible after washing. Stimulation of the GABAergic neuron at 80 Hz for 1 s was accompanied by tetanic depression of the IPSCs by 52+/-6% and was followed by post-tetanic potentiation (PTP), reaching a peak value of 71+/-21% and lasting about 100 s. IPSCs evoked after tetanic stimulation were depressed and PTP was absent when tetanic stimulation was applied within 3 min after starting injection of GTPgammaS into the presynaptic neuron. At longer times, basal release underlying a single IPSC was depressed. This affected the ratios recorded in response to tetanic stimulations such that tetanic depression was abolished, while PTP increased to 117+/-34%. In conclusion, GTPgammaS reduces the probability of GABA-release in both a use- and time-dependent manner, most likely through an inhibitory action on presynaptic Ca2+-influx through voltage-gated Ca2+ channels or an interaction with small GTP-binding proteins in the nerve terminals.     

GABA-Mediated inhibition between amacrine cells in the goldfish retina

Retinal amacrine cells have abundant dendro-dendritic synapses between neighboring amacrine cells. Therefore an amacrine cell has both presynaptic and postsynaptic aspects. To understand these synaptic interactions in the amacrine cell, we recorded from amacrine cells in the goldfish retinal slice preparation with perforated- and whole cell-patch clamp techniques. As the presynaptic element, 19% of the cells recorded (15 of 78 cells) showed spontaneous tetrodotoxin (TTX)-sensitive action potentials. As the postsynaptic element, all amacrine cells (n = 9) were found to have GABA-evoked responses and, under perforated patch clamp, 50 microM GABA hyperpolarized amacrine cells by activating a Cl(-) conductance. Bicuculline-sensitive spontaneous postsynaptic currents, carried by Cl(-), were observed in 82% of the cells (64 of 78 cells). Since the source of GABA in the inner plexiform layer is amacrine cells alone, these events are likely to be inhibitory postsynaptic currents (IPSCs) caused by GABA spontaneously released from neighboring amacrine cells. IPSCs were classified into three groups. Large amplitude IPSCs were suppressed by TTX (1 microM), indicating that presynaptic action potentials triggered GABA release. Medium amplitude IPSCs were also TTX sensitive. Small amplitude IPSCs were TTX insensitive (miniature IPSCs; n = 26). All of spike-induced, medium amplitude, and miniature IPSCs were Ca(2+) dependent and blocked by Co(2+). Blocking of glutamatergic inputs by DL-2-amino-phosphonoheptanoate (AP7; 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2 microM) had almost no effect on spontaneous GABA release from presynaptic amacrine cells. We suggest that these dendro-dendrotic inhibitory networks contribute to receptive field spatiotemporal properties.     

Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels

Presynaptic GABA(B) receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABA(B)-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by omega-conotoxin GVIA (< or = 72%), mibefradil (< or = 52%), and omega-agatoxin TK (< or = 15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP-sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.     

Cannabinoids depress inhibitory synaptic inputs received by layer 2/3 pyramidal neurons of the neocortex

Using whole cell voltage-clamp recordings we investigated the effects of a synthetic cannabinoid (WIN55,212-2) on inhibitory inputs received by layer 2/3 pyramidal neurons in slices of the mouse auditory cortex. Activation of the type 1 cannabinoid receptor (CB1R) with WIN55,212-2 reliably reduced the amplitude of GABAergic inhibitory postsynaptic currents evoked by extracellular stimulation within layer 2/3. The suppression of this inhibition was blocked and reversed by the highly selective CB1R antagonist AM251, confirming a CB1R-mediated inhibition. Pairing evoked inhibitory postsynaptic currents (IPSCs) at short interstimulus intervals while applying WIN55,212-2 resulted in an increase in paired-pulse facilitation suggesting that the probability of GABA release was reduced. A presynaptic site of cannabinoid action was verified by an observed decrease in the frequency with no change in the amplitude or kinetics of action potential-independent postsynaptic currents (mIPSCs). When Cd(2+) was added or Ca(2+) was omitted from the recording solution, the remaining fraction of Ca(2+)-independent mIPSCs did not respond to WIN55,212-2. These data suggest that cannabinoids are capable of suppressing the inhibition of neocortical pyramidal neurons by depressing Ca(2+)-dependent GABA release from local interneurons.     

Tonic and synaptically evoked presynaptic inhibition of sensory input to the rat olfactory bulb via GABA(B) heteroreceptors

Olfactory receptor neurons of the nasal epithelium send their axons, via the olfactory nerve (ON), to the glomeruli of the olfactory bulb (OB), where the axon terminals form glutamatergic synapses with the apical dendrites of mitral and tufted cells, the output cells of the OB, and with juxtaglomerular (JG) interneurons. Many JG cells are GABAergic. Here we show that, despite the absence of conventional synapses, GABA released from JG cells activates GABA(B) receptors on ON terminals and inhibits glutamate release both tonically and in response to ON stimulation. Field potential recordings and current-source density analysis, as well as intracellular and whole cell recording techniques were used in rat OB slices. Baclofen (2-5 microM), a GABA(B) agonist, completely suppressed ON-evoked synaptic responses of both mitral/tufted cells and JG cells, with no evidence for postsynaptic effects. Baclofen (0.5-1 microM) also reversed paired-pulse depression (PPD) of mitral/tufted cell responses to paired-pulse facilitation (PPF), and reduced depression of JG cell excitatory postsynaptic currents (EPSCs) during repetitive ON stimulation. These results suggest that baclofen reduced the probability of glutamate release from ON terminals. The GABA(B) antagonists CGP35348 or CGP55845A increased mitral/tufted cell responses evoked by single-pulse ON stimulation, suggesting that glutamate release from ON terminals is tonically suppressed via GABA(B) receptors. The same antagonists reduced PPD of ON-evoked mitral/tufted cell responses at interstimulus intervals 50-400 ms. This finding suggests that a single ON impulse evokes sufficient GABA release, presumably from JG cells, to activate GABA(B) receptors on ON terminals. Thus GABA(B) heteroreceptors on ON terminals are activated by ambient levels of extrasynaptic GABA, and by ON input to the OB. The time course of ON-evoked, GABA(B) presynaptic inhibition suggests that neurotransmission to M/T cells and JG cells will be significantly suppressed when ON impulses arrive in glomeruli at 2.5-20 Hz. GABA(B) receptor-mediated presynaptic inhibition of sensory input to the OB may play an important role in shaping the activation pattern of the OB glomeruli during olfactory coding.     

Dihydropyridines modulate K+-evoked amino acid and adenosine release from cerebellar neuronal cultures

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, gamma-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 microM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.     

Activation of extrasynaptic NMDA receptors induces a PKC-dependent switch in AMPA receptor subtypes in mouse cerebellar stellate cells

The repetitive activation of synaptic glutamate receptors can induce a lasting change in the number or subunit composition of synaptic AMPA receptors (AMPARs). However, NMDA receptors that are present extrasynaptically can also be activated by a burst of presynaptic activity, and thus may be involved in the induction of synaptic plasticity. Here we show that the physiological-like activation of extrasynaptic NMDARs induces a lasting change in the synaptic current, by changing the subunit composition of AMPARs at the parallel fibre-to-cerebellar stellate cell synapse. This extrasynaptic NMDAR-induced switch in synaptic AMPARs from GluR2-lacking (Ca²⁺-permeable) to GluR2-containing (Ca²⁺-impermeable) receptors requires the activation of protein kinase C (PKC). These results indicate that the activation of extrasynaptic NMDARs by glutamate spillover is an important mechanism that detects the pattern of afferent activity and subsequently exerts a remote regulation of AMPAR subtypes at the synapse via a PKC-dependent pathway.     

Differential activation of GABAA and GABAB receptors by spontaneously released transmitter.

1. Whole-cell patch-clamp techniques were used to record from dentate gyrus granule cells in adult rat brain slices when N-methyl-D-aspartate (NMDA) and non-NMDA type glutamate receptors were blocked by D-2-amino-5-phosphonovaleric acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. Spontaneous inhibitory postsynaptic currents (sIPSCs), each presumably due to vesicular release of gamma-aminobutyric acid (GABA), selectively activated GABAA-type receptors. None of the individual sIPSCs showed a slow-onset potassium current characteristic of GABAB receptor activation. 2. In contrast, stimulation in the molecular layer with a bipolar stimulating electrode or bath application of the convulsant drug 4-aminopyridine (4-AP, 10-30 microM) elicited fast GABAA IPSCs followed by slower outward currents that were sensitive to the selective GABAB antagonist CGP 35348 (0.1-1 mM) and that reversed polarity near the potassium equilibrium potential. 3. CGP 35348 (0.5-1 mM) or the GABAB agonist (-)baclofen (1 microM) had no significant effect on the frequency or average amplitude of sIPSCs. However, either bath application of (-)baclofen (1 microM) or a preceding conditioning stimulus caused large reductions in the amplitude of stimulus-evoked IPSCs, suggesting a strong GABAB-mediated presynaptic inhibition of stimulus-evoked GABA release. 4. We conclude that under normal conditions spontaneous transmitter release does not activate GABAB receptors in dentate gyrus slices. These findings are consistent with either of two general possibilities. Separate groups of interneurons with different basal firing rates may selectively form GABAA and GABAB synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of spontaneous but not Ca2+ -dependent release machinery by presynaptic group II mGluRs in rat cerebellar slices

Two main forms of neurotransmitter release are known: action potential-evoked and spontaneous release. Action potential-evoked release depends on Ca2+ entry through voltage-gated Ca2+ channels, whereas spontaneous release is thought to be Ca2+ -independent. Generally, spontaneous and action potential-evoked release are believed to use the same release machinery to release neurotransmitter. This study shows, using the whole cell patch-clamp technique in rat cerebellar slices, that at the interneuron- Purkinje cell synapse activation of presynaptic group II metabotropic glutamate receptors suppresses spontaneous GABA release through a mechanism independent of voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Ca2+ release from intracellular Ca2+ stores, suggesting that the metabotropic receptors target the release machinery directly. Voltage gated Ca2+ channel-independent release following increased presynaptic cAMP production is similarly inhibited by these metabotropic receptors. In contrast, both voltage-gated Ca2+ channel-dependent and presynaptic N-methyl-D-aspartate receptor-dependent GABA release were unaffected by activation of group II metabotropic glutamate receptors. Hence, the mechanisms underlying spontaneous and Ca2+ -dependent GABA release are distinct in that only the former is blocked by group II metabotropic glutamate receptors. Thus the same neurotransmitter, glutamate, can activate or inhibit neurotransmitter release by selecting different receptors that target different release machineries.     

Parvalbumin-deficiency facilitates repetitive IPSCs and gamma oscillations in the hippocampus

In the hippocampus, the calcium-binding protein parvalbumin (PV) is expressed in interneurons that innervate perisomatic regions. PV in GABAergic synaptic terminals was proposed to limit repetitive GABA release by buffering of "residual calcium." We assessed the role of presynaptic PV in Ca(2+)-dependent GABA release in the hippocampus of PV-deficient (PV-/-) mice and wild-type (PV+/+) littermates. Pharmacologically isolated inhibitory postsynaptic currents (IPSCs) were evoked by low-intensity stimulation of the stratum pyramidale and recorded from voltage-clamped CA1 pyramidal neurons. The amplitude and decay time constant of single IPSCs were similar for both genotypes. Under our experimental conditions of reduced release probability and minimal presynaptic suppression, paired-pulse facilitation of IPSCs occurred at intervals from 2 to 50 ms, irrespective of the presence of PV. The facilitation of IPSCs induced by trains of 10 stimuli at frequencies >20 Hz was enhanced in cells from PV-/- mice, the largest difference between PV-/- and PV+/+ animals (220%) being observed at 33 Hz. The effect of IPSC facilitation at sustained gamma frequencies was assessed on kainate-induced rhythmic IPSC-paced neuronal oscillations at gamma frequencies, recorded with dual field potential recordings in area CA3. The maximum power of the oscillation was 138 microV(2) at 36 Hz in slices from PV+/+ mice and was trebled in slices from PV-/- mice. PV deficiency caused a similar increase in gamma power under conditions used to study IPSC facilitation and can be explained by an increased facilitation of GABA release at sustained high frequencies. The dominant frequency and coherence were not affected by PV deficiency. These observations suggest that PV deficiency, due to an increased short-term facilitation of GABA release, enhances inhibition by high-frequency burst-firing PV-expressing interneurons and may affect the higher cognitive functions associated with gamma oscillations.