Different pools of postsynaptic GABAA receptors mediate inhibition evoked by low‐ and high‐frequency presynaptic stimulation at hippocampal synapses

Patterns of short‐term synaptic plasticity could considerably differ between synapses of the same axon. This may lead to separation of synaptic receptors transmitting either low‐ or high‐frequency signals and, therefore, may have functional consequences for the information transfer in the brain. Here, we estimated a degree of such separation at hippocampal GABAergic synapses using a use‐dependent GABA_A receptor antagonist, picrotoxin, to selectively suppress a pool of GABA_A receptors monosynaptically activated during the low‐frequency stimulation. The relative changes in postsynaptic responses evoked by the high‐frequency stimulation before and after such block were used to estimate the contribution of this GABA_A receptor pool to synaptic transmission at high frequencies. Using this approach, we have shown that IPSCs evoked by low‐frequency (0.2 Hz) stimulation and asynchronous currents evoked by high‐frequency (20–40 Hz) stimulation are mediated by different pools of postsynaptic GABA_A receptors. Thus, our findings suggest that inhibition produced by a single hippocampal interneuron may be selectively routed to different postsynaptic targets depending on the presynaptic discharge frequency. Synapse 68:344–354, 2014 . © 2014 Wiley Periodicals, Inc.     

GABAB receptor‐dependent modulation of network activity in the rat prefrontal cortex in vitro

GABA (γ‐aminobutyric acid) can mediate inhibition via pre‐ and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA_B receptor‐dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage‐clamp experiments the presence of a tonic GABA_B receptor‐dependent outward current in these neurons. This GABA_Bergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA_B current was similar at different temperatures. In addition to the tonic GABA_B current, we found presynaptic GABA_B effects, GABA_B‐mediated inhibitory postsynaptic currents and tonic GABA_A currents. Second, we performed current‐clamp experiments to evaluate the functional impact of GABA_B receptor‐mediated inhibition in the mPFC. Activating or inactivating GABA_B receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input–output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca²⁺‐imaging that GABA_B receptor‐mediated tonic inhibition is capable of regulating network activity. Blocking GABA_B receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA_B receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.     

Cocaine withdrawal reduces GABABR transmission at entopeduncular nucleus – lateral habenula synapses

Lateral habenula (LHb) hyperactivity plays a pivotal role in the emergence of negative emotional states, including those occurring during withdrawal from addictive drugs. We have previously implicated cocaine‐driven adaptations at synapses from the entopeduncular nucleus (EPN) to the LHb in this process. Specifically, ionotropic GABA_A receptor (R)‐mediated neurotransmission at EPN‐to‐LHb synapses is reduced during cocaine withdrawal, due to impaired vesicle filling. Recent studies have shown that metabotropic GABA_BR signaling also controls LHb activity, although its role at EPN‐to‐LHb synapses during drug withdrawal is unknown. Here, we predicted that cocaine treatment would reduce GABA_BR‐mediated neurotransmission at EPN‐to‐LHb synapses. We chronically treated mice with saline or cocaine, prepared brain slices after two days of withdrawal and performed voltage‐clamp recordings from LHb neurons whilst optogenetically stimulating EPN terminals. Compared with controls, mice in cocaine withdrawal exhibited reduced GABA_AR‐mediated input to LHb neurons, and a reduced occurrence of GABA_BR‐signaling at EPN‐to‐LHb synapses. We then assessed the underlying mechanism of this decrease. Application of GABA_BR agonist baclofen evoked similar postsynaptic responses in EPN‐innervated LHb neurons in saline‐ and cocaine‐treated mice. Release probability at EPN‐to‐LHb GABAergic synapses was also comparable between groups. However, incubating brain slices in glutamine to facilitate GABA vesicle filling, normalized GABA_BR‐currents at EPN‐to‐LHb synapses in cocaine‐treated mice. Overall, we show that during cocaine withdrawal, together with reduced GABA_AR transmission, also GABA_BR‐mediated inhibitory signaling is diminished at EPN‐to‐LHb synapses, likely via the same presynaptic deficit. In concert, these alterations are predicted to contribute to the emergence of drug withdrawal symptoms, facilitating drug relapse.     

Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals

In presynaptic terminals, membrane-delimited G_i/o-mediated presynaptic inhibition is ubiquitous and acts via Gβγ to inhibit Ca²⁺ entry, or directly at SNARE complexes to inhibit Ca²⁺-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT_1B and GABA_B receptors colocalize. GABA_B receptors inhibit Ca²⁺ entry, whereas 5-HT_1B receptors target SNARE complexes. We demonstrate in male and female rats that GABA_B receptors alter P_r, whereas 5-HT_1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of P_r changes, consistent with GABA_B receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT_1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca²⁺ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT_1B receptor activation. This is consistent with competition between Ca²⁺-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT_1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABA_B receptors, which alter presynaptic Ca²⁺. In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one (a GABA_B receptor) inhibits Ca²⁺ entry whereas another (a 5-HT_1B receptor) competes with Ca²⁺-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABA_B receptors alter P_r leaving synaptic properties unchanged, whereas 5-HT_1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABA_B receptor alteration on Ca²⁺ and the effect of this altered Ca²⁺ signal on 5-HT_1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.     

GABA transporter subtype 1 and GABA transporter subtype 3 modulate glutamatergic transmission via activation of presynaptic GABA(B) receptors in the rat globus pallidus

The intra‐pallidal application of γ‐aminobutyric acid (GABA) transporter subtype 1 (GAT‐1) or GABA transporter subtype 3 (GAT‐3) transporter blockers [1‐(4,4‐diphenyl‐3‐butenyl)‐3‐piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1‐[2‐[tris(4‐methoxyphenyl)methoxy]ethyl]‐(S)‐3‐piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA_B heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole‐cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA_A receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA_B receptor blockade by the intra‐cellular application of N‐(2,6‐dimethylphenylcarbamoylmethyl)‐triethylammonium bromide (OX314), bath application of SKF 89976A (10 μm ) or SNAP 5114 (10 μm ) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)‐3‐[[(1S)‐1‐(3,4‐dichlorophenyl)ethyl]amino‐2‐hydroxypropyl](phenylmethyl)phosphinic acid, a GABA_B receptor antagonist. The paired‐pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA_B heteroreceptors following GAT‐1 or GAT‐3 blockade. In conclusion, our findings demonstrate that presynaptic GABA_B heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced by GAT inactivation, thereby suggesting that GAT blockade represents a potential mechanism by which overactive subthalamopallidal activity may be reduced in parkinsonism.     

Long‐term depression of inhibitory synaptic transmission induced by spike‐timing dependent plasticity requires coactivation of endocannabinoid and muscarinic receptors

The precise timing of pre‐postsynaptic activity is vital for the induction of long‐term potentiation (LTP) or depression (LTD) at many central synapses. We show in synapses of rat CA1 pyramidal neurons in vitro that spike timing dependent plasticity (STDP) protocols that induce LTP at glutamatergic synapses can evoke LTD of inhibitory postsynaptic currents or STDP‐iLTD. The STDP‐iLTD requires a postsynaptic Ca²⁺ increase, a release of endocannabinoids (eCBs), the activation of type‐1 endocananabinoid receptors and presynaptic muscarinic receptors that mediate a decreased probability of GABA release. In contrast, the STDP‐iLTD is independent of the activation of nicotinic receptors, GABA_BRs and G protein‐coupled postsynaptic receptors at pyramidal neurons. We determine that the downregulation of presynaptic Cyclic adenosine monophosphate/protein Kinase A pathways is essential for the induction of STDP‐iLTD. These results suggest a novel mechanism by which the activation of cholinergic neurons and retrograde signaling by eCBs can modulate the efficacy of GABAergic synaptic transmission in ways that may contribute to information processing and storage in the hippocampus. © 2013 Wiley Periodicals, Inc.     

Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP‐mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. In this study, using whole‐cell patch‐clamp techniques on Drosophila primary neuronal cultures, we demonstrate that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). We observed a dual regulatory role of FSK on GABAergic transmission, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, we show that cAMP decreased GABAergic IPSCs in a PKA‐dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short‐term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, our study suggests that alterations in cAMP‐mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning.     

GABA receptors and prepulse inhibition of acoustic startle in mice and rats

The acoustic startle reflex is strongly inhibited by a moderate‐intensity acoustic stimulus that precedes the startling stimulus by roughly 10–1000 ms (prepulse inhibition, PPI). At long interstimulus intervals (ISIs) of 100–1000 ms, PPI in rats is reduced by the muscarinic receptor antagonist scopolamine. Here, we studied the role of GABA receptors in PPI at full ISI ranges in both mice and rats. In B6 mice, PPI begins and ends at shorter ISIs (4 and 1000 ms, respectively) than in Wistar rats (8 and 5000 ms). The GABA_A antagonist bicuculline (1 mg/kg i.p.) reduced PPI at ISIs near the peak of PPI in both rats and mice. The GABA_B antagonist phaclofen (10 or 30 mg/kg i.p. in rats or mice, respectively) reduced PPI only at long ISIs, similar to the effects of the muscarinic antagonist scopolamine (1 mg/kg i.p.). The effects of phaclofen and scopolamine were additive in rats, suggesting independent effects of GABA_B and muscarinic receptors. Patch‐clamp recordings of startle‐mediating PnC (nucleus reticularis pontis caudalis) giant neurons in rat slices show that EPSCs evoked by either trigeminal or auditory fiber stimulation were inhibited by the GABA_A/C agonist muscimol or the GABA_B agonist baclofen via postsynaptic mechanisms. Hyperpolarization of PnC neurons by muscimol was reversed with bicuculline, indicating that postsynaptic GABA_A receptors strongly inhibit PnC giant neurons needed for startle. Therefore, GABA receptors on PnC giant neurons mediate a substantial part of PPI, with GABA_A receptors contributing at the peak of PPI, and GABA_B receptors adding to muscarinic effects on PPI at long ISIs.     

GABAB receptor‐mediated presynaptic inhibition reverses inter‐columnar covariability of synaptic actions by intracortical axons in the rat barrel cortex

Intracortical axons originating from pyramidal cells in layer 3 of the rat somatosensory cortex are shared between adjacent columns, and receive the presynaptic inhibition that is mediated by the GABA_B receptor. Synaptic actions by intracortical axons of single layer 3 pyramidal cells covary between the two adjacent columns in response to stimulation of layer 3 of either column. We examined whether GABA_B receptor‐mediated presynaptic inhibition affects the covariability of synaptic actions by intracortical axons between adjacent columns in slice preparations of the rat barrel cortex. Paired stimulations of superficial layer 3 evoked first and second excitatory postsynaptic currents (EPSCs) of varying amplitudes, yielding varying paired‐pulse depression of EPSCs in layer 3 pyramidal cells that were located in the stimulated column, but not in its adjacent column. The amplitude of the second EPSC was inversely proportional to that of the first EPSC in layer 3 pyramidal cells in the stimulated column, yielding a negative correlation coefficient between the first and second EPSCs. Baclofen and CGP55845 attenuated paired‐pulse depression and abolished the inverse relationship. Simultaneous recordings from two layer 3 pyramidal cells in the stimulated and adjacent columns revealed a positive correlation between the paired first EPSC amplitudes and a negative correlation between the paired second EPSC amplitudes, which, respectively, indicate the positive and negative covariability of synaptic actions by intracortical axons between the two adjacent columns. These results suggest that GABA_B receptor‐mediated presynaptic inhibition can reverse the positive covariability of inter‐columnar synaptic actions, which may serve as a basis for inter‐columnar desynchronisation.     

GABAA receptors can initiate the formation of functional inhibitory GABAergic synapses

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABA_A receptors (GABA_ARs) themselves – the essential functional postsynaptic components of GABAergic synapses – can be sufficient to initiate formation of synaptic contacts, a novel co‐culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2‐GABA_ARs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse‐like contacts in these co‐cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle‐luminal domain‐specific antibody; and they supported spontaneous and action potential‐driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N‐methyl‐d ‐aspartate receptor‐expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin‐2 was co‐expressed with GABA_ARs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABA_ARs can promote the adhesion of inhibitory axons and the development of functional synapses.     

Impaired function of GABAB receptors in tissues from pharmacoresistant epilepsy patients

Purpose: Effects of pre‐ and postsynaptic γ‐aminobutyric acid B (GABA_B) receptor activation were characterized in human tissue from epilepsy surgery. Methods: Slices of human cortical tissue were investigated in a submerged‐type chamber with intracellular recordings in layers II/III. Parallel experiments were performed in rat neocortical slices with identical methods. Synaptic responses were elicited with single or paired stimulations of incrementing intervals. Results: Neurons in human epileptogenic tissue exhibited usually small inhibitory postsynaptic potentials (IPSP) mediated by GABA_B receptor, verified by the sensitivity to the selective antagonist CGP 55845A. The IPSP_B conductance averaged 5.8 nS in neurons from epileptogenic tissues and 15.9 nS in neurons from nonepileptogenic tissues (p < 0.0001). Application of baclofen caused small conductance increases in human neurons, which were linearly related to IPSP_B conductances. Paired‐pulse stimulation revealed constant synaptic responses in human temporal lobe epilepsy (TLE) slices at all interstimulus intervals (ISIs). Pharmacologically isolated IPSP_A in the human tissue exhibited a small paired‐pulse depression (average 10% at 500 ms ISI). Bicuculline‐induced paroxysmal depolarization shifts (PDSs) were transiently depressed by 24% in human TLE tissue; and by 74% in rat neocortical slices (200 ms ISI; p = 0.015). The depressions of bicuculline‐induced PDSs were antagonized by CGP 55845A in both species. Staining for GABA_B receptors revealed significantly smaller numbers of immunopositive dots in human epileptogenic neurons versus human control neurons. Discussion: The small IPSP_B, baclofen‐conductances, and paired‐pulse depression of PDSs and IPSPs in human TLE tissue indicate a reduced density of post‐ and presynaptic GABA_B receptors. The reduced efficacy of presynaptic GABA_B receptors facilitates the occurrence of repetitive synaptic activity.     

Involvement of GABAB receptors in convulsant‐induced epileptiform activity in rat neocortex in vitro

The role of γ-aminobutyric acid B (GABA_B) receptors in the generation and maintenance of bicuculline-induced epileptiform activity in rat neocortical slices was studied using electrophysiological methods. A block of GABA_B receptors in the presence of functional GABA_A receptor-mediated inhibition was not sufficient to induce epileptiform activity. In the presence of the GABA_A receptor antagonist bicuculline (10 μm ) and at suprathreshold stimulation, the GABA_B receptor antagonist CGP 35348 (10–300 μm ) significantly potentiated epileptiform activity. With stimulation at threshold intensity, low concentrations of CGP 35348 (10–30 μm ) potentiated bicuculline-induced activity, whereas higher concentrations (100–300 μm ) invariably led to a reversible suppression of stimulus-evoked epileptiform discharges. CGP 35348 also enhanced picrotoxin-induced epileptiform activity, but at higher concentrations it was considerably less effective in suppressing such epileptiform discharges. The GABA uptake inhibitor nipecotic acid partially mimicked the actions of CGP 35348: with stimulation at threshold intensity, it reversibly suppressed bicuculline-induced epileptiform field potentials, but it did not influence epileptiform activity induced by picrotoxin. We conclude that a postsynaptic blockade of GABA_B receptors induces an amplification of epileptiform activity in neocortical slices disinhibited by GABA_A receptor antagonists. An additional blockade of presynaptic GABA_B receptors, especially under conditions of weak stimulation of the neurons, reduces the inhibitory auto-feedback control of GABA release, leading to a displacement of competitive antagonists from the postsynaptic GABA_A receptor and hence, to a suppression of epileptiform activity induced by competitive GABA_A receptor antagonists.     

Protein kinase C regulates tonic GABAA receptor‐mediated inhibition in the hippocampus and thalamus

Tonic inhibition mediated by extrasynaptic GABA_A receptors (GABA_ARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA_ARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole‐cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC‐mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA_AR‐mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA_AR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA_ARs, which represent a key extrasynaptic GABA_AR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA_ARs. The inhibitory effects of PKC activation on α4β2δ GABA_AR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC‐mediated phosphorylation can be an important physiological regulator of tonic GABA_AR‐mediated inhibition.     

N‐type calcium channels mediate a GABAB presynaptic modulation in the corticostriatal synapse in turtle's paleostriatum augmentatum

Spikes population evoked by a paired pulse protocol were used to assess the influence of GABA_A and GABA_B receptors agonists and antagonists on the synaptic potentials and in the S₂/S₁ ratio in a paired pulse (PP) protocol in the cortico‐paleostriatum augmentatum synapses of the turtle. GABA_A agonist, muscimol, decreased the amplitude of synaptic responses whereas the facilitation produced with the PP protocol did not change, suggesting a postsynaptic action for GABA_A receptors. GABA_B agonist, baclofen, enhanced paired pulse ratio indicating a presynaptic modulation through the GABA_B receptor. Selective antagonists for N‐ and P/Q‐type Ca²⁺‐channels also enhanced paired pulse ratio, suggesting that any of these channel types may be involved in neurotransmitter release. However, the strong paired pulse facilitation produced by baclofen was occluded by blocking the N‐type Ca²⁺ channels with ω‐conotoxin GVIA (1 μM), but not by the blockage of P/Q‐type Ca²⁺ channels with ω‐agatoxin TK (400 nM). These data suggest that N and P/Q channels participate in the neurotransmitter release, whereas only N‐type Ca²⁺ channels are involved in the presynaptic modulation of GABA_B in the corticostriatal synapse of the turtle. Synapse 63:855–862, 2009. © 2009 Wiley‐Liss, Inc.     

Presynaptic and postsynaptic GABAB receptors of neocortical neurons of the rat in vitro: Differences in pharmacology and ionic mechanisms

The properties of pre- and postsynaptic GABA_B receptors were investigated with intracellular recordings from rat neocortical neurons in vitro. An antagonist of the GABA_B receptor (CGP 35348) and ions or drugs interfering with GABA_B receptor-mediated K⁺ conductance (Ba²⁺, QX 314) were employed to delineate possible differences. CGP 35348 reduced the conductance of the late inhibitory postsynaptic potential (IPSP_B) in a dose-dependent manner. Neither the early GABA_A receptor-mediated inhibitory postsynaptic potential (IPSP_A), nor resting membrane potential or direct excitability, were consistently affected by CGP 35348. Bath application of 100 μmol/l Ba²⁺ decreased IPSP_B conductance to about 40% and increased IPSP_A conductance to 130% of control. The depression of a second IPSP by a pair of stimuli (paired pulse depression, or PPD) was used as an index for presynaptic GABA_B receptor activation. Neither CGP 35348 nor Ba²⁺ exerted significant effects on the PPD at intervals of 400 msec. The dependence of PPD on the latency of the interval of the stimulus pair was investigated after intracellular application of QX 314 had virtually abolished the IPSP_B. Decreasing the stimulus interval from 500 msec to 100 msec revealed a stronger depression of the second IPSP_A. Application of CGP 35348 alleviated PPD for stimulus intervals below 300 msec. The data indicate a distinct pharmacological difference between pre- and postsynaptic GABA_B receptors. Moreover, we suggest that two temporally distinct presynaptic GABA_B receptor effects contribute to PPD: a short-lasting effect, sensitive to CGP 35348, and a long-lasting effect, insensitive to CGP 35348. The latter is insensitive to Ba²⁺, implying that this component is not associated with a K⁻ conductance mechanism. Synapse 25:62–72, 1997. © 1997 Wiley-Liss, Inc.     

Segmental disinhibition suppresses C‐fiber inputs to the rat superficial medullary dorsal horn via the activation of GABAB receptors

Specialized primary afferents, although they terminate in different laminae within the dorsal horn (DH), are known to interact through local circuit excitatory and inhibitory neurons. That a loss of segmental inhibition probably contributes to persistent pain hypersensitivity during chronic pain raises the question as to how disinhibition‐induced changes in cross‐modal interactions account for chronic pain symptoms. We sought to characterize how pharmacological blockade of glycine and gamma‐aminobutyric acid (GABA) receptors modifies synaptic transmission between primary afferent fibers and second‐order neurons by recording field potentials in the superficial medullary dorsal horn (MDH) of anesthetized rats. Transcutaneous electrical stimulation evokes three negative field potentials elicited by, from earliest to latest, Aβ‐, Aδ‐ and C‐fiber primary afferents. Blocking segmental glycine and/or GABA_A receptors, with strychnine and bicuculline, respectively, strongly facilitates Aβ‐ and Aδ‐fiber‐evoked polysynaptic field potentials but, conversely, inhibits, or even abolishes, the whole C‐fiber field potential. Blocking segmental GABA_B receptors, with phaclofen, reverses such suppression of C‐fiber field potentials. Interestingly, it also potentiates C‐fiber field potentials under control conditions. Finally, activation of segmental GABA_B receptors, with baclofen, preferentially inhibits C‐fiber field potentials. Our results suggest that activation of A‐fiber primary afferents inhibits C‐fiber inputs to the MDH by the way of polysynaptic excitatory pathways, last‐order GABAergic interneurons and presynaptic GABA_B receptors on C‐fiber primary afferents. Under physiological conditions, activation of such local DH circuits is closely controlled by segmental inhibition but it might contribute to paradoxically reduced pain hypersensitivity under pathological disinhibition.     

Developmental increase in D1‐like dopamine receptor‐mediated inhibition of glutamatergic transmission through P/Q‐type channel regulation in the basal forebrain of rats

Whole‐cell patch‐clamp recordings of non‐N‐methyl‐d ‐aspartate glutamatergic excitatory postsynaptic currents (EPSCs) were carried out from cholinergic neurons in slices of basal forebrain (BF) of developing rats aged 21–42 postnatal days to elucidate postnatal developmental change in Ca²⁺ channel subtypes involved in the transmission as well as that in dopamine D₁‐like receptor‐mediated presynaptic inhibition. The amplitude of EPSCs was inhibited by bath application of ω‐conotoxin GVIA (ω‐CgTX; 3 μm ) or ω‐agatoxin‐TK (ω‐Aga‐TK; 200 nm ) throughout the age range examined, suggesting that multiple types of Ca²⁺ channel are involved in the transmission. The EPSC fraction reduced by ω‐CgTX decreased with age, whereas that reduced by ω‐Aga‐TK increased. Inhibition of the EPSCs by a D₁‐like receptor agonist, SKF 81297 (SKF; 30 μm ) increased with age in parallel with the increase in ω‐Aga‐TK‐induced inhibition. An activator of the adenylyl cyclase (AC) pathway, forskolin (FK; 10 μm ) inhibited the EPSCs, and FK‐induced inhibition also increased with age in parallel with the increase in SKF‐induced inhibition. Throughout the age range examined, SKF showed no further inhibitory effect on the EPSCs after ω‐Aga‐TK‐ or FK‐induced effect had reached steady‐state. These findings suggest that D₁‐like receptor‐mediated presynaptic inhibition of glutamate release onto cholinergic BF neurons increases with age, and that the change is coupled with a developmental increase in the contribution of P/Q‐type Ca²⁺ channels as well as a developmental increase in AC pathway contribution.     

PACAP induces plasticity at autonomic synapses by nAChR-dependent NOS1 activation and AKAP-mediated PKA targeting

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide found at synapses throughout the central and autonomic nervous system. We previously found that PACAP engages a selective G-protein coupled receptor (PAC₁R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca^2 + influx. Ca^2 +-permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC₁R signaling and were required for PACAP-induced NO production and synaptic plasticity since both outcomes were drastically reduced following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric α3*-nAChRs that generate the bulk of synaptic activity. NOS1–nAChR physical association could facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC₁R signaling are localized by PKA anchoring proteins (AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC₁R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such coordination has broad relevance for understanding the control of autonomic synapses and consequent visceral functions.     

A specific GABAergic synapse onto oligodendrocyte precursors does not regulate cortical oligodendrogenesis

In the brain, neurons establish bona fide synapses onto oligodendrocyte precursor cells (OPCs), but the function of these neuron‐glia synapses remains unresolved. A leading hypothesis suggests that these synapses regulate OPC proliferation and differentiation. However, a causal link between synaptic activity and OPC cellular dynamics is still missing. In the developing somatosensory cortex, OPCs receive a major type of synapse from GABAergic interneurons that is mediated by postsynaptic γ2‐containing GABA_A receptors. Here we genetically silenced these receptors in OPCs during the critical period of cortical oligodendrogenesis. We found that the inactivation of γ2‐mediated synapses does not impact OPC proliferation and differentiation or the propensity of OPCs to myelinate their presynaptic interneurons. However, this inactivation causes a progressive and specific depletion of the OPC pool that lacks γ2‐mediated synaptic activity without affecting the oligodendrocyte production. Our results show that, during cortical development, the γ2‐mediated interneuron‐to‐OPC synapses do not play a role in oligodendrogenesis and suggest that these synapses finely tune OPC self‐maintenance capacity. They also open the interesting possibility that a particular synaptic signaling onto OPCs plays a specific role in OPC function according to the neurotransmitter released, the identity of presynaptic neurons or the postsynaptic receptors involved.