Morphological heterogeneity of the GABAergic network in the suprachiasmatic nucleus, the brain's circadian pacemaker

GABA (gamma-amino-butyric acid) is the predominant neurotransmitter in the mammalian suprachiasmatic nucleus (SCN), with a central role in circadian time-keeping. We therefore undertook an ultrastructural analysis of the GABA-containing innervation in the SCN of mice and rats using immunoperoxidase and immunogold procedures. GABA-immunoreactive (GABA-ir) neurons were identified by use of anti-GABA and anti-GAD (glutamic acid decarboxylase) antisera. The relationship between GABA-ir elements and the most prominent peptidergic neurons in the SCN, containing vasopressin-neurophysin (VP-NP) or vasoactive intestinal polypeptide (VIP), was also studied. Within any given field in the SCN, approximately 40–70% of the neuronal profiles were GABA-ir. In GABA-ir somata, immunogold particles were prominent over mitochondria, sparse over cytoplasm, and scattered as aggregates over nucleoplasm. In axonal boutons, gold particles were concentrated over electron-lucent synaptic vesicles (diameter 40–60 nm) and mitochondria, and in some instances over dense-cored vesicles (DCVs, diameter 90–110 nm). GABA-ir boutons formed either symmetric or asymmetric synaptic contacts with somata, dendritic shafts and spines, and occasionally with other terminals (axo-axonic). Homologous or autaptic connections (GABA on GABA, or GAD on GAD) were common. Although GABA appeared to predominate in most neuronal profiles, colocalisation of GABA within neurons that were predominantly neuropeptide-containing was also evident. About 66% of the VIP-containing boutons and 32% of the vasopressinergic boutons contained GABA. The dense and complex GABAergic network that pervades the SCN is therefore comprised of multiple neuronal phenotypes containing GABA, including a wide variety of axonal boutons that impinge on heterologous and homologous postsynaptic sites.     

Co-localization of vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities in the nerve fibers of the rat adrenal gland

The co-localization of Vasoactive Intestinal Polypeptide (VIP) with Neuropeptide Y (NPY) or its C-flanking peptide (C-PON) was investigated with immunocytochemistry methods in the adrenal gland of the rat. Most of the VIP immunoreactive (+) nerve fibers found in the capsule/glomerular zone also exhibited NPY or C-PON immunoreactivity (IR). We found that at least two populations of VIP varicose nerve fibers can be observed, the most prevalent exhibited both VIP/NPY or VIP/C-PON IR and the other which was rather scarce lacked NPY or C-PON IR. In the superficial cortex VIP/NPY or VIP/C-PON IR nerve fibers were often associated with capsular or subcapsular vascularization and extended into the zona glomerulosa. In the deeper layers of the adrenal cortex radial fibers were closely associated with the inner vascularization of the zona fasciculata and reticularis. In the adrenal medulla NPY or C-PON immunoreactivity was associated with ganglion neurons as well as chromaffin cells; these last cells were always VIP (-). VIP and NPY/C-PON IR could be co-localized in catecholaminergic nerve terminals of the adrenal cortex but not in the adrenal medulla.     

Ultrastructural relationships of serotonin and GABA terminals in the rat suprachiasmatic nucleus. Evidence for a close interconnection between the two afferent systems

Summary Serotonin (5-HT) and -aminobutyric acid (GABA) nerve endings were identified in the rat suprachiasmatic nucleus (SCN) by combined [³H]5-HT uptake radioautography and glutamate decarboxylase (GAD) immunocytochemistry at the electron microscope level. In areas of good overlap between radioautographic and immunocytochemical labellings, there were no axonal varicosities exhibiting both labellings, indicating that 5-HT and GABA are not co-localized in the SCN. The systematic survey in these areas of all profiles that had accumulated [³H]5-HT and of all GAD-immunoreactive varicosities allowed the analysis of 247 of the former and 896, i.e. an almost four-fold greater number, of the latter. This seems concordant with the view that GABA endings would be the most numerous of all classes of nerve terminals so far identified in the SCN. More than 22% of the [³H]5-HT labelled profiles showed the membrane specialization typically associated with synap'tic junctions. Thereby, it was possible to evaluate that about 45% of the 5-HT terminals actually form a synapse in the SCN. Some 37% of the GAD-positive varicose profiles which could be formally interpreted also showed well differentiated synaptic contacts, suggesting that the GABAergic innervation of the SCN could be entirely junctional. Whereas 5-HT terminals usually innervated only one dendritic or somatic element, a convergence of several GABAergic terminals onto the same postsynaptic target also receiving a 5-HT input was frequently observed. Of all the [³H]5-HT labelled varicose profiles scanned, as much as 41% were directly apposed to at least one GAD-immunoreactive profile, indicating that these 5-HT/GABA axonal interfaces could well represent privileged sites of interactions between the two transmitters. Taken together, these data could be of potential value in determining the neurochemical mechanisms subserving cellular integration of rhythmic signals in the SCN.     

Catecholaminergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat

Summary The coexistence of immunoreactivities for tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in various brain regions in colchicine-injected and untreated rats, using the peroxidase-antiperoxidase method. Consecutive 40 m thick Vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for TH and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the olfactory bulb, retina, diencephalon, mesencephalic central grey and cerebral cortex, many TH-like immunoreactive neurons also showed GAD-like or GABA-like immunoreactivity, whereas in the substantia nigra, ventral tegmental area and locus ceruleus none of TH-like immunoreactive neurons showed either GAD-like or GABA-like immunoreactivity. In the olfactory bulb, retina and cerebral cortex, the majority of the TH-like immunoreactive neurons were also GAD-like or GABA-like immunoreactive. In the diencephalon of colchicine-injected rats, at least one-third of the TH-like immunoreactive neurons were GAD-like immunoreactive. Using serial 0.5 m thick plasticembedded sections, it was shown that immunoreactivities for three antigens, GAD, GABA and TH could occur in the same neurons in the olfactory bulb. These observations indicate the possible coexistence of two classical transmitters, GABA and catecholamine, in various brain regions of the rat.     

Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat

Previous studies have shown that neurons in the sacral dorsal commissural nucleus (SDCN) express neurokinin-1 receptor (NK1R) and can be modulated by the co-release of GABA and glycine (Gly) from single presynaptic terminal. These results raise the possibility that GABA/Gly-cocontaining terminals might make synaptic contacts with NK1R-expressing neurons in the SDCN. In order to provide morphological evidence for this hypothesis, the triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical study showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). In electron microscopic dual- and triple-immunohistochemistry for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, or GAD/GlyT2/NK1R also revealed dually labeled (GAD/GlyT2-ip) synaptic terminals upon SDCN neurons, as well as GAD- and/or GlyT2-ip axon terminals in synaptic contact with NK1R-ip SDCN neurons. These results suggested that some synaptic terminals upon NK1R-expressing SDCN neurons co-released both GABA and Gly.     

Cell-type specific distribution of chloride transporters in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA_A receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl⁻ influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K⁺/Cl⁻ cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K⁺/Cl⁻ cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine–threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl⁻ levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.     

Coexistence of GABA and peptide immunoreactivity in non-pyramidal neurons of the basolateral amygdala

Colocalization of γ-aminobutyric acid (GABA) immunoreactivity with somatostatin (SOM), neuropeptide Y (NPY), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP) immunoreactivity was demonstrated in non-pyramidal neurons of the basolateral amygdala using a two-color immunoperoxidase procedure. Approximately 80–90% of SOM- and NPY-positive neurons in the basolateral amygdala were also immunoreactive for GABA. Virtually all large CCK-positive neurons also exhibited GABA-like immunoreactivity. About one-half of VIP-positive neurons and small CCK-positive cells were also immunoreactive for GABA.     

Demonstration of GABAergic cell bodies in the suprachiasmatic nucleus: in situ hybridization of glutamic acid decarboxylase (GAD) mRNA and immunocytochemistry of GAD and GABA

The existence of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) was demonstrated by three specific markers; mRNA coding for glutamic acid decarboxylase (GAD) and visualized by in situ hybridization using a 35S-labelled cDNA probe, and GAD protein and GABA were identified by immunocytochemistry using specific antisera. In situ hybridization demonstrated well labelled GAD mRNA positive cells throughout SCN, and GABA and GAD immunoreactive cells showed similar distributions. These results indicate that GABA is a transmitter of a large portion of the SCN neuronal population.     

Neurons and terminals in the retrohippocampal region in the rat''s brain identified by anti-gamma-aminobutyric acid and anti-glutamic acid decarboxylase immunocytochemistry

Summary The distribution of gamma-aminobutyric acid (GABA) containing nerve cells and terminals was studied at the light and electron microscopic levels in the retrohippocampal region of the rat by using anti-glutamic acid decarboxylase (GAD) and anti-GABA antibodies in immunocytochemistry. Large numbers of GAD and GABA stained cells were found in all retrohippocampal structures. At the ultrastructural level, the immunoreactivity against GABA and against the synthesizing enzyme GAD was localized to cytoplasmic structures, including loose clumps of rough endoplasmic reticulum, ribosomal arrays, outer mitochondrial surfaces and in axonal boutons.The GAD- and GABA-immunorective(-i) cells were found in all subfields of the retrohippocampal region (e.g., the subicular complex, the entorhinal area). Within the entorhinal area a slightly larger number of immunoreactive cells could be detected in layers II and III than in the other layers. In the subiculum, pre- and parasubiculum the GAD and GABA-i cells were present in relatively large numbers in all layers, except the molecular layer, which contained only a small number of GABA cells. Within the entorhinal area, GAD and GABA stained cells ranged in size from small (13 m in diameter) to large (22 m in diameter). A large number of different morphological classes of cells were found, except pyramidal and stellate cells. In the pre- and parasubiculum, on the other hand, the GABA cells were generally small to medium in size and morphologically more homogeneous than in the subiculum and entorhinal area.The entire retrohippocampal region was densely innervated by GABA preterminal processes, with little variation in the regional density of innervation. Within the entorhinal area, presubiculum and subiculum, a clear difference was found in the laminar pattern of innervation. In all three subfields the densest innervation was in layer II. In the entorhinal area both GAD- and GABA-i axons form palisades of fibers around the somata of neurons, which are tightly packed together in this layer. In the electron microscope both GAD-i and GABA-i were demonstrated in these axons. Axosomatic synaptic contacts were common between axons and the stellate neurons and other cells of this layer. Layers IV and VI appeared less dense in GAD-i terminals but appeared more densely innervated than layers III and V. The lamina dessicans was relatively poor in GAD-i. In the subiculum and presubiculum, as well as all other subfields of the hippocampal region, the innervation is dominated by axo-somatic innervation of layer II cells. The outer third of the molecular layer was more densely innervated than the inner part. Taken together, the present study has shown that the retrohippocampal region is rich in GABAergic neurons as well as axon terminals, some of which form numerous synapic contacts with cells of the region. GABAergic neurotransmission is an important mechanism in retrohippocampal circuits not only for the resident interneuronal population but in the surround as well.     

Cholinergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat

Summary The coexistence of immunoreactivities for choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in some brain regions of the rat, using the peroxidase-antiperoxidase method. Consecutive 40 m thick vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for ChAT and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the retina, cerebral cortex, basal forebrain and spinal cord, colocalization of ChAT-like and GAD-like or GABA-like immunoreactivities was observed in some cell types, whereas no such colocalization was observed in cells in the striatum or brainstem. In the retina, the majority of ChAT-like immunoreactive (ChAT-LI) amacrine cells contained GABA-like or GAD-like immunoreactivity. About half of the ChAT-LI neurons in the cerebral cortex showed GABA-like immunoreactivity. In the basal forebrain only a small proportion of ChAT-LI neurons (0.6%) contained GAD-like immunoreactivity. In the spinal cord, about one-third of ChAT-LI central canal cluster cells and about half of ChAT-LI dorsal horn cells showed GAD-like and/or GABA-like immunoreactivities. These observations indicate the possible coexistence of two classical transmitters, GABA and acetylcholine, in various brain regions and spinal cord of the rat.This paper is dedicated to Prof. Hama on the occasion of his 66th birthday     

Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase

Summary Glutamic acid decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter -aminobutyric acid (GABA), has been localized in the rat visual cortex by immunocytochemical methods with both light and electron microscopy. In both colchicine-injected and non-injected preparations of the visual cortex, GAD-positive reaction product was observed in somata, proximal dendrites and axon terminals of non-pyramidal neurons. The GAD-positive terminals were observed to form symmetric synaptic junctions most commonly with dendritic shafts and somata of pyramidal and stellate neurons and less frequently with initial axon segments of pyramidal neurons and dendritic spines. In colchicine-injected preparations, GAD-positive somata were located in all cortical layers including the immediately subjacent white matter. In contrast, sections from non-injected rats displayed GAD-positive somata within a superficial and a deep cortical band. The GAD-positive somata observed in both types of preparations received both symmetric and asymmetric synaptic junctions, lacked apical dendrites, and had radially oriented dendrites of small diameter. These characteristics of GAD-positive neurons indicate that they are aspinous and sparsely-spinous stellate neurons. The localization of GAD within these neurons in combination with physiological and pharmacological data indicate that these local circuit neurons mediate GABA-ergic inhibition in the neocortex.     

The relationship between GABA immunoreactivity and labelling by local uptake of [3H]GABA in the striate cortex of monkey

Summary An antiserum to GABA was used in the macaque monkey to determine whether neurons that accumulate exogenously applied [³H]GABA in vivo are also immunoreactive for GABA. Following the injection of [³H]GABA into different laminae of striate cortex in two untreated animals and in one animal treated with amino-oxyacetic acid, selective accumulation of the labelled amino acid was demonstrated in perikarya by autoradiography. Radiographically labelled neurons (n, 519) and their unlabelled neighbours were tested in consecutive 0.5 m thick sections by immunocytochemistry for GABA immunoreacitivity. Injection of [³H]GABA did not increase the number of neurons showing GABA immunoreactivity. On the contrary many of the cells that accumulated [³H]GABA were immunonegative. These neurons were mostly located in layers IVC and VA following [³H]GABA injection into layers II–III, and in layers upper III and II following injection into layers V and VI. A comparison of the position of these neurons with known local projection patterns in the striate cortex of monkey suggests that GABA-immunonegative neurons may nevertheless become labelled by [³H]GABA if most of their local axon terminals fall within the injection site. The interlaminar projection of GABA-immunopositive neurons, which probably contain endogenous GABA, could be deduced from the position of the [³H]GABA injection site that leads to their autoradiographic labelling. Although the present study confirmed our previous results on the interlaminar connections of neurons that accumulate [³H]GABA, it demonstrated that [³H]GABA labelling alone may not be a sufficient criterion to assess the GABAergic nature of neurons in the striate cortex of monkey.     

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 m transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of -aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing -motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei cocontained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of -motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26–42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5–9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface. A colocalization of GABA and glycine immunoreactivities in putative nerve terminals could be shown both in the neuropil and in close relation to cell bodies of motoneurons. These results suggest that among the studied amino acids probably only three, namely GABA, glycine, and glutamate, can be considered to be neurotransmitter candidates in the ventral horn of the cat spinal cord.     

Possible coexistence of amino acid (γ-aminobutyric acid), amine (dopamine) and peptide (substance P); neurons containing immunoreactivities for glutamic acid decarboxylase,tyrosine hydroxylase and substance P in the hamster main olfactory bulb

Summary The coexistence of immunoreactivities for glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH) and substance P (SP) was revealed in the hamster main olfactory bulb, using the peroxidase-antiperoxidase immunohistochemical method. Adjacent 40 m thick Vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning were identified at the paired surfaces of two consecutive sections. The coexistence of the immunoreactivities for 1) TH and GAD, 2) TH and SP and 3) GAD and SP in the same cells could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. About 70% of TH-like immunoreactive (TH-LI) neurons in the periglomerular region also contained GAD-like immunoreactivity, whereas about 45% of GAD-LI ones were also TH-like immunoreactive. Furthermore, almost all (more than 95%) of SP-LI neurons contained both GAD-like and TH-like immunoreactivities. These observations indicate that in the periglomerular region of the hamster main olfactory bulb, some neurons (about 9% of all neurons containing TH-like and/or GAD-like immunoreactivities) may contain three different categories of neuroactive substances, that is, amino acid (GABA), amine (dopamine) and peptide (SP).     

Simultaneous demonstration of choline acetyltransferase and glutamic acid decarboxylase immunoreactivity in the rat interpeduncular nucleus

Double antigen immunohistochemistry was employed to simultaneously examine the distribution of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunoreactivity in the rat interpeduncular nucleus (IPN) at the subnuclear and ultrastructural level. ChAT-immunoreactive axons of the fasciculus retroflexus (FR) innervated specific subnuclear divisions of the IPN that possessed GAD-immunoreactive somata and a high density of GAD-immunoreactive axons and terminals. At the ultrastructural level, each of the cholinoceptive subnuclei possessed a characteristic axodendritic synaptic contact. These morphologically distinct synapses were composed of terminals of ChAT-positive FR axons forming asymmetric contacts with dendritic profiles of GAD-positive neurons. An array of symmetric axodendritic contacts with GAD immunoreactivity located pre- and/or postsynaptically was also present in the cholinoceptive subnuclear divisions. The present study provides direct evidence for synaptic interactions between ChAT-immunoreactive FR axons and dendritic processes of GAD-immunoreactive neurons in the rat IPN. Also, GAD-positive terminals arising from possible intrinsic projections contact dendritic profiles of GAD-immunoreactive neurons in receipt of ChAT-positive FR terminals. These results reveal that putative cholinergic afferent inputs and GABAergic intranuclear projections simultaneously innervate a subpopulation of IPN neurons that possess GAD immunoreactivity.     

Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN), the brain's circadian clock, is composed mainly of GABAergic neurons, that are interconnected via synapses with GABA(A) receptors. Here we report on the subcellular localization of these receptors in the SCN, as revealed by an extensively characterized antibody to the alpha 3 subunit of GABA(A) receptors in conjunction with pre- and postembedding electron microscopic immunocytochemistry. GABA(A) receptor immunoreactivity was observed in neuronal perikarya, dendritic processes and axonal terminals. In perikarya and proximal dendrites, GABA(A) receptor immunoreactivity was expressed mainly in endoplasmic reticulum and Golgi complexes, while in the distal part of dendrites, immunoreaction product was associated with postsynaptic plasma membrane. Many GABAergic axonal terminals, as revealed by postembedding immunogold labeling, displayed GABA(A) receptor immunoreactivity, associated mainly with the extrasynaptic portion of their plasma membrane. The function of these receptors was studied in hypothalamic slices using whole-cell patch-clamp recording of the responses to minimal stimulation of an area dorsal to the SCN. Analysis of the evoked inhibitory postsynaptic currents showed that either bath or local application of 100 microM of GABA decreased GABAergic transmission, manifested as a two-fold increase in failure rate. This presynaptic effect, which was detected in the presence of the glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione and the selective GABA(B) receptor blocker CGP55845A, appears to be mediated via activation of GABA(A) receptors. Our results thus show that GABA(A) receptors are widely distributed in the SCN and may subserve both pre- and postsynaptic roles in controlling the mammalian circadian clock.     

Neurochemical properties of the middle cervical ganglion in the sheep

The neurochemical properties of the ovine middle cervical ganglion (MCG) were studied using antibodies raised against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL). Double-labelling immunocytochemistry revealed that the vast majority (95.5 +/- 0.8%) of postganglionic sympathetic MCG neurons expressed simultaneously both catecholamine-synthesizing enzymes (neurons were TH/DbetaH-positive). A large population of noradrenergic neurons exhibited immunoreactivity (IR) to NPY (62.2 +/- 2.2%), but single NPY-positive perikarya-lacking noradrenergic markers were also observed (2.0 +/- 0.3%). None of the examined MCG neuronal somata contained SP, CGRP, GAL or VIP. A moderate number of noradrenergic nerve fibres located amongst neuronal cell bodies was also found. In small number of these terminals the presence of NPYor GAL (but not CGRP or VIP) was detected. The ovine MCG was numerously innervated with SP-immunoreactive nerve fibres which sometimes formed basket-like formations around postganglionic neurons. The MCG exhibited a sparse CGRP-immunoreactive innervation and lacked VIP-positive nerve terminals. In many aspects the chemical coding of MCG postganglionic neurons and nerve terminals resembles that found in other mammalian cervico-thoracic paravertebral ganglia, but some important species-dependent differences exist. The functional implications of these differences remain to be elucidated.     

GABA-immunoreactivity in processes presynaptic to the terminals of afferents from a locust leg proprioceptor

Summary Individually labelled sensory neurons from the femoral chordotonal organ, a proprioceptor at the femoro-tibial joint of a locust hindleg, were analysed by intracellular recording, and by electron microscopical immunocytochemistry to reveal the arrangement of their input and output synapses and to determine whether the input synapses were GABAergic. Intracellular recordings from these sensory neurons show spikes superimposed on a barrage of synaptic potentials during movements of the femoro-tibial joint. These synaptic inputs can be mimicked by GABA. Input synapses are made onto the vesicle-containing terminals of afferents and are often closely associated with the output synapses. By contrast, the axons of the afferents in the neuropil have no vesicles and neither make nor receive synapses. The input synapses to the afferent terminals are made from processes typically a few microns in diameter, whereas the output synapses are made onto much smaller processes of only 0.1–0.2 m. Input synapses at which an afferent terminal is the only postsynaptic element are common. Where the synapse is dyadic the second postsynaptic element does not usually appear to be a chordotonal afferent. The output synapses from the afferent terminals are usually dyadic. At 78% of the input synapses, the presynaptic neurite showed immunoreactivity to a GABA antibody, supporting the physiological evidence that the presynaptic effects can be mediated by the release of GABA. The remaining (22%) immunonegative synapses are intermingled with those showing GABA immunoreactivity, but their putative transmitter is unknown. These morphological observations suggest that the presynaptic control of the chordotonal afferents is largely mediated by GABAergic neurons, but because other types of neuron also appear to be involved, presynaptic modulation may be more complex than has yet been revealed by the physiology.     

VIP neurons as prime synaptic targets for serotonin afferents in rat suprachiasmatic nucleus: a combined radioautographic and immunocytochemical study

Summary Cellular relationships between serotonin (5-HT) axon terminals and neurons containing vasoactive intestinal peptide (VIP) were characterized by combined radioautography and immunocytochemistry in rat suprachiasmatic nucleus (SCN). Light microscopic immunoradioautographs showed significant overlap between (³H)5-HT uptake sites and VIP-immunoreactive elements in the ventral half of the SCN. Of the 255 (³H)5-HT-labelled axonal profiles detected in a systematic electron microscopic survey of single thin sections from this area, 75 (30%) were directly apposed to VIP-immunoreactive nerve cell bodies and/or dendrites. Radioautographically labelled 5-HT varicosities often showed well-differentiated, symmetrical or asymmetrical synaptic junctions, 60% of which were established on VIP-immunoreactive nerve cell bodies or dendrites. In a separate sampling of 198 (³H)5-HT-labelled terminals seen in apposition with VIP-immunoreactive elements, 50 showed a junctional complex at the site of contact. Postsynaptic immunoreactive elements were mostly dendrites but also included nerve cell bodies. Despite the methodological limitations inherent to the present double labelling approach, these data strongly support the view that VIP neurons are prime synaptic targets for 5-HT afferents in the SCN. VIP/5-HT interactions are thus likely to play an important functional role in this nucleus and may in particular subserve the 5-HT mediated regulation of certain circadian rhythms, including that of pituitary hormone secretion.     

Glutamic acid decarboxylase isoforms are differentially distributed in the septal region of the rat

The septal region of the brain consists of a heterogeneous population of GABAergic neurons that play an important role in the generation of hippocampal theta rhythms. While GABAergic neurons employ two isoforms of the enzyme glutamic acid decarboxylase (GAD) for the synthesis of GABA, distribution of GAD isoforms has not been investigated in the septum. Immunohistochemical techniques were used to investigate the expression of GAD enzymes in medial and lateral septum. GAD65 and GAD67 immunohistochemistry revealed dense fibers and punctuated immunoreactivity in septal regions. While few GAD65-positive neuronal somas were detected in medial septum, a significantly higher number of immunoreactive neurons were detected in lateral septum. GAD65- and GAD67-positive neurons in the lateral septum exhibit higher complexity of dendritic arborizations than in the medial septum where staining was mainly restricted to the soma. Presumptive axon terminals (puncta) showed abundant immunoreactivity predominantly for GAD65 isoforms in all septal regions. This suggests that septal GABAergic neurons differentially express GAD enzymes thereby potentially reflecting functional differences. Differences found between medial and lateral septal GABAergic neuronal populations are in agreement with the concept that medial and lateral septum are brain structures with highly different connectivity and function despite anatomical proximity.