Differences in synaptic GABA(A) receptor number underlie variation in GABA mini amplitude.

In many neurons, responses to individual quanta of transmitter exhibit large variations in amplitude. The origin of this variability, although central to our understanding of synaptic transmission and plasticity, remains controversial. To examine the relationship between quantal amplitude and postsynaptic receptor number, we adopted a novel approach, combining patch-clamp recording of synaptic currents with quantitative immunogold localization of synaptic receptors. Here, we report that in cerebellar stellate cells, where variability in GABA miniature synaptic currents is particularly marked, the distribution of quantal amplitudes parallels that of synaptic GABA(A) receptor number. We also show that postsynaptic GABA(A) receptor density is uniform, allowing synaptic area to be used as a measure of relative receptor content. Flurazepam, which increases GABA(A) receptor affinity, prolongs the decay of all miniature currents but selectively increases the amplitude of large events. From this differential effect, we show that a quantum of GABA saturates postsynaptic receptors when <80 receptors are present but results in incomplete occupancy at larger synapses.

The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.

An antiserum to mGluR1 alpha labeled a 160 kd protein in immunoblots of membranes derived from rat brain or cells transfected with mGluR1 alpha. Immunoreactivity for mGluR1 alpha was present in discrete subpopulations of neurons. The GABAergic neurons of the cerebellar cortex were strongly immunoreactive; only some Golgi cells were immunonegative. Somatostatin/GABA-immunopositive cells in the neocortex and hippocampus were enriched in mGluR1 alpha. The hippocampal cells had spiny dendrites that were precisely codistributed with the local axon collaterals of pyramidal and granule cells. Electron microscopic immunometal detection of mGluR1 alpha showed a preferential localization at the periphery of the extensive postsynaptic densities of type 1 synapses in both the cerebellum and the hippocampus. The receptor was also present at sites in the dendritic and somatic membrane where synapses were not located.

Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.

It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.

AMPA and NMDA receptors: similarities and differences in their synaptic distribution.

Recent technical developments, including antigen-retrieval and electron microscopic immunogold methods, are making it possible to determine some of the basic principles governing the subcellular distribution of ionotropic glutamate receptors. Distinct AMPA and NMDA receptor subtypes are selectively targeted to functionally different synapses of a single cell, resulting in an input-selective fine-tuning and regulation of the postsynaptic responses. The amount, density and variability of AMPA receptors at a given glutamatergic synapse is governed by both pre- and postsynaptic factors, resulting in functionally distinct glutamatergic connections that display characteristic patterns of receptor expression.

Release-independent short-term facilitation at GABAergic synapses in the olfactory bulb.

Neurones of the olfactory bulb are innervated by GABA-releasing axons and dendrites of diverse origin. Here, I studied GABAergic neurotransmission in juxtaglomerular cells using whole-cell voltage-clamp recordings in acute olfactory bulb slices. Spontaneous IPSCs were fully blocked by the GABA(A) receptor antagonist SR95531 (40 microM) and the sodium channel blocker tetrodotoxin (1 microM). The IPSCs had mean amplitudes of 125+/-86 pA and relatively slow biexponential decay times (tau(1)=4.3+/-1.0 ms (67+/-12%), tau(2)=16.9+/-2.7 ms) at physiological temperatures. Short-term plasticity of evoked IPSCs showed two distinct patterns: depressing (n=4 cells) and facilitating-depressing (n=9). In two cells, postsynaptic responses were mediated by single functional release sites. During a train of stimuli (4 stimuli at 20 Hz), the release probability increased by two-fold, whereas the potency (postsynaptic responses excluding failures) decreased by ~15%. The increase in release probability for the second stimulus in the train also occurred when the first action potential failed to release transmitter. However, the decrease in the potency was only observed if the preceding action potential released transmitter. These results reveal a heterogeneity in the short-term plasticity of evoked IPSCs in juxtaglomerular cells and demonstrate that the short-term facilitation at some GABAergic synapses is independent of release.

Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization.

Glutamate is a major neurotransmitter in the brain that acts both through fast ionotropic receptors and through slower metabotropic receptors coupled to G proteins. Both receptors are present throughout the somatodendritic domain of neurons as shown by immunohistochemical and patch clamp recording studies. Immunogold labelling revealed a concentration of metabotropic receptors at the edge, but not within the main body of anatomically defined synapses, raising the possibility that ionotropic and metabotropic receptors are segregated. We applied double immunogold labelling to study glutamatergic parallel and climbing fibre synapses in the cerebellar cortex. The ionotropic AMPA type receptors occupy the membrane opposite the release site in the main body of the synaptic junction, whereas the metabotropic receptors are located at the periphery of the same synapses. Furthermore, immunoreactivity for AMPA receptors is at least twice as high in the parallel fibre synapses as in glutamatergic mossy fibre synapses. We suggest that the spatial segregation of ionotropic and metabotropic glutamate receptors permits the differential activation of these receptors according to the amount of glutamate released presynaptically, whereas the different densities of the ionotropic receptor at distinct synapses could allow the same amount of glutamate to evoke fast responses of different magnitude.

High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus.

The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the GluRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the GluRD subunit. At the subcellular level, silver intensified immunogold particles for the GluRA, GluRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CA1 and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type 1 synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata. The results demonstrate that the GluRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

Synapse-specific contribution of the variation of transmitter concentration to the decay of inhibitory postsynaptic currents.

Synaptic transmission is characterized by a remarkable trial-to-trial variability in the postsynaptic response, influencing the way in which information is processed in neuronal networks. This variability may originate from the probabilistic nature of quantal transmitter release, from the stochastic behavior of the receptors, or from the fluctuation of the transmitter concentration in the cleft. We combined nonstationary noise analysis and modeling techniques to estimate the contribution of transmitter fluctuation to miniature inhibitory postsynaptic current (mIPSC) variability. A substantial variability (approximately 30%) in mIPSC decay was found in all cell types studied (neocortical layer2/3 pyramidal cells, granule cells of the olfactory bulb, and interneurons of the cerebellar molecular layer). This large variability was not solely the consequence of the expression of multiple types of GABA(A) receptors, as a similar mIPSC decay variability was observed in cerebellar interneurons that express only a single type (alpha(1)beta(2)gamma(2)) of GABA(A) receptor. At large synapses on these cells, all variance in mIPSC decay could be accounted for by the stochastic behavior of approximately 36 pS channels, consistent with the conductance of alpha(1)beta(2)gamma(2) GABA(A) receptors at physiological temperatures. In contrast, at small synapses, a significant amount of variability in the synaptic cleft GABA transient had to be present to account for the additional variance in IPSC decay over that produced by stochastic channel openings. Thus, our results suggest a synapse-specific contribution of the variation of the spatiotemporal profile of GABA to the decay of IPSCs.

Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells.

Two types of GABAA receptor-mediated inhibition (phasic and tonic) have been described in cerebellar granule cells, although these cells receive GABAergic input only from a single cell type, the Golgi cell. In adult rats, granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta), which are coassembled into at least four to six distinct GABAA receptor subtypes. We tested whether a differential distribution of GABAA receptors on the surface of granule cells could play a role in the different forms of inhibition, assuming that phasic inhibition originates from the activation of synaptic receptors, whereas tonic inhibition is provided mainly by extrasynaptic receptors. The alpha1, alpha6, beta2/3, and gamma2 subunits have been found by immunogold localizations to be concentrated in GABAergic Golgi synapses and also are present in the extrasynaptic membrane at a lower concentration. In contrast, immunoparticles for the delta subunit could not be detected in synaptic junctions, although they were abundantly present in the extrasynaptic dendritic and somatic membranes. Gold particles for the alpha6, gamma2, and beta2/3, but not the alpha1 and delta, subunits also were concentrated in some glutamatergic mossy fiber synapses, where their colocalization with AMPA-type glutamate receptors was demonstrated. The exclusive extrasynaptic presence of the delta subunit-containing receptors, together with their kinetic properties, suggests that tonic inhibition could be mediated mainly by extrasynaptic alpha6beta2/3delta receptors, whereas phasic inhibition is attributable to the activation of synaptic alpha1beta2/3gamma2, alpha6beta2/3gamma2, and alpha1alpha6beta2/3gamma2 receptors.

Differential regulation of synaptic GABAA receptors by cAMP-dependent protein kinase in mouse cerebellar and olfactory bulb neurones.

1. It has been demonstrated that the regulation of recombinant GABAA receptors by phosphorylation depends on the subunit composition. Here we studied the regulation of synaptic GABAA receptor function by cAMP-dependent protein kinase (PKA) in neurones expressing distinct receptor subtypes. 2. Light microscopic immunocytochemistry revealed that granule cells of the olfactory bulb express only the beta3 as the beta subunit variant, whereas cerebellar stellate and basket cells express only the beta2 as the beta subunit. 3. In cerebellar interneurones, intracellular application of 20 microM microcystin, a protein phosphatase 1/2A inhibitor, prolonged (63 +/- 14 %; mean +/- s.e.m.) the decay time course of miniature IPSCs (mIPSCs) without significantly affecting their amplitude, rise time and frequency. The effect of microcystin could be blocked by co-applying PKA inhibitory peptide (PKA-I, 1 microM). 4. No significant changes in any of the mIPSC parameters could be detected after intracellular application of PKA-I alone or following the inhibition of calcineurin with FK506 (50 nM). 5. In granule cells of the olfactory bulb expressing the beta3 subunit fast and slowly rising mIPSCs were detected, resulting in a bimodal distribution of the 10-90 % rise times, suggesting two distinct populations of events. Fast rising mIPSCs (mIPSCFR) had a 10-90 % rise time of 410 +/- 50 micros, an amplitude of 68 +/- 6 pA, and a weighted decay time constant (tauw) of 15.8 +/- 2.9 ms. In contrast, slowly rising mIPSCs (mIPSCSR) displayed an approximately threefold slower rise time (1.15 +/- 0.12 ms), 57 % smaller amplitude (29 +/- 1.7 pA), but had a tauw (16.8 +/- 3.0 ms) similar to that of the fast events. 6. mIPSCs in olfactory granule cells were not affected by the intracellular perfusion of microcystin. In spite of this, intracellular administration of constitutively active PKA caused a small, gradual, but significant increase (18 +/- 5 %) in the amplitude of the events without changing their time course. 7. These findings demonstrate a cell-type-dependent regulation of synaptic inhibition by protein phosphorylation. Furthermore, our results show that the effect of PKA-mediated phosphorylation on synaptic inhibition depends upon the subunit composition of postsynaptic GABAA receptors.

The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells.

Although three distinct subunits seem to be sufficient to form a functional pentameric GABAA receptor channel, cerebellar granule cells express nRNA for nine subunits. They receive GABAergic input from a relatively homogenous population of Golgi cells. It is not known whether all subunits are distributed similarly on the surface of granule cells or whether some of them have differential subcellular distribution resulting in distinct types of synaptic and/or extrasynaptic channels. Antibodies to different parts of the alpha 6 and alpha 1 subunits of the GABAA receptor and electron microscopic immunogold localization were used to determine the precise subcellular distribution of these subunits in relation to specific synaptic inputs. Both subunits were present in the extrasynaptic dendritic and somatic membranes at lower densities than in synaptic junctions. The alpha 6 and alpha 1 subunits were colocalized in many GABAergic Golgi synapses, demonstrating that both subunits are involved in synaptic transmission in the same synapse. Synapses immunopositive for only one of the alpha subunits were also found. The alpha 6, but not the alpha 1, subunit was also concentrated in glutamatergic mossy fiber synapses, indicating that the alpha 6 subunit may have several roles depending on its different locations. The results demonstrate a partially differential synaptic targeting of two distinct GABAA receptor subunits on the surface of the same type of neuron.

Increased number of synaptic GABA(A) receptors underlies potentiation at hippocampal inhibitory synapses.

Changes in synaptic efficacy are essential for neuronal development, learning and memory formation and for pathological states of neuronal excitability, including temporal-lobe epilepsy. At synapses, where there is a high probability of opening of postsynaptic receptors, all of which are occupied by the released transmitter, the most effective means of augmenting postsynaptic responses is to increase the number of receptors. Here we combine quantal analysis of evoked inhibitory postsynaptic currents with quantitative immunogold localization of synaptic GABA(A) receptors in hippocampal granule cells in order to clarify the basis of inhibitory synaptic plasticity induced by an experimental model of temporal-lobe epilepsy (a process known as kindling). We find that the larger amplitude (66% increase) of elementary synaptic currents (quantal size) after kindling results directly from a 75% increase in the number of GABA(A) receptors at inhibitory synapses on somata and axon initial segments. Receptor density was up by 34-40% and the synaptic junctional area was expanded by 31%. Presynaptic boutons were enlarged, which may account for the 39% decrease in the average number of released transmitter packets (quantal content). Our findings establish the postsynaptic insertion of new GABA(A) receptors and the corresponding increase in postsynaptic responses augmenting the efficacy of mammalian inhibitory synapses.

Cell type- and synapse-specific variability in synaptic GABAA receptor occupancy.

The degree of postsynaptic type A gamma-aminobutyric acid receptor (GABAA receptor) occupancy was investigated by using the benzodiazepine agonist zolpidem. This drug increases the affinity of GABAA receptors for gamma-aminobutyric acid (GABA) at room temperature (Perrais & Ropert 1999, J. Neurosci., 19, 578) leading to an enhancement of synaptic current amplitudes if receptors are not fully occupied by the released transmitter. We recorded miniature inhibitory postsynaptic currents (mIPSCs) from eight different cell types in three brain regions of rats and mice. Receptors in every cell type were benzodiazepine sensitive, as 10-20 microM zolpidem prolonged the decays of mIPSCs (151-184% of control). The amplitude of the GABAA receptor-mediated events was significantly enhanced in dentate granule cells, CA1 pyramidal cells, hippocampal GABAergic interneurons, cortical layer V pyramidal cells, cortical layer V interneurons, and in cortical layer II/III interneurons. An incomplete postsynaptic GABAA receptor occupancy is thus predicted in these cells. In contrast, zolpidem induced no significant change in mIPSC amplitudes recorded from layer II/III pyramidal cells, suggesting full GABAA receptor occupancy. Moreover, different degrees of receptor occupancy could be found at distinct GABAergic synapses on a given cell. For example, of the two distinct populations of zolpidem-sensitive mIPSCs recorded in olfactory bulb granule cells, the amplitude of only one type was significantly enhanced by the drug. Thus, at synapses that generate mIPSCs, postsynaptic receptor occupancy is cell type and synapse specific, reflecting local differences in the number of receptors or in the transmitter concentration in the synaptic cleft.

Immunocytochemical localization of the alpha 1 and beta 2/3 subunits of the GABAA receptor in relation to specific GABAergic synapses in the dentate gyrus.

Dentate granule cells receive spatially segregated GABAergic innervation from at least five types of local circuit neurons, and express mRNA for at least 11 subunits of the GABAA receptor. At most two to four different subunits are required to make a functional pentamer, raising the possibility that cells have on their surface several types of GABAA receptor channel, which may not be uniformly distributed. In order to establish the subcellular location of GABAA receptors on different parts of dentate neurons, the distribution of immunoreactivity for the alpha 1 and beta 2/3 subunits of the receptor was studied using high-resolution immunocytochemistry. Light microscopic immunoperoxidase reactions revealed strong GABAA receptor immunoreactivity in the molecular layer of the dentate gyrus. Pre-embedding immunogold localization of the alpha 1 and beta 2/3 subunits consistently showed extrasynaptic location of the GABAA receptor on the somatic, dendritic and axon initial segment membrane of granule cells, but failed to show receptors in synaptic junctions. Using a postembedding immunogold technique on freeze-substituted, Lowicryl-embedded tissue, synaptic enrichment of immunoreactivity for these subunits was found on both granule and non-principal cells. Only the postembedding immunogold method is suitable for revealing relative differences in receptor density at the subcellular level, giving approximately 20 nm resolution. The immunolabelling for GABAA receptor occupied the whole width of synaptic junctions, with a sharp decrease in labelling at the edge of the synaptic membrane specialization. Both subunits have been localized in the synaptic junctions between basket cell terminals and somata, and between axo-axonic cell terminals and axon initial segments of granule cells, with no qualitative difference in labelling. Receptor-immunopositive synapses were found at all depths of the molecular layer. Some of the boutons forming these dendritic synapses have been shown to contain GABA, providing evidence that some of the GABAergic cells that terminate only on the dendrites of granule cells also act through GABAA receptors. Double immunolabelling experiments demonstrated that a population of GABA-immunopositive neurons expresses a higher density of immunoreactive GABAA receptor on their surface than principal cells. Interneurons were found to receive GABAA receptor-positive synapses on their dendrites in the hilus, molecular and granule cell layers. Receptor-immunopositive synapses were also present throughout the hilus on presumed mossy cells. The results demonstrate that both granule cells and interneurons exhibit a compartmentalized distribution of the GABAA receptor on their surface, the postjunctional membrane to GABAergic terminals having the highest concentration of receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane topology of the GluR1 glutamate receptor subunit: epitope mapping by site-directed antipeptide antibodies.

In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N-terminal residues 253-267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877-889 increased about 1.6-fold following permeabilization. Our data suggest that the N- and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross-reactivities in different species and their effect on ligand binding activity were also established.

Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method.

Ion channels gated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are thought to be located in synaptic junctions, but they have also been found throughout the somatodendritic membrane of neurons independent of synapses. To test whether synaptic junctions are enriched in GABAA receptors, and to determine the relative densities of synaptic and extrasynaptic receptors, the alpha 1 and beta 2/3 subunits of the GABAA receptor were localized on cerebellar granule cells using a postembedding immunogold method in cats. Immunoparticle density for the alpha 1 and beta 2/3 subunits was approximately 230 and 180 times more concentrated, respectively, in the synaptic junction made by GABAergic Golgi cell terminals with granule cell dendrites than on the extrasynaptic somatic membrane. Quantification of immunoreactivity revealed one synapse population for the beta 2/3, but appeared to show two populations for the alpha 1 subunit immunoreactivity. The concentration of these subunits on somatic membrane was significantly lower than on the extrasynaptic dendritic membrane. Synaptic junctions with glutamatergic mossy fiber terminals were immunonegative. The results demonstrate that granule cells receiving GABAergic synapses at a restricted location on their distal dendrites exhibit a highly compartmentalized distribution of GABAA receptor in their plasma membrane.

Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus.

Ionotropic and metabotropic (mGluR1a) glutamate receptors were reported to be segregated from each other within the postsynaptic membrane at individual synapses. In order to establish whether this pattern of distribution applies to the hippocampal principal cells and to other postsynaptic metabotropic glutamate receptors, the mGluR1a/b/c and mGluR4 subtypes were localized by immunocytochemistry. Principal cells in all hippocampal fields were reactive for mGluR5, the strata oriens and radiatum of the CA1 area being most strongly immunolabelled. Labelling for mGluR1b/c was strongest on some pyramids in the CA3 area, weaker on granule cells and absent on CA1 pyramids. Subpopulations of non-principal cells showed strong mGluR1 or mGluR5 immunoreactivity. Electron microscopic pre-embedding immunoperoxidase and both pre- and postembedding immunogold methods consistently revealed the extrasynaptic location of both mGluRs in the somatic and dendritic membrane of pyramidal and granule cells. The density of immunolabelling was highest on dendritic spines. At synapses, immunoparticles for both mGluR1 and mGluR5 were found always outside the postsynaptic membrane specializations. Receptors were particularly concentrated in a perisynaptic annulus around type 1 synaptic junctions, including the invaginations at 'perforated' synapses. Measurements of immunolabelling on dendritic spines showed decreasing levels of receptor as a function of distance from the edge of the synaptic specialization. We propose that glutamergic synapses with an irregular edge develop in order to increase the circumference of synaptic junctions leading to an increase in the metabotropic to ionotropic glutamate receptor ratio at glutamate release sites. The perisynaptic position of postsynaptic metabotropic glutamate receptors appears to be a general feature of glutamatergic synaptic organization and may apply to other G-protein-coupled receptors.

Disruption of GABA(A) receptors on GABAergic interneurons leads to increased oscillatory power in the olfactory bulb network.

Synchronized neural activity is believed to be essential for many CNS functions, including neuronal development, sensory perception, and memory formation. In several brain areas GABA(A) receptor-mediated synaptic inhibition is thought to be important for the generation of synchronous network activity. We have used GABA(A) receptor beta3 subunit deficient mice (beta3-/-) to study the role of GABAergic inhibition in the generation of network oscillations in the olfactory bulb (OB) and to reveal the role of such oscillations in olfaction. The expression of functional GABA(A) receptors was drastically reduced (>93%) in beta3-/- granule cells, the local inhibitory interneurons of the OB. This was revealed by a large reduction of muscimol-evoked whole-cell current and the total current mediated by spontaneous, miniature inhibitory postsynaptic currents (mIPSCs). In beta3-/- mitral/tufted cells (principal cells), there was a two-fold increase in mIPSC amplitudes without any significant change in their kinetics or frequency. In parallel with the altered inhibition, there was a significant increase in the amplitude of theta (80% increase) and gamma (178% increase) frequency oscillations in beta3-/- OBs recorded in vivo from freely moving mice. In odor discrimination tests, we found beta3-/- mice to be initially the same as, but better with experience than beta3+/+ mice in distinguishing closely related monomolecular alcohols. However, beta3-/- mice were initially better and then worse with practice than control mice in distinguishing closely related mixtures of alcohols. Our results indicate that the disruption of GABA(A) receptor-mediated synaptic inhibition of GABAergic interneurons and the augmentation of IPSCs in principal cells result in increased network oscillations in the OB with complex effects on olfactory discrimination, which can be explained by an increase in the size or effective power of oscillating neural cell assemblies among the mitral cells of beta3-/- mice.

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.