Loss of the major GABA(A) receptor subtype in the brain is not lethal in mice.

The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

The 5HT(1B) receptor agonist, CP-93129, inhibits [(3)H]-GABA release from rat globus pallidus slices and reverses akinesia following intrapallidal injection in the reserpine-treated rat.

This study examined whether activation of 5HT(1B) receptors in the rodent globus pallidus (GP) could reduce GABA release in vitro and reverse reserpine-induced akinesia in vivo. Microdissected slices of GP from male Sprague Dawley rats (300-350 g) were preloaded with [(3)H]-GABA. During subsequent superfusion, 4 min fractions were collected for analysis of release. The effects of the 5HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3, 2-b]pyrid-5-one (CP-93129), on 25 mM KCl-evoked release were examined using a standard dual stimulation paradigm. Male Sprague Dawley rats (270 - 290 g), stereotaxically cannulated above the GP, were rendered akinetic by injection of reserpine (5 mg kg(-1) s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of CP-93129 was examined. CP-93129 (0.6-16.2 microM) produced a concentration-dependent inhibition of 25 mM KCl-evoked [(3)H]-GABA release reaching a maximum inhibition of 52.5+/-4.5%. The effect of a submaximal concentration of CP-93129 (5.4 microM) was fully inhibited by the 5HT(1B) receptor antagonist, isamoltane (10 microM). Following intrapallidal injection, CP-93129 (30-330 nmol in 0.5 microl) produced a dose-dependent increase in net contraversive rotations reaching a maximum of 197+/-32 rotations in 240 min at 330 nmol. Pre-treatment with isamoltane (10 nmol in 1 microl) inhibited the effects of a submaximal dose of CP-93129 (220 nmol) by 84+/-6%. These data suggest that at least some 5HT(1B) receptor function as heteroreceptors in the GP, reducing the release of GABA. Moreover, CP-93129-mediated activation of these receptors in the GP provides relief of akinesia in the reserpine-treated rat model of PD.

Density and pharmacology of alpha5 subunit-containing GABA(A) receptors are preserved in hippocampus of Alzheimer's disease patients.

The anatomical localization and pharmacology of alpha5 subunit-containing GABA type-A receptors in the human hippocampal formation of Alzheimer's disease patients were studied with an alpha5 receptor selective ligand, [3H]L-655,708 and compared to age-matched human controls. Autoradiographic analyses revealed a heterogeneous distribution of [3H]L-655,708 binding sites in CA1-CA3 areas with high levels in stratum oriens, stratum pyramidale and stratum radiatum contrasting with low levels in stratum lacunosum. The highest quantity of alpha5 receptors was found in the molecular layer of the dentate gyrus. This pattern of expression was identical in both hippocampus from control and Alzheimer's disease subjects. Quantitative studies demonstrated that the number of [3H]L-655,708 binding sites is well preserved in Alzheimer's disease with only a moderate reduction (25-30%) in the CA1 subfield and entorhinal cortex. Furthermore, saturation and competition experiments with [3H]L-655,708 and representative benzodiazepine site ligands revealed that alpha5 receptors in Alzheimer's hippocampus have an alpha5beta2/3gamma2 pharmacology and structure as in control human brain.Overall, the data reported here provide evidence for a specific expression and relative sparing of alpha5 subunit-containing gamma-aminobutyric acid type-A receptors in the hippocampus of Alzheimer's patients.

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Changes in [3H]zolpidem and [3H]Ro 15-1788 binding in rat globus pallidus and substantia nigra pars reticulata following a nigrostriatal tract lesion.

Changes in GABA(A) receptor alpha(1) subunit gene expression occur in the globus pallidus and substantia nigra pars reticulata following lesions of the nigrostriatal tract. To determine whether these changes are translated at the protein level, we performed quantitative autoradiography with the alpha(1) selective ligand, [3H]zolpidem, and the non-selective benzodiazepine site ligand, [3H]Ro 15-1788. Binding of both [3H]zolpidem and [3H]Ro 15-1788 was significantly increased in the substantia nigra pars reticulata (13. 5+/-4.1 and 26.3+/-2.9%, respectively) and significantly reduced in the globus pallidus (20.9+/-0.8 and 18.3+/-1.3%, respectively). These changes in alpha(1) subunit protein expression may help to compensate for the pathological changes in GABAergic activity that occur after striatal dopamine depletion.

Preferential coassembly of alpha4 and delta subunits of the gamma-aminobutyric acidA receptor in rat thalamus.

Pharmacological study of rat thalamic gamma-aminobutyric acidA (GABAA) receptors revealed the presence of two distinct populations, namely, diazepam-sensitive and diazepam-insensitive [3H]Ro15-4513 binding sites accounting for 94 +/- 2% (1339 +/- 253 fmol/mg protein) and 6 +/- 2% (90 +/- 44 fmol/mg protein) of total sites, respectively. Thalamic diazepam-insensitive sites exhibited a pharmacology that was distinct from diazepam-sensitive sites but comparable to that of the alpha4beta3gamma2 subtype of the GABAA receptor stably expressed in L(tk-) cells. Immunoprecipitation experiments with a specific anti-alpha4-antiserum immunoprecipitated 20 and 7% of total thalamic [3H]muscimol and [3H]Ro15-4513 sites, respectively. Combinatorial immunoprecipitation using antisera against the alpha4, gamma2, and delta subunit revealed that alpha4delta- and alpha4gamma2-containing receptors account for 13 +/- 2 and 8 +/- 3% of [3H]muscimol sites from thalamus, respectively. It also indicated that all delta subunits coexist with an alpha4 subunit in this brain region. In conclusion, our results show that in rat thalamus both alpha4betagamma2 and alpha4betadelta subtypes are expressed but alpha4betadelta is the major alpha4-containing GABAA receptor population.

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain.

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics.

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.

Distinct effects of imipramine on 5-hydroxytryptamine uptake mediated by the recombinant rat serotonin transporter SERT1.

Tricyclic and nontricyclic serotonin [5-hydroxytryptamine (5-HT)] uptake inhibitors are widely used for the treatment of depression. Here, we show that both the tricyclic antidepressant imipramine and the nontricyclic antidepressant citalopram competitively inhibit 5-HT transport mediated by the recombinant rat 5-HT transporter SERT1. For citalopram, the concentration producing half-maximal transport inhibition was in the same order of magnitude as its K(D) value determined by equilibrium binding. In contrast, the inhibitory potency of imipramine was more than one order of magnitude lower than its K(D) value. Our data are consistent with low-affinity imipramine binding occurring at or close to the substrate recognition site, which also binds citalopram. Occupation of the high-affinity imipramine binding site on SERT1 did not affect 5-HT transport but allosterically displaced citalopram from the substrate recognition site. Consequently, low concentrations of imipramine partially protected 5-HT transport from citalopram inhibition. This protection was only observed in the presence of Na+ because high-affinity imipramine binding is strictly sodium-dependent. Thus, depending on which of its binding sites on SERT1 is occupied, imipramine may exert distinct effects on 5-HT uptake mediated by the recombinant rat 5-HT transporter.

A single serine residue controls the cation dependence of substrate transport by the rat serotonin transporter.

The serotonin transporter (SERT) is a member of the Na+/Cl--dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. Here, replacement of serine-545 in the recombinant rat SERT by alanine was found to alter the cation dependence of serotonin uptake. Substrate transport was now driven as efficiently by LiCl as by NaCl without significant changes in serotonin affinity. Binding of the antidepressant [3H]imipramine occurred with 1/5th the affinity, whereas [3H]citalopram binding was unchanged. These results indicate that serine-545 is a crucial determinant of both the cation dependence of serotonin transport by SERT and the imipramine binding properties of SERT.

The rat serotonin transporter: identification of cysteine residues important for substrate transport.

Reduction and alkylation of disulfide bonds are known to affect substrate translocation by and antidepressant binding to the serotonin transporter (SERT). To identify functionally relevant cysteine residues, we substituted 16 cysteins of the rat SERT by alanine or serine residues and analyzed the transport and binding properties of the respective mutant transporters after heterologous expression in a mammalian cell line. Replacement of cysteine 209 by serine resulted in a marked reduction of the maximal transport rate, loss of positive cooperativity, and insensitivity to treatment with disulfide reducing agents, indicating that cysteine 209 participates in a structurally important disulfide bridge. Replacement of cysteine residues 147, 200, 369, and 540 caused a complete loss of both substrate transport and antidepressant binding, a result that is likely to reflect impaired processing and/or cell surface expression of the mutated polypeptides.

Localization of the serotonin transporter in rat spinal cord.

The spinal cord is richly innervated by serotoninergic fibres originating from the raphe nuclei. The localization of the terminating component of serotoninergic neurotransmission, the serotonin transporter SERT1, was found in both the dorsal and ventral horns, especially at the level of the cervical and lumbar segments. Within the thoracic region, we observed a heavily labelled bundle in the intermediolateral nucleus of lamina VII. A low density of stained fibres was encountered in the sacral spinal cord. In contrast to homogeneous staining of motor nuclei, a differential labelling of laminae was seen in the dorsal horn, with laminae I, III and IV exhibiting a higher density of immunopositive terminals than the medial part of lamina II. High magnification revealed a preferential accumulation of serotonin transporter staining within nerve endings and varicosities of thin fibres. Double immunofluorescence staining demonstrated a co-localization of serotonin and its uptake system within these varicosities. These results show that the serotonin transporter is highly expressed in the rat spinal cord and that its distribution parallels the serotoninergic innervation. They also reinforce the view that varicosities are important neuronal structures, which modulate the function of dorsal and ventral horn neurons by releasing serotonin.

Immunocytochemical detection of the serotonin transporter in rat brain.

The regional distribution of the serotonin uptake system was studied in rat brain using a specific polyclonal antibody raised against the putative extracellular loop between transmembrane domains 7 and 8 of the cloned rat serotonin transporter. Light microscope analysis with fluorescence and avidin-biotin-peroxidase techniques revealed a punctate staining as well as numerous labelled thin fibres, which exhibited accumulation of reaction end-product deposit over varicosities. These immunopositive processes were widely and heterogeneously distributed in the rat brain. High densities of immunoreactivity were seen within the caudate-putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, Islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nucleus and olfactory nuclei. We also found strong expression of serotonin transporter in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum molecular of CA1-CA3 regions of the hippocampus. Within the raphe nuclei, a moderate to high incidence of stained processes was observed, and immunopositive cell bodies were detected in the dorsal raphe nucleus. In addition, some immunoreactive fibres were present in the molecular and granular layers of the cerebellum as well as in the cochlear and olivary nuclei. In none of the regions analysed was evidence for glial staining obtained. The present immunocytochemical data reveal a widespread and heterogeneous distribution of the serotonin transporter in rat brain and suggest that serotoni transporter is preferentially sorted into axons, where it appears concentrated at varicosities and terminal boutons.

Subcellular localization of the GABAA receptor gamma 2 subunit in the rat spinal cord.

The fine subcellular organization of the GABAA receptor complex in the adult rat spinal ventral horn was analysed by immunocytochemistry using a specific polyclonal antiserum raised against the gamma 2 subunit. This subunit confers benzodiazepine sensitivity on the chloride channel of the GABAA receptor. With both fluorescent and peroxidase staining, the immunoreactivity was mainly observed in the grey matter and more specifically in the dorsal and ventral horns on medium and large neurons. A high number of immunostained somata were clustered in regions corresponding to motor nuclei. On the neuronal surface, labelling appeared as fluorescent dots over the more diffuse staining that was present on the soma and proximal part of dendrites. At the ultrastructural level, peroxidase end product was in most cases associated with the internal side of postsynaptic differentiations facing terminal boutons enriched with pleiomorphic small clear vesicles. The positively stained synapses were encountered on proximal dendrites of neurons and throughout the neuropil of the ventral horn (layers VII-IX). An immunoreactivity on the postsynaptic membrane was occasionally found to decorate large pieces of membrane not directly apposed to presynaptic active zones. In addition, presynaptic labelling was observed at axoaxonic contacts and at extrasynaptic sites on membranes within boutons, sometimes themselves apposed to gamma 2 immunoreactivity. Finally, we also observed gamma 2 immunoreactivity at the cytosolic face of the plasma membrane of some glial elements. These results give morphological evidence for the involvement of GABAA receptors in both post- and presynaptic inhibition in the rat spinal ventral horn. The presence of gamma 2 subunit immunoreactivity at these different synaptic contacts suggests that the two types of inhibition can be modulated by benzodiazepine drugs. The findings also provide anatomical evidence for the possible regulation of GABA release through an autoreceptor, and for GABAergic communication between neuronal and glial components.

GABAA receptor-like immunoreactivity in the goldfish brainstem with emphasis on the Mauthner cell.

The distribution of the GABAA receptor in the goldfish brainstem and on the Mauthner cell membrane was investigated with both optical and electron microscopy using a polyclonal antibody raised against the intracellular loop of the rat gamma 2 subunit. At the optical level, immunofluorescent dots were detected on small and large neurons belonging to vestibular and reticular nuclei. On the Mauthner cell plasmalemma, a gamma 2-like immunoreactivity was observed predominantly on the tip of the lateral dendrite. Fluorescent parches were intermingled with a more diffuse staining. Immunoreactive spots of weaker intensity were also present on the soma and some were also observed inside and within the periphery of the axon-cap as well. Observations at the electron microscopic level revealed that the peroxidase end-product predominates postsynaptically in front of release sites in the studied nuclei and on the Mauthner cell. On the lateral dendrite of the neuron, numerous immunopositive postsynaptic differentiations were encountered on spines. Stained glial elements were encountered in the different areas studied. These results demonstrate that the GABAA receptor gamma 2 subunit has a precise distribution on neuronal membranes and suggest that it could be involved in the remote dendritic inhibition of the Mauthner cell and in the control of input-output properties of both vestibular and reticular nuclei.

Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron.

Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed.

The inhibitory neuronal glycine receptor.

Glycine is a major inhibitory neurotransmitter in the spinal cord and in the brain stem, where it acts by activating a chloride conductance. The postsynaptic glycine receptor has been purified and contains two transmembrane subunits of 48 kDa (alpha) and 58 kDa (beta), and a peripheral membrane protein of 93 kDa. cDNA sequencing of the alpha and beta subunits has revealed a common structural organization and a strong homology between these polypeptides and the nicotinic acetylcholine and GABAA receptor proteins. The glycine receptor exhibits a heterogeneity resulting from the existence of several alpha subtypes with distinct functional properties and different developmental expressions. When present in the central nervous system in situ, as well as in primary cultures of spinal cord neurons, these receptors are localized at the postsynaptic membrane adjacent to the presynaptic release sites, thus forming functional microdomains at the neuronal surface. This distribution raises the question of the formation and the maintenance of the heterogeneity of the somato-dendritic plasma membrane.

Synchronous bursting in a subset of interneurons inhibitory to the goldfish Mauthner cell: synaptic mediation and plasticity.

1. Presynaptic activity in the inhibitory network impinging on the Mauthner (M-) cell was investigated in the goldfish medulla in vivo using extra- and intracellular recordings. The inhibitory presynaptic volley elicited by stimulation of the contralateral vestibular nerve consisted of multiple successive peaks at high frequency (up to 1,000 Hz). Less pronounced multicomponent responses were recorded after antidromic activation of the M-cell. Such high-frequency "oscillatory" field potentials also occurred spontaneously. 2. In intracellular recordings, a subset of inhibitory interneurons showed evoked and spontaneous burst discharge. Burst action potentials were correlated with the peaks in the extracellular volley, suggesting that repetitive firing of these cells is synchronized. Nonbursting cells, on the other hand, fired single action potentials in response to vestibular stimuli and were not activated via the M-cell collateral network. 3. Bursting cells were determined morphologically to be part of the feedback inhibitory circuit. Their responses to stimulation of the contralateral vestibular nerve thus suggest the existence of a crossed excitatory pathway to these interneurons. 4. Vestibular-evoked excitatory postsynaptic potentials (EPSPs) in bursting interneurons had a short latency of 0.781 +/- 0.08 ms (mean +/- SD, n = 18) but reached threshold at 2.25 +/- 1 ms (n = 21). These characteristics are suggestive of a chemically mediated EPSP. Indeed, the evoked synchronous repetitive activity of these cells was prevented by superfusion with excitatory amino-acid receptor antagonists. 5. Bursting neurons showed several characteristics that differentiate them from nonbursting cells, including brief action potentials, plateau responses, and intense spontaneous subthreshold activity. 6. With extracellular recordings, tetanization of contralateral vestibular primary afferents evoked a long-lasting potentiation of oscillatory population responses in 11 of 27 cases. Furthermore in three experiments, the frequency of occurrence of spontaneous bursts was enhanced and a similar facilitation was detected at the intracellular level. 7. We conclude that a subset of interneurons in this inhibitory network is capable of repetitive discharges and that evoked as well as spontaneous firing in this population is synchronized. Although electrical coupling between interneurons may mediate synchronization and intrinsic membrane properties may promote burst activity, our data suggest strongly that repetitive firing requires chemically mediated transmission. Furthermore they indicate that the mechanisms underlying evoked as well as spontaneous bursting in this population show activity-dependent plasticity.

Colocalization of somatostatin with GABA or glutamate in distinct afferent terminals presynaptic to the Mauthner cell.

The presence of somatostatin in afferent fibers impinging on the goldfish Mauthner (M-) cell was determined using immunohistochemical methods, combined with confocal and electron microscopy, and the relationship of this peptide with inhibitory and excitatory terminals was studied. Somatostatin-reactive boutons were present only on the distal part of the M-cell's lateral dendrite. Somatostatin immunoreactivity was observed in typical large myelinated club endings (LMCEs) corresponding to mixed (electrical and chemical) eighth nerve primary afferent fibers. The axoplasm of these fibers contained dense-core vesicles (DCVs) dispersed among round vesicles. We have made a novel finding that the excitatory transmitter glutamate is present in LMCEs. Colocalization of this amino acid with somatostatin was detected in 75% of these endings using postembedding staining with gold particles of various sizes. The other structures labeled by somatostatin antibody were found to be small vesicle boutons (SVBs), which establish symmetrical synapses and contain a population of pleiomorphic vesicles with DCVs scattered among them. Double labeling with antibodies against glutamic acid decarboxylase and GABA allowed the definition of three types of biochemically characterized terminals: [somatostatin-GABA], [GABA], and [somatostatin]. However, the occurrence of DCVs in SVBs stained for GABA alone suggests that neuropeptides other than somatostatin may also coexist with GABA in this class of boutons. The coexistence of somatostatin with both inhibitory and excitatory neurotransmitters acting on the same region of a postsynaptic cell is discussed in relation to the role postulated for this peptide in synaptic plasticity.