GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro

Synapse formation in CNS neurons requires appropriate sorting and clustering of neurotransmitter receptors and associated proteins at postsynaptic sites. In GABAergic synapses, clustering of GABA(A) receptors requires gephyrin, but it is not known whether presynaptic signals are also involved in this process. To investigate this issue, we analyzed the subcellular distribution of GABA(A) receptors and gephyrin in primary cultures of cerebellar granule cells, by comparing cells receiving GABAergic input with cells devoid of such afferents. Using immunofluorescence staining, we show that the GABA(A) receptor alpha1 and gamma2 subunit, but not alpha6 or delta subunit, form clusters co-localized with gephyrin in granule cell neurites, irrespective of the presence of GABAergic axons. GABAergic terminals typically were surrounded by groups of gephyrin clusters, pointing to the presence of multiple synaptic sites. In contrast, in neurites devoid of GABAergic input, gephyrin clusters were distributed at random and apposed to glutamatergic terminals, suggesting the formation of mismatched synapses. Both populations of gephyrin clusters were co-localized with GABA(A) receptor subunits, indicating that these proteins are associated also in non-GABAergic synapses. To determine whether signaling mediated by GABA(A) receptors is required for the formation of appropriately matched gephyrin clusters, cultures were treated chronically with bicuculline, or with either muscimol or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. All these treatments failed to influence the distribution of gephyrin clusters. We conclude that although GABAergic presynaptic terminals have a preponderant influence on the distribution of gephyrin clusters in dendrites of cerebellar granule cells, GABA transmission is dispensable for postsynaptic clustering of gephyrin and GABA(A) receptors and for the formation of appropriately matched GABAergic synapses.     

alpha7 nicotinic acetylcholine receptors on GABAergic interneurons evoke dendritic and somatic inhibition of hippocampal neurons.

GABAergic interneurons in the hippocampus express high levels of alpha7 nicotinic acetylcholine receptors, but because of the diverse roles played by hippocampal interneurons, the impact of activation of these receptors on hippocampal output neurons (i.e., CA1 pyramidal cells) is unclear. Activation of hippocampal interneurons could directly inhibit pyramidal neuron activity but could also produce inhibition of other GABAergic cells leading to disinhibition of pyramidal cells. To characterize the inhibitory circuits activated by these receptors, exogenous acetylcholine was applied directly to CA1 interneurons in hippocampal slices, and the resulting postsynaptic responses were recorded from pyramidal neurons or interneurons. Inhibitory currents mediated by GABA(A) receptors were observed in 27/131 interneuron/pyramidal cell pairs, but no instances of disinhibition of spontaneous inhibitory events or GABA(B) receptor-mediated responses were observed. Two populations of bicuculline-sensitive GABA(A) receptor-mediated currents could be distinguished based on their kinetics and amplitude. Anatomical reconstructions of the interneurons in a subset of connected pairs support the hypothesis that these two populations correspond to inhibitory synapses located either on the somata or dendrites of pyramidal cells. In 11 interneuron/interneuron cell pairs, one presynaptic neuron was observed that produced strong inhibitory currents in several nearby interneurons, suggesting that disinhibition of pyramidal neurons may also occur. All three types of inhibitory responses (somatic-pyramidal, dendritic-pyramidal, and interneuronal) were blocked by the alpha7 receptor-selective antagonist methyllycaconitine. These data suggest activation of these functionally distinct circuits by alpha7 receptors results in significant inhibition of both hippocampal pyramidal neurons as well as interneurons.     

NMDA receptor activation enhances inhibitory GABAergic transmission onto hippocampal pyramidal neurons via presynaptic and postsynaptic mechanisms

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.     

Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.     

Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus

Psychoactive effects of cannabinoids are thought to be mediated, at least in part, by suppression of both glutamate and GABA release via CB1 cannabinoid receptor. Two types of cannabinoid receptor (CB1 and CB2) have been cloned so far. The CB1 receptors are abundantly expressed in the nervous system, whereas CB2 receptors are limited to lymphoid organs (Matsuda et al., 1990; Munro et al., 1993). Immunocytochemical and electrophysiological studies revealed that in the hippocampus CB1 receptors are expressed on axon terminals of GABAergic inhibitory interneurons (Tsou et al., 1999; Katona et al., 1999) and activation of these receptors decreases GABA release (Hájos et al., 2000). Other physiological studies pointed out the involvement of CB1 receptors in the modulation of hippocampal glutamatergic synaptic transmission and long-term potentiation (Stella et al., 1997; Misner and Sullivan, 1999), but anatomical studies could not confirm the existence of CB1 receptors on glutamatergic terminals. Here we examined cannabinoid actions on both glutamatergic and GABAergic synaptic transmission in the hippocampus of wild type (CB1+/+) and CB1 receptor knockout mice (CB1-/-). The synthetic cannabinoid agonist WIN55,212-2 reduced the amplitudes of excitatory postsynaptic currents in both wild type and CB1-/- mice, while inhibitory postsynaptic currents were decreased only in wild type mice, but not in CB1-/- animals. Our findings are consistent with a CB1 cannabinoid receptor-dependent modulation of GABAergic postsynaptic currents, but a novel cannabinoid-sensitive receptor must be responsible for the inhibition of glutamatergic neurotransmission.     

Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus

The aim of this study was to determine whether age-associated alterations in the GABAergic input to pyramidal neurons in the hippocampus are due to a dysfunction of GABAergic interneurons, and/or a decrease in their cholinergic control via nicotinic receptors (nAChRs). Electrophysiological recordings were obtained from pyramidal cells in the CA1 area of hippocampal slices from young (3-4 months old) and aged (25-30 months old) Sprague-Dawley rats. Synaptic GABA(A) receptor-mediated inhibitory postsynaptic currents and inhibitory postsynaptic potentials induced by stimulation of the stratum oriens were significantly smaller in aged rats. The frequency (but not amplitude) of spontaneous and miniature GABA inhibitory postsynaptic currents (IPSCs) was reduced in aged rats, suggesting a presynaptic alteration. Tetanic stimulation of cholinergic afferents to release endogenous acetylcholine, or an exogenous application of the nAChR agonist cytisine, increased the frequency of spontaneous IPSCs in young rats; however these effects were not evident in aged rats, indicating that the nicotinic control of GABA release is lowered during aging. None of these age-related alterations were reversed by a chronic treatment with donepezil, a cholinesterase inhibitor. Immunofluorescent labeling of GABA interneurons with somatostatin (SOM), parvalbumin (PV) or calbindin (CB), together with the vesicular acetylcholine transporter VAChT, revealed a selective loss of subpopulations of SOM and CB positive interneurons. This loss was associated with a general decrease in density of the cholinergic network in aged rats. Thus, the lower GABAergic inhibition observed in the aged rat hippocampus is due to a selective loss/dysfunction of subpopulations of GABAergic interneurons, associated with a widespread cholinergic deficit.     

Inhibition and disinhibition of pyramidal neurons by activation of nicotinic receptors on hippocampal interneurons

Nicotinic acetylcholine receptors (nAChRs) are expressed in the hippocampus, and their functional roles are beginning to be delineated. The effect of nAChR activation on the activity of both interneurons and pyramidal neurons in the CA1 region was studied in rat hippocampal slices. In CA1 stratum radiatum with muscarinic receptors inhibited, local pressure application of acetylcholine (ACh) elicited a nicotinic current in 82% of the neurons. The majority of the ACh-induced currents were sensitive to methyllycaconitine, which is a specific inhibitor of alpha7-containing nAChRs. Methyllycaconitine-insensitive nicotinic currents also were present as detected by a nonspecific nAChR inhibitor. The ACh-sensitive neurons in the s. radiatum were identified as GABAergic interneurons by their electrophysiological properties. Pressure application of ACh induced firing of action potentials in approximately 70% of the interneurons. The ACh-induced excitation of interneurons could induce either inhibition or disinhibition of pyramidal neurons. The inhibition was recorded from the pyramidal neuron as a burst of GABAergic synaptic activity. That synaptic activity was sensitive to bicuculline, indicating that GABA(A) receptors mediated the ACh-induced synaptic currents. The disinhibition was recorded from the pyramidal neuron as a reduction of spontaneous GABAergic synaptic activity when ACh was delivered onto an interneuron. Both the inhibition and disinhibition were sensitive to either methyllycaconitine or mecamylamine, indicating that activation of nicotinic receptors on interneurons was necessary for the effects. These results show that nAChRs are capable of regulating hippocampal circuits by exciting interneurons and, subsequently, inhibiting or disinhibiting pyramidal neurons.     

Alterations in excitatory and GABAergic inhibitory connections in hippocampal transplants

Solid pieces of embryonic hippocampal tissue were implanted in a cavity formed by aspiration of the fimbria-fornix and the overlying cingulate cortex in adult rats. Six to 8 months after the transplantation, chronic recording electrodes were implanted into the graft and the host hippocampi for the recording of electroencephalogram and unit activity in the freely moving animal. Irregularly occurring sharp waves or electroencephalogram spikes and concurrent synchronous discharge of large groups of neurons dominated the electrical activity of the grafts, in contrast to the situation in normal animals. Light microscopy and GABA immunocytochemistry in the grafts revealed that the three major cell types of the hippocampal formation, i.e. pyramidal neurons, dentate granule cells and GABA-immunoreactive interneurons were present in the hippocampal grafts. At the ultrastructural level, however, significant alterations in connectivity were observed. The most striking finding was the absence or sparse occurrence of synapses on the axon initial segments of pyramidal neurons. The axon initial segments are normally densely covered by GABAergic synapses derived from a specialized type of interneuron, the chandelier or axo-axonic cell. On the other hand, numerous GABA-immunoreactive terminals were found in synaptic contact with somata of pyramidal neurons, suggesting that other types of GABAergic interneurons and their efferent connections may have developed in a normal manner. The cell bodies of pyramidal neurons received, in addition, several asymmetric synapses from GABA-negative terminals. These presumably excitatory synapses are not present on the somata of pyramidal cells in the normally developing hippocampus. We hypothesize that the somatic excitatory synapses originate, at least in part, from the axon collaterals of the neighbouring pyramidal cells in the graft. We suggest that the hyperexcitability of the neuronal circuitry within the graft is due to reduced inhibition (lack of axo-axonic synapses) coupled with increased collateral excitation of the pyramidal neurons.     

Cannabinoid CB1 receptors in the paraventricular nucleus and central control of penile erection: immunocytochemistry, autoradiography and behavioral studies

[N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxyamide] (SR 141716A), a selective cannabinoid CB1 receptor antagonist, injected into the paraventricular nucleus of the hypothalamus (PVN) of male rats, induces penile erection. This effect is mediated by the release of glutamic acid, which in turn activates central oxytocinergic neurons mediating penile erection. Double immunofluorescence studies with selective antibodies against CB1 receptors, glutamic acid transporters (vesicular glutamate transporters 1 and 2 (VGlut1 and VGlut2), glutamic acid decarboxylase-67 (GAD67) and oxytocin itself, have shown that CB1 receptors in the PVN are located mainly in GABAergic terminals and fibers surrounding oxytocinergic cell bodies. As GABAergic synapses in the PVN impinge directly on oxytocinergic neurons or on excitatory glutamatergic synapses, which also impinge on oxytocinergic neurons, these results suggest that the blockade of CB1 receptors decreases GABA release in the PVN, increasing in turn glutamatergic neurotransmission to activate oxytocinergic neurons mediating penile erection. Autoradiography studies with [(3)H](-)-CP 55,940 show that chronic treatment with SR 141716A for 15 days twice daily (1 mg/kg i.p.) significantly increases the density of CB1 receptors in the PVN. This increase occurs concomitantly with an almost twofold increase in the pro-erectile effect of SR 141716A injected into the PVN as compared with control rats. The present findings confirm that PVN CB1 receptors, localized mainly in GABAergic synapses that control in an inhibitory fashion excitatory synapses, exert an inhibitory control on penile erection, demonstrating for the first time that chronic blockade of CB1 receptors by SR 141716A increases the density of these receptors in the PVN. This increase is related to an enhanced pro-erectile effect of SR 141716A, which is still present 3 days after the end of the chronic treatment.     

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.     

Nicotinic acetylcholine receptor alpha7 and alpha4beta2 subtypes differentially control GABAergic input to CA1 neurons in rat hippocampus.

The hippocampus, a limbic brain region involved in the encoding and retrieval of memory, has a well-defined structural network assembled from excitatory principal neurons and inhibitory interneurons. Because the GABAergic interneurons form synapses onto both pyramidal neurons and interneurons, the activation of nicotinic acetylcholine receptors (nAChRs) present on certain interneurons could induce either inhibition or disinhibition in the hippocampal circuitry. To understand the role of nAChRs in controlling synaptic transmission in the hippocampus, we evaluated the magnitude of nAChR-modulated GABAergic postsynaptic currents (PSCs) in pyramidal neurons and various interneurons of the CA1 region. Using whole cell patch-clamp recording and post hoc identification of neuronal types in rat hippocampal slices, we show that brief (12-s) nAChR activation by ACh (1 mM) or choline (10 mM) enhances the frequency of GABAergic PSCs in both pyramidal neurons and CA1 interneurons. The magnitude of alpha7 nAChR-mediated GABAergic inhibition, as assessed by the net charge of choline-induced PSCs, was highest in stratum lacunosum moleculare interneurons followed by pyramidal neurons and s. radiatum interneurons. In contrast, the magnitude of alpha4beta2 nAChR-mediated GABAergic inhibition, as assessed by the difference between the net charge of PSCs induced by ACh and choline, was highest in pyramidal neurons followed by s. lacunosum moleculare and s. radiatum interneurons. The present results suggest that cholinergic cues transmitted via specific subtypes of nAChRs modify the synaptic function in the hippocampus by inducing a differential degree of GABAergic inhibition in the target neurons.     

Action of tachykinins in the hippocampus: facilitation of inhibitory drive to GABAergic interneurons

By acting on neurokinin 1 (NK1) receptors, neuropeptides of the tachykinin family can powerfully excite rat hippocampal GABAergic interneurons located in the CA1 region and by this way indirectly inhibit CA1 pyramidal neurons. In addition to contact pyramidal neurons, however, GABAergic hippocampal interneurons can also innervate other interneurons. We thus asked whether activation of tachykinin-sensitive interneurons could indirectly inhibit other interneurons. The study was performed in hippocampal slices of young adult rats. Synaptic events were recorded using the whole-cell patch clamp technique. We found that substance P enhanced GABAergic inhibitory postsynaptic currents in a majority of the interneurons tested. Miniature, action potential-independent inhibitory postsynaptic currents were unaffected by substance P, as were evoked inhibitory synaptic currents. This suggests that the peptide acted at the somatodendritic membrane of interneurons, rather than at their axon terminals. The effect of substance P was mimicked by a selective NK1 receptor agonist, but not by neurokinin 2 (NK2) or neurokinin 3 (NK3) receptor agonists, and was suppressed by a NK1 selective receptor antagonist. In contrast to substance P, oxytocin, another peptide capable of activating hippocampal interneurons, had no effect on the inhibitory synaptic drive onto interneurons. We conclude that tachykinins, by acting on NK1 receptors, can influence the hippocampal activity by indirectly inhibiting both pyramidal neurons and GABAergic interneurons. Depending on the precise balance between these effects, tachykinins may either activate or depress hippocampal network activity.     

Prolongation of hippocampal miniature inhibitory postsynaptic currents in mice lacking the GABA(A) receptor alpha1 subunit

GABA(A) receptors (GABA(A)-Rs) are pentameric structures consisting of two alpha, two beta, and one gamma subunit. The alpha subunit influences agonist efficacy, benzodiazepine pharmacology, and kinetics of activation/deactivation. To investigate the contribution of the alpha1 subunit to native GABA(A)-Rs, we analyzed miniature inhibitory postsynaptic currents (mIPSCs) in CA1 hippocampal pyramidal cells and interneurons from wild-type (WT) and alpha1 subunit knock-out (alpha1 KO) mice. mIPSCs recorded from interneurons and pyramidal cells obtained from alpha1 KO mice were detected less frequently, were smaller in amplitude, and decayed more slowly than mIPSCs recorded in neurons from WT mice. The effect of zolpidem was examined in view of its reported selectivity for receptors containing the alpha1 subunit. In interneurons and pyramidal cells from WT mice, zolpidem significantly increased mIPSC frequency, prolonged mIPSC decay, and increased mIPSC amplitude; those effects were diminished or absent in neurons from alpha1 KO mice. Nonstationary fluctuation analysis of mIPSCs indicated that the zolpidem-induced increase in mIPSC amplitude was associated with an increase in the number of open receptors rather than a change in the unitary conductance of individual channels. These data indicate that the alpha1 subunit is present at synapses on WT interneurons and pyramidal cells, although differences in mIPSC decay times and zolpidem sensitivity suggest that the degree to which the alpha1 subunit is functionally expressed at synapses on CA1 interneurons may be greater than that at synapses on CA1 pyramidal cells.     

大鼠杏仁体基底外侧核中含D2受体的γ氨基丁酸神经元受多巴胺能末梢支配(英文)

杏仁体中的多巴胺(DA)和γ -氨基丁酸(GABA)递质系统均参与精神分裂症的病理过程,临床上一般用多巴胺II型受体(D2)阻断剂予以治疗。然而,目前尚不清楚GABA与D2受体是否共存,也不清楚DA能神经末梢与GABA能神经元之间的联系方式。本实验用共聚焦激光扫描显微镜(CLSM)和免疫电镜(IEM)研究了杏仁体关键性核团基底外侧核中GABA与D2受体的共存关系以及DA神经能末梢与GABA能神经元之间的突触关系。CLSM显示由谷氨酸脱羧酶(GAD)标记的GABA能神经元全部对D2受体呈免疫阳性反应,表明GABA能神经元含有D2受体。IEM显示,在 980个DA能神经末梢形成的突触中,45%的突触是由DA免疫反应阳性神经末梢直接(36% )或间接(9% )与GAD免疫反应阳性神经元的树突形成,另 55%是由DA免疫反应阳性神经末梢与未标记的神经元成分形成。DA GABA的直接性突触进而可区分为单突触 (16% )、汇聚突触 (14% )及轴 轴突触(6% )。而DA- GABA的间接性突触是个突触复合体。在该复合体中,DA免疫反应阳性末梢在一个未标记的末梢上形成对称性突触,而该未标记末梢又与GAD免疫反应阳性树突形成非对称性突触。在DA与未标记神经元成分之间的突触中,AD免疫反应阳性末梢分别与未标记胞体(4% )、树突(42% )及轴突末梢(9% )形成突触。所有DA突触无一例外均为?     

GABA(B) receptors at glutamatergic synapses in the rat striatum

Although multiple effects of GABA(B) receptor activation on synaptic transmission in the striatum have been described, the precise locations of the receptors mediating these effects have not been determined. To address this issue, we carried out pre-embedding immunogold electron microscopy in the rat using antibodies against the GABA(B) receptor subunits, GABA(B1) and GABA(B2). In addition, to investigate the relationship between GABA(B) receptors and glutamatergic striatal afferents, we used antibodies against the vesicular glutamate transporters, vesicular glutamate transporter 1 and vesicular glutamate transporter 2, as markers for glutamatergic terminals. Immunolabeling for GABA(B1) and GABA(B2) was widely and similarly distributed in the striatum, with immunogold particles localized at both presynaptic and postsynaptic sites. The most commonly labeled structures were dendritic shafts and spines, as well as terminals forming asymmetric and symmetric synapses. In postsynaptic structures, the majority of labeling associated with the plasma membrane was localized at extrasynaptic sites, although immunogold particles were also found at the postsynaptic specialization of some symmetric, putative GABAergic synapses. Labeling in axon terminals was located within, or at the edge of, the presynaptic active zone, as well as at extrasynaptic sites. Double labeling for GABA(B) receptor subunits and vesicular glutamate transporters revealed that labeling for both GABA(B1) and GABA(B2) was localized on glutamatergic axon terminals that expressed either vesicular glutamate transporter 1 or vesicular glutamate transporter 2. The patterns of innervation of striatal neurons by the vesicular glutamate transporter 1- and vesicular glutamate transporter 2-positive terminals suggest that they are selective markers of corticostriatal and thalamostriatal afferents, respectively. These results thus provide evidence that presynaptic GABA(B) heteroreceptors are in a position to modulate the two major excitatory inputs to striatal spiny projection neurons arising in the cortex and thalamus. In addition, presynaptic GABA(B) autoreceptors are present on the terminals of spiny projection neurons and/or striatal GABAergic interneurons. Furthermore, the data indicate that GABA may also affect the excitability of striatal neurons via postsynaptic GABA(B) receptors.     

CB1 modulation of temporally distinct synaptic facilitation among local circuit interneurons mediated by N-type calcium channels in CA1

One of the critical factors in determining network behavior of neurons is the influence of local circuit connections among interneurons. The short-term synaptic plasticity and the subtype of presynaptic calcium channels used at local circuit connections of inhibitory interneurons in CA1 were investigated using dual whole-cell recordings combined with biocytin and double immunofluorescence labeling in acute slices of P18- to 21-day-old rat stratum radiatum (SR) and stratum lacunosum molecular (SLM). Two forms of temporally distinct synaptic facilitation were observed among interneuron connections involving presynaptic cholecystokinin (CCK)-positive cells in SR, frequency-dependent facilitation, and a delayed onset of release (45-80 ms) with subsequent facilitation (DORF). Inhibition at both these synapses was under tonic cannabinoid-type 1 (CB1) receptor activity. DORF synapses did not display conventional release-dependent properties; however, blocking CB1 receptors with antagonist AM-251 (10 μM) altered the synaptic transmission to frequency-dependent depression with a fast onset of release (2-4 ms). Presynaptic CCK-negative interneurons in SLM elicited inhibitory postsynaptic potentials (IPSPs) insensitive to CB1 receptor pharmacology displayed frequency-dependent depression. Release of GABA at facilitating synapses was solely mediated via N-type presynaptic calcium channels, whereas depressing synapses utilized P/Q-type channels. These data reveal two distinct models of neurotransmitter release patterns among interneuron circuits that correlate with the subtype of presynaptic calcium channel. These data suggest that endocannabinoids act via CB1 receptors to selectively modulate N-type calcium channels to alter signal transmission.     

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [³H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [³H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A.

This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.     

Immunohistochemical characterization of parvalbumin-containing interneurons in the monkey basolateral amygdala

Interneurons expressing the calcium-binding protein parvalbumin (PV) are a critical component of the inhibitory circuitry of the basolateral nuclear complex (BLC) of the mammalian amygdala. These neurons form interneuronal networks interconnected by chemical and electrical synapses, and provide a strong perisomatic inhibition of local pyramidal projection neurons. Immunohistochemical studies in rodents have shown that most parvalbumin-positive (PV+) cells are GABAergic interneurons that co-express the calcium-binding protein calbindin (CB), but exhibit no overlap with interneuronal subpopulations containing the calcium-binding protein calretinin (CR) or neuropeptides. Despite the importance of identifying interneuronal subpopulations for clarifying the major players in the inhibitory circuitry of the BLC, very little is known about these subpopulations in primates. Therefore, in the present investigation dual-labeling immunofluorescence histochemical techniques were used to characterize PV+ interneurons in the basal and lateral nuclei of the monkey amygdala. These studies revealed that 90–94% of PV+ neurons were GABA+, depending on the nucleus, and that these neurons constituted 29–38% of the total GABAergic population. CB+ and CR+ interneurons constituted 31–46% and 23–27%, respectively, of GABAergic neurons. Approximately one quarter of PV+ neurons contained CB, and these cells constituted one third of the CB+ interneuronal population. There was no colocalization of PV with the neuropeptides somatostatin or cholecystokinin, and virtually no colocalization with CR. These data indicate that the neurochemical characteristics of the PV+ interneuronal subpopulation in the monkey BLC are fairly similar to those seen in the rat, but there is far less colocalization of PV and CB in the monkey. These findings suggest that PV+ neurons are a discrete interneuronal subpopulation in the monkey BLC and undoubtedly play a unique functional role in the inhibitory circuitry of this brain region.     

GABA能神经成分对大鼠咀嚼肌本体觉传入进行初级整合的形态学基础──Ricin跨节溃变、HRP逆行追踪与抗GABA免疫组化法结合的电镜观察

本文作者曾证明,三叉神经感觉主核背内侧区和三叉上核尾外侧部是大鼠三又神经本体觉三级传入通路中继站。为证实此二处的ＧＡＢＡ能丘脑投射神经元和中间神经元是否参与三叉神经本体觉的传导和初级整合并阐明其整合机制,用Ｒｉｃｉｎ毁损三叉神经中脑核神经元及其终末,ＨＲＰ逆行标记此区的丘脑投射神经元以及抗ＧＡＢＡ免疫组化三者结合的方法,在电镜下发现：（１）中脑核神经元的溃变终末与该二区神经毯中的ＧＡＢＡ能三叉—丘脑投射神经元的胞体和树突形成轴—体和轴—树突触。（２）溃变终末与ＧＡＢＡ样中间神经元形成突触。（３）ＧＡ—ＢＡ样终末与ＧＡＢＡ能三叉—丘脑投射神经元形成突触。（４）ＧＡＢＡ样终末也与ＧＡＢＡ样神经元的胞体和树突形成自突触。此外还观察到ＧＡＢＡ能三叉—丘脑投射神经元的回返侧支与ＧＡＢＡ能中间神经元间的突触连接和大的“贝壳状”ＧＡＢＡ样终末与周围的神经成分形成复杂的突触复合体。证实该二区的ＧＡＢＡ能三叉—丘脑投射神经元直接参与三叉神经本体党的向心传导;同时,ＧＡＢＡ能中间神经元和其它来源的ＧＡＢＡ能神经成分也参与本体觉传导的初级整合作用,其间存在着突触前、后抑制,去抑制,突触前易化,回返抑制等复杂整合机制的结构基础。