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.     

Activation of postsynaptic Ca(2+) stores modulates glutamate receptor cycling in hippocampal neurons

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP(3)Rs) with the IP(3)R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal synaptic transmission. The regulation of postsynaptic AMPAR trafficking involves synaptotagmin-SNARE-mediated vesicle exocytosis and interactions between AMPARs and the PDZ domains in GRIP/PICK1. We show that inhibitors of synaptotagmin-SNARE-mediated exocytosis, or interactions between AMPARs and GRIP/PICK1, attenuated AdA-enhanced increases in EPSC amplitudes. These results suggest that IP(3)R-mediated Ca(2+) release can enhance AMPAR EPSC amplitudes through mechanisms that involve AMPAR-PDZ interactions and/or synaptotagmin-SNARE-mediated receptor trafficking.     

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.     

Adrenergic facilitation of GABAergic transmission in rat entorhinal cortex

Whereas the entorhinal cortex (EC) receives noradrenergic innervations from the locus coeruleus of the pons and expresses adrenergic receptors, the function of norepinephrine (NE) in the EC is still elusive. We examined the effects of NE on GABA(A) receptor-mediated synaptic transmission in the superficial layers of the EC. Application of NE dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III through activation of alpha(1) adrenergic receptors. NE increased the frequency and not the amplitude of miniature IPSCs (mIPSCs) recorded in the presence of TTX, suggesting that NE increases presynaptic GABA release with no effects on postsynaptic GABA(A) receptors. Application of Ca(2+) channel blockers (Cd(2+) and Ni(2+)), omission of Ca(2+) in the extracellular solution, or replacement of extracellular Na(+) with N-methyl-D-glucamine (NMDG) failed to alter NE-induced increase in mIPSC frequency, suggesting that Ca(2+) influx through voltage-gated Ca(2+) or other cationic channels is not required. Application of BAPTA-AM, thapsigargin, and ryanodine did not change NE-induced increase in mIPSC frequency, suggesting that Ca(2+) release from intracellular stores is not necessary for NE-induced increase in GABA release. Whereas alpha(1) receptors are coupled to G(q/11) resulting in activation of the phospholipase C (PLC) pathway, NE-mediated facilitation of GABAergic transmission was independent of PLC, protein kinase C, and tyrosine kinase activities. Our results suggest that NE-mediated facilitation of GABAergic function contributes to its antiepileptic effects in the EC.     

Mechanisms underlying LTP of inhibitory synaptic transmission in the deep cerebellar nuclei

Whole-cell recordings were used to investigate long-term potentiation of inhibitory synaptic currents (IPSCs) in neurons of deep cerebellar nuclei (DCN) in slices. IPSCs were evoked by electrical stimulation of the white matter surrounding the DCN in the presence of non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (20 microM). High-frequency stimulation induced a long-term potentation (LTP) of the IPSC amplitude without changing its reversal potential, rise time, and decay-time constant. This LTP did not require the activation of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors but depended on the activation of NMDA receptors. LTP of IPSCs in DCN neurons could also be induced by voltage-depolarizing pulses in postsynaptic neurons and appeared to depend on an increase in intracellular calcium as the LTP was blocked when the cells were loaded with a calcium chelator, 1,2-bis-(2-amino-phenoxy)-N,N,N', N'-tetraacetic acid (BAPTA, 10 mM). LTP of IPSCs was accompanied by an increase in the frequency of spontaneous IPSCs and miniature IPSCs (recorded in the presence of tetrodotoxin 1 microM), but there was no significant change in their amplitude. In addition, during the LTP, the amplitude of response to exogenously applied GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride was increased. Intracellular application of tetanus toxin, a powerful blocker of exocytosis, in DCN neuron prevented the induction of LTP of IPSCs. Our results suggest that the induction of LTP of IPSCs in the DCN neurons likely involves a postsynaptic locus. Plasticity of inhibitory synaptic transmission in DCN neurons may play a crucial role in cerebellar control of motor coordination and learning.     

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.     

Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal CA3 pyramidal neurons in an isolated neuron/bouton preparation

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABA_A receptors to the GABA_A receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55212-2 (mesylate(R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid 2-arachidonoylglycerol also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-βS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.     

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.     

Activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells

Amacrine cells in the vertebrate retina receive glutamatergic input from bipolar cells and make synapses onto bipolar cells, ganglion cells, and other amacrine cells. Recent studies indicate that amacrine cells express metabotropic glutamate receptors (mGluRs) and that signaling within the inner plexiform layer (IPL) of the retina might be modulated by mGluR activity. This study tests the hypothesis that activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells. Whole cell voltage-clamp recordings were combined with pharmacology to establish the identity of the ionotropic GABA receptors expressed in primary cultures of chick amacrine cells and to determine how mGluR5 activity affected the behavior of those receptors. Application of GABA (20 microM) produced currents that reversed at -58.2 +/- 0.9 mV, near the predicted Cl(-) reversal potential of -59 mV. The GABA(A) receptor antagonist, bicuculline (50 microM), completely blocked the GABA-gated currents. cis-4-Aminocrotonic acid (CACA; 100 microM), a GABA(C) receptor agonist, produced small currents that were not blocked by the GABA(C) antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA; 20 microM), but were completely blocked by bicuculline. These results indicate that cultured amacrine cells express GABA(A) receptors exclusively. Activating mGluR5 with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 300 microM) enhanced GABA-gated currents by 10.0 +/- 1.5%. Buffering internal Ca(2+) with BAPTA (10 mM) blocked the CHPG-dependent enhancement. Activation of PKC with the cell-permeable PKC activators (-)-7-octylindolactam V, phorbol 12-myristate 13 acetate (PMA), or 1-oleoyl-2-acetyl-sn-glycerol (OAG) also enhanced GABA-gated currents in a dose-dependent manner. Preactivation of PKC occluded the mGluR5-dependent enhancement, and inhibition of Ca-dependent PKC isotypes with G?6976 (35 nM) suppressed the effects of mGluR5 activation, suggesting that mGluR5 and PKC are part of the same pathway. To determine if mGluR5-dependent enhancement occurred at synaptic GABA(A) receptors, postsynaptic currents were recorded in the presence of CHPG. On average, the mean amplitudes of the quantal events were increased by about 18% when mGluR5 was activated. These results indicate that activation of mGluR5 enhances GABA-gated current in cultured amacrine cells in a manner that is both Ca(2+)- and PKC-dependent. These results support the possibility that glutamate released from bipolar cells can modulate the function of GABAergic amacrine cells and alter signaling in the inner plexiform layer.     

Retrograde signaling changes the venue of postsynaptic inhibition in rat substantia nigra

Both endocannabinoids through cannabinoid receptor type I (CB1) receptors and dopamine through dopamine receptor type D1 receptors modulate postsynaptic inhibition in substantia nigra by changing GABA release from striatonigral terminals. By recording from visually identified pars compacta and pars reticulata neurons we searched for a possible co-release and interaction of endocannabinoids and dopamine. Depolarization of a neuron in pars reticulata or in pars compacta transiently suppressed evoked synaptic currents which were blocked by GABA(A) receptor antagonists (inhibitory postsynaptic currents [IPSCs]). This depolarization-induced suppression of inhibition (DSI) was abrogated by the cannabinoid CB1 receptor antagonist AM251 (1 microM). A correlation existed between the degree of DSI and the degree of reduction of evoked IPSCs by the CB1 receptor agonist WIN55,212-2 (1 microM). The cholinergic receptor agonist carbachol (0.5-5 microM) enhanced DSI, but suppression of spontaneous IPSCs was barely detectable pointing to the existence of GABA release sites without CB1 receptors. In dopamine, but not in GABAergic neurons DSI was enhanced by the dopamine D1 receptor antagonist SCH23390 (3-10 microM). Both the antagonist for CB1 receptors and the antagonist for dopamine D1 receptors enhanced or reduced, respectively, the amplitudes of evoked IPSCs. This tonic influence persisted if the receptor for the other ligand was blocked. We conclude that endocannabinoids and dopamine can be co-released. Retrograde signaling through endocannabinoids and dopamine changes inhibition independently from each other. Activation of dopamine D1 receptors emphasizes extrinsic inhibition and activation of CB1 receptors promotes intrinsic inhibition.     

Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures.

In vitro ischemic preconditioning induced by subjecting rat cortical cultures to nonlethal oxygen-glucose deprivation protects against a subsequent exposure to otherwise lethal oxygen-glucose deprivation. We provide evidence that attenuation of the postsynaptic N-methyl-D-aspartate (NMDA) receptor- and Ca(2+)-dependent neurotoxicity underlies oxygen-glucose deprivation tolerance. It is demonstrated that extended tolerance to otherwise lethal NMDA or oxygen-glucose deprivation can be induced by either of their sublethal forms of preconditioning. These four pathways are linked, since NMDA receptor blockade during preconditioning by oxygen-glucose deprivation eliminates tolerance. These results suggest that NMDA tolerance, induced by nonlethal activation of these receptors during oxygen-glucose deprivation preconditioning, underlies oxygen-glucose deprivation tolerance. Several neurotoxic downstream Ca(2+)-dependent signaling events specifically linked to NMDA receptor activation are attenuated during otherwise lethal oxygen-glucose deprivation in preconditioned cultures. Specifically, calpain activation, as well as degradation of microtubule-associated protein-2 and postsynaptic density-95, are attenuated 2 h following otherwise lethal NMDA treatment alone or oxygen-glucose deprivation in preconditioned cultures. Formation of microtubule-associated protein-2-labeled dendritic varicosities is also attenuated in preconditioned cultures within 1 h of lethal oxygen-glucose deprivation or NMDA application. Intracellular Ca(2+) levels, measured using the high- or low-affinity dyes Fluo-4 (K(d) approximately equal 345 nM) or Fluo-4FF (K(d) approximately equal 9.7 microM) respectively, are markedly attenuated during lethal oxygen-glucose deprivation in preconditioned cultures.Collectively, the results suggest the attenuation of the postsynaptic NMDA-mediated component of otherwise lethal oxygen-glucose deprivation through the suppression of Ca(2+)-dependent neurotoxic signaling, a mechanism that is initially induced by transient nonlethal activation of this receptor during ischemic preconditioning.     

Distinctive glycinergic currents with fast and slow kinetics in thalamus

We examined functional properties of inhibitory postsynaptic currents (IPSCs) evoked by medial lemniscal stimulation, spontaneous IPSCs (sIPSCs), and single-channel, extrasynaptic currents evoked by glycine receptor agonists or gamma-aminobutyric acid (GABA) in rat ventrobasal thalamus. We identified synaptic currents by reversal at E(Cl) and sensitivity to elimination by strychnine, GABA(A) antagonists, or combined application. Glycinergic IPSCs featured short (about 12 ms) and long (about 80 ms) decay time constants. These fast and slow IPSCs occurred separately with monoexponential decays, or together with biexponential decay kinetics. Glycinergic sIPSCs decayed monoexponentially with time constants, matching fast and slow IPSCs. These findings were consistent with synaptic responses generated by two populations of glycine receptors, localized under different nerve terminals. Glycine, taurine, or beta-alanine applied to excised membrane patches evoked short- and long-duration current bursts. Extrasynaptic burst durations resembled fast and slow IPSC time constants. The single, intermediate time constant (about 22 ms) of GABA(A)ergic IPSCs cotransmitted with glycinergic IPSCs approximated the burst duration of extrasynaptic GABA(A) channels. We noted differences between synaptic and extrasynaptic receptors. Endogenously activated glycine and GABA(A) receptor channels had higher Cl- permeability than that of their extrasynaptic counterparts. The beta-amino acids activated long-duration bursts at extrasynaptic glycine receptors, consistent with a role in detection of ambient taurine or beta-alanine. Heterogeneous kinetics and permeabilities implicate molecular and functional diversity in thalamic glycine receptors. Fast, intermediate, and slow inhibitory postsynaptic potential decays, mostly attributed to cotransmission by glycinergic and GABAergic pathways, allow for discriminative modulation and integration with voltage-dependent currents in ventrobasal neurons.     

Multiple types of GABAA receptors mediate inhibition in brain stem parasympathetic cardiac neurons in the nucleus ambiguus

Recent work suggests neurons can have different types of gamma-aminobutyric acid type A (GABA(A)) receptors that mediate phasic inhibitory postsynaptic currents (IPSCs) and tonic currents. This study examines the diversity of GABAergic synaptic currents in parasympathetic cardioinhibitory neurons that receive rhythmic bursts of GABAergic neurotransmission. Focal application of gabazine (25 microM) to cardiac vagal neurons in vitro did not change the frequency of firing in spontaneously active neurons or the resting membrane potential; however, picrotoxin (100 microM) significantly depolarized cardiac vagal neurons and increased their firing. Similarly, gabazine (25 microM) selectively blocked GABAergic IPSCs but did not change holding current in cardiac vagal neurons, whereas picrotoxin (100 microM) not only blocked GABAergic IPSCs but also rapidly decreased the tonic current. Because the tonic current could be attributable to activation of GABA receptors by ambient GABA or, alternatively, spontaneous opening of constitutively active GABA channels, an antagonist for the GAT-1 GABA transporter NO-711 (10 microM) was applied to distinguish between these possibilities. NO-711 did not significantly alter the holding current in these neurons. The benzodiazepine flunitrazepam (1 microM) significantly increased the tonic current and GABAergic IPSC decay time; surprisingly, however, in the presence of gabazine flunitrazepam failed to elicit any change. These results suggest cardiac vagal neurons possess gabazine-sensitive GABA(A) receptors that mediate phasic synaptic currents, a gabazine-insensitive but picrotoxin-sensitive extrasynaptic tonic current that when blocked depolarizes and increases the firing rate of cardiac vagal neurons, and benzodiazepines recruit a third type of GABA(A) receptor that is sensitive to gabazine and augments the extrasynaptic tonic current.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in cerebellar inhibitory interneurons.

The inhibitory interneurons in the molecular layer of the cerebellar cortex form a complex network, interconnected by both chemical and electrotonic synapses. Previous work, using voltage optical imaging in an isolated cerebellum, has indicated that these interneurons also form presynaptic inhibitory interconnections. Here we examine the participation of GABA(B) receptors in the proposed presynaptic inhibition by recording from the molecular layer interneurons (MLI) in cerebellar slices. The GABA(B) agonist, baclofen, profoundly depressed synaptic transmission; a concentration of 10 microM decreased the frequency of spontaneous inhibitory synaptic potentials by 82 +/- 15% and of miniature synaptic potentials by 75 +/- 13%. In simultaneous recording from two synaptically interconnected MLIs, baclofen (10 microM) increased the failure rate of synaptic transmission by a factor of 3, confirming a presynaptic mechanism, most likely mediated by a decrease in calcium conductance. A postsynaptic effect of baclofen was also found; 10 microM decreased the spontaneous firing rate by 55 +/- 19% even in the presence of synaptic blockers. One hundred micromolar baclofen induced an averaged hyperpolarization of 6 +/- 2 mV or an averaged 7.8 +/- 3 pA net outward current that can account for the decrease in firing rate. The outward current reflects a reduction in a tonic Ca(2+) current, since it was abolished by blocking Ca(2+) currents and remained unchanged in the presence of Ba(2+). Application of the specific GABA(B) blocker, CGP 55845A (1 microM), not only reversed the effects of baclofen but also increased the spontaneous firing rate and synaptic activity when applied alone. Thus in slice preparations, GABA(B) receptors are tonically activated by endogenous GABA. The temporal role of GABA(B) receptors was tested using the paired-pulse paradigm. Recording from two synaptically interconnected MLIs showed a 3.5 times lower probability of release for the second stimulus. In the isolated cerebellar preparation, a robust depression of the second inhibitory response was observed. This depression was partially blocked by CGP 55845A (2 microM). We conclude that both the pre- and postsynaptic effects of baclofen are mediated by GABA(B) receptors that decrease Ca(2+) currents. These can serve a modulatory role as well as participating in shaping the temporal interactions between consecutive inputs.     

Receptor-stimulated phospholipase A(2) liberates arachidonic acid and regulates neuronal excitability through protein kinase C

Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca(2+) increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca(2+), co-factors sufficient to stimulate conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A(2) (PLA(2)), which is thought to regulate the liberation of arachidonic acid (AA), an "atypical" activator of PKC. Here, we directly assess whether GABA binding or PLA(2) stimulation liberates AA in these cells and whether free AA potentiates the stimulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). In response to 5 microM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA(2) inhibitor arachidonyltrifluoromethyl ketone (50 microM). Furthermore, direct stimulation of PLA(2) by melittin (10 microM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 microM) and light (to elevate intracellular Ca(2+)), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 microM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for > or =90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 microm), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA(2) signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca(2+) to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca(2+) may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.     

Endogenous zinc inhibits GABA(A) receptors in a hippocampal pathway

Depending on their subunit composition, GABA(A) receptors can be highly sensitive to Zn(2+). Although a pathological role for Zn(2+)-mediated inhibition of GABA(A) receptors has been postulated, no direct evidence exists that endogenous Zn(2+) can modulate GABAergic signaling in the brain. A possible explanation is that Zn(2+) is mainly localized to a subset of glutamatergic synapses. Hippocampal mossy fibers are unusual in that they are glutamatergic but have also been reported to contain GABA and Zn(2+). Here, we show, using combined Timm's method and post-embedding immunogold, that the same mossy fiber varicosities can contain both GABA and Zn(2+). Chelating Zn(2+) with either calcium-saturated EDTA or N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine had no effect on stratum-radiatum-evoked inhibitory postsynaptic currents (IPSCs), but enhanced IPSCs evoked by stimuli designed to recruit dentate granule cells. We also show that IPSCs recorded in CA3 pyramidal neurons in acute hippocampal slices are depressed by exogenous Zn(2+). This depression was of similar amplitude whether the IPSCs were evoked by stimulation in s. radiatum (to recruit local interneurons) or in the s. granulosum of the dentate gyrus (to recruit mossy fibers). These results show for the first time that GABAergic IPSCs can be modulated by endogenous Zn(2+) and are consistent with GABA release at Zn(2+)-containing mossy fiber synapses.     

GABAC receptors in the rat superior colliculus and pretectum participate in synaptic neurotransmission

In mammals, GABA(C) receptors seem to be specifically expressed in the retina and the subcortical visual system, with highest extraretinal expression levels in the superior colliculus (SC). Although its presence in the superficial SC has been demonstrated physiologically, a direct involvement of this receptor type in fast synaptic neurotransmission still awaits verification. We addressed the question of a possible synaptic localization of GABA(C) receptors by performing in vitro whole-cell patch-clamp recordings of inhibitory postsynaptic currents (IPSCs) in single neurons of the rat SC and the neighboring pretectal nuclear complex, where GABA(C) receptors are also expressed at significant levels. To increase the likelihood to record IPSCs we induced spontaneous activity by application of the potassium channel blocker 4-aminopyridine (4-AP) and blocked glutamate-mediated excitatory neurotransmission with kynurenic acid. All 4-AP-induced postsynaptic currents were of synaptic origin because they were completely suppressed by lidocaine or by substitution of extracellular calcium with cobalt. In 40% of the SC cells and in 60% of the pretectal neurons, IPSCs in the presence of 4-AP and kynurenic acid were only partly blocked by the selective GABA(A) receptor antagonist bicuculline. Inhibitory currents that were insensitive to bicuculline, however, could be blocked by coapplication of either the specific GABA(C) receptor antagonist 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid or picrotoxin, an unselective GABA(A) and GABA(C) receptor antagonist. We conclude that GABA(C) receptors are, at least partially, located synaptically in SC and pretectal neurons in the rat, which indicates a direct function of this receptor type for synaptic processing in both structures.     

Kappa opioid receptor activation in the nucleus accumbens inhibits glutamate and GABA release through different mechanisms

Through their actions in the nucleus accumbens (NAc), kappa opioid (KOP) receptors and their endogenous ligand, dynorphin, modify behaviors associated with the administration of drugs of abuse and are regulated by exposure to such drugs. Despite their demonstrated behavioral significance, the synaptic actions of KOP receptor ligands in the NAc are not clearly understood. Using whole-cell voltage-clamp recordings of NAc medium spiny neurons, we have found that, in addition to suppressing glutamate release, the KOP receptor agonist also inhibits GABA release. Interestingly, the mechanism of inhibition of the release of glutamate differs from that controlling GABA. reduces the frequency of Ca(2+)-independent miniature excitatory postsynaptic currents, but not miniature inhibitory postsynaptic currents. Furthermore, while the inhibition of GABAergic transmission is blocked by the N-type Ca(2+) channel blocker omega-CgTx, the inhibition of excitatory glutamatergic transmission by is unaffected by N-type Ca(2+) channel blockade. These results indicate that KOP receptor activation inhibits GABA release by reducing Ca(2+) influx, but inhibits glutamate release at a step downstream of Ca(2+) entry.     

Major role for tonic GABAA conductances in anesthetic suppression of intrinsic neuronal excitability

Anesthetics appear to produce neurodepression by altering synaptic transmission and/or intrinsic neuronal excitability. Propofol, a widely used anesthetic, has proposed effects on many targets, ranging from sodium channels to GABA(A) inhibition. We examined effects of propofol on the intrinsic excitability of hippocampal CA1 neurons (primarily interneurons) recorded from adult rat brain slices. Propofol strongly depressed action potential production induced by DC injection, synaptic stimulation, or high-potassium solutions. Propofol-induced depression of intrinsic excitability was completely reversed by bicuculline and picrotoxin but was strychnine-insensitive, implicating GABA(A) but not glycine receptors. Propofol strongly enhanced inhibitory postsynaptic currents (IPSCs) and induced a tonic GABA(A)-mediated current. We pharmacologically differentiated tonic and phasic (synaptic) GABA(A)-mediated inhibition using the GABA(A) receptor antagonist SR95531 (gabazine). Gabazine (20 microM) completely blocked both evoked and spontaneous IPSCs but failed to block the propofol-induced depression of intrinsic excitability, implicating tonic, but not phasic, GABA(A) inhibition. Glutamatergic synaptic responses were not altered by propofol (< or =30 microM). Similar results were found in both interneurons and pyramidal cells and with the chemically unrelated anesthetic thiopental. These results suggest that suppression of CA1 neuron intrinsic excitability, by these anesthetics, is largely due to activation of tonic GABA(A) conductances; although other sites of action may play important roles in affecting synaptic transmission, which also can produce strong neurodepression. We propose that for some anesthetics, suppression of intrinsic excitability, mediated by tonic GABA(A) conductances, operates in conjunction with effects on synaptic transmission, mediated by other mechanisms, to depress hippocampal function during anesthesia.     

Mu opioid receptor-mediated modulation of synaptic currents in dentate granule cells of rat hippocampus.

1. The effect of a selective mu opioid agonist, [N-MePhe3-D-Pro4]morphiceptin (PL017), on synaptic transmission in the dentate gyrus was examined in hippocampal slices. Synaptic currents were evoked by stimulation of the outer molecular layer and recorded from granule cells using whole-cell voltage-clamp techniques. 2. Monosynaptic inhibitory postsynaptic currents (IPSCs) were evoked in the presence of D(-)-2-amino-5-phosphonovaleric acid (D-APV), and N-methyl-D-aspartate (NMDA) receptor antagonist, and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA type of glutamate receptor antagonist. The IPSCs consisted of a gamma-aminobutyric acid (GABA)A receptor-mediated early component and a GABAB receptor-mediated late component. 3. Bath application of PL017 (0.3-3 microM) induced a dose-dependent reduction in the amplitude of both early IPSCs (21-56%) and late IPSCs (43-81%). These effects could be reversed by the opiate antagonist naloxone (1 microM) or prevented by the selective mu antagonist beta-funaltrexamine hydrochloride (10 microM). 4. NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were revealed in the presence of DNQX and the GABAA antagonist bicuculline methiodide. PL017 (3 microM) caused a 35% reduction in the amplitude of NMDA EPSCs. NMDA receptor-mediated population EPSPs recorded extracellularly were also inhibited by 3 microM PL017 to a similar degree. 5. Non-NMDA receptor-mediated EPSCs were demonstrated in the presence of D-APV and bicuculline methiodide. The amplitude of non-NMDA EPSCs was not affected by PL017.(ABSTRACT TRUNCATED AT 250 WORDS)