GABAergic Profiles Research Articles

Experience-related reorganization of giant synapses in the lateral complex: Potential role in plasticity of the sky-compass pathway in the desert ant Cataglyphis fortis.

Cataglyphis desert ants undergo an age-related polyethism from interior workers to relatively short-lived foragers with remarkable visual navigation capabilities, predominantly achieved by path integration using a polarized skylight-based sun compass and a stride-integrating odometer. Behavioral and physiological experiments revealed that the polarization (POL) pattern is processed via specialized UV-photoreceptors in the dorsal rim area of the compound eye and POL sensitive optic lobe neurons. Further information about the neuronal substrate for processing of POL information in the ant brain has remained elusive. This work focuses on the lateral complex (LX), known as an important relay station in the insect sky-compass pathway. Neuroanatomical results in Cataglyphis fortis show that LX giant synapses (GS) connect large presynaptic terminals from anterior optic tubercle neurons with postsynaptic GABAergic profiles of tangential neurons innervating the ellipsoid body of the central complex. At the ultrastructural level, the cup-shaped presynaptic structures comprise many active zones contacting numerous small postsynaptic profiles. Three-dimensional quantification demonstrated a significantly higher number of GS (∼ 13%) in foragers compared with interior workers. Light exposure, as opposed to age, was necessary and sufficient to trigger a similar increase in GS numbers. Furthermore, the increase in GS numbers was sensitive to the exclusion of UV light. As previous experiments have demonstrated the importance of the UV spectrum for sky-compass navigation in Cataglyphis, we conclude that plasticity in LX GS may reflect processes involved in the initial calibration of sky-compass neuronal circuits during orientation walks preceding active foraging.

Ultrastructural examination of the corticocollicular pathway in the guinea pig: a study using electron microscopy, neural tracers, and GABA immunocytochemistry

Projections from auditory cortex (AC) can alter the responses of cells in the inferior colliculus (IC) to sounds. Most IC cells show excitation and inhibition after stimulation of the AC. AC axons release glutamate and excite their targets, so inhibition is presumed to result from cortical activation of GABAergic IC cells that inhibit other IC cells via local projections. However, it is not known whether cortical axons contact GABAergic IC cells directly. We labeled corticocollicular axons by injecting fluorescent dextrans into the AC in guinea pigs. We visualized the tracer with diaminobenzidine and processed the tissue for electron microscopy. We identified presumptive GABAergic profiles with post-embedding anti-GABA immunogold histochemistry on ultrathin sections. We identified dextran-labeled cortical boutons in the IC and identified their postsynaptic targets according to morphology (e.g., spine, dendrite) and GABA-reactivity. Cortical synapses were observed in all IC subdivisions, but were comparatively rare in the central nucleus. Cortical boutons contain round vesicles and few mitochondria. They form asymmetric synapses with spines (most frequently), dendritic shafts and, least often, with cell bodies. Excitatory boutons in the IC can be classified as large, medium or small; most cortical boutons belong to the small excitatory class, while a minority (~14%) belong to the medium excitatory class. Approximately 4% of the cortical targets were GABA-positive; these included dendritic shafts, spines, and cell bodies. We conclude that the majority of cortical boutons contact non-GABAergic (i.e., excitatory) IC cells and a small proportion (4%) contact GABAergic cells. Given that most IC cells show inhibition (as well as excitation) after cortical stimulation, it is likely that the majority of cortically-driven inhibition in the IC results from cortical activation of a relatively small number of IC GABAergic cells that have extensive local axons.

Ultrastructural examination of diffuse and specific tectopulvinar projections in the tree shrew

Two pathways from the superior colliculus (SC) to the tree shrew pulvinar nucleus have been described, one in which the axons terminate in dense (or specific) patches and one in which the axon arbors are more diffusely organized (Luppino et al. [1988] J. Comp. Neurol. 273:67-86). As predicted by Lyon et al. ([2003] J. Comp. Neurol. 467:593-606), we found that anterograde labeling of the diffuse tectopulvinar pathway terminated in the acetylcholinesterase (AChE)-rich dorsal pulvinar (Pd), whereas the specific pathway terminated in the AChE-poor central pulvinar (Pc). Injections of retrograde tracers in Pd labeled non-gamma-aminobutyric acid (GABA)-ergic wide-field vertical cells located in the lower stratum griseum superficiale and stratum opticum of the medial SC, whereas injections in Pc labeled similar cells in more lateral regions. At the ultrastructural level, we found that tectopulvinar terminals in both Pd and Pc contact primarily non-GABAergic dendrites. When present, however, synaptic contacts on GABAergic profiles were observed more frequently in Pc (31% of all contacts) compared with Pd (16%). Terminals stained for the type 2 vesicular glutamate transporter, a potential marker of tectopulvinar terminals, also contacted more GABAergic profiles in Pc (19%) compared with Pd (4%). These results provide strong evidence for the division of the tree shrew pulvinar into two distinct tectorecipient zones. The potential functions of these pathways are discussed.

The role of the dorsomedial part of the prefrontal cortex serotonergic innervation in rat responses to the aversively conditioned context: Behavioral, biochemical and immunocytochemical studies

In this study we have explored differences in animal reactivity to conditioned aversive stimuli using the conditioned fear test (a contextual fear—freezing response), in rats subjected to the selective lesion of the prefrontal cortex serotonergic innervation, and differing in their response to the acute painful stimulation, a footshock (HS – high sensitivity rats, and LS – low sensitivity rats, selected arbitrarily according to their behavior in the ‘flinch-jump’ pre-test). Local administration of serotonergic neurotoxin (5,7-dihydroxytryptamine) to the dorsomedial part of the prefrontal cortex caused a very strong, structure and neurotransmitter selective depletion of serotonin concentration. In HS rats, the serotonergic lesion significantly disinhibited rat behavior controlled by fear, enhanced c-Fos expression in the dorsomedial prefrontal area, and increased the concentration of GABA in the basolateral amygdala, measured in vivo after the testing session of the conditioned fear test. The LS animals revealed an opposite pattern of behavioral and biochemical changes after serotonergic lesion: an increase in the duration of a freezing response, and expression of c-Fos in the basolateral and central nuclei of amygdala, and a lower GABA concentration in the basolateral amygdala. In control conditions, c-Fos expression did not differ in LS and HS, naïve, not conditioned and not exposed to the test cage animals. The present study adds more arguments for the controlling role of serotonergic innervation of the dorsomedial part of the prefrontal cortex in processing emotional input by other brain centers. Moreover, it provides experimental data, which may help to better explain the anatomical and biochemical basis of differences in individual reactivity to stressful stimulation, and, possibly, to anxiolytic drugs with serotonergic or GABAergic profiles of action.

Involvement of the GABAergic System in Dimethoate-induced Intoxication

The alteration of the GABAergic system in dimethoate intoxication was explored. A rat brain AChE (acetylcholinesterase) activity and GABAergic profiles, including the level of gamma-aminobutyric acid (GABA) and the densities and affinities of gamma-aminobutyric acid A (GABAA )r eceptors in the hippocampus, were examined 2h r after dimethoate administration (0, 38.9, 83.7 and 180.0 mg/kg bw po). The AChE activity was reduced by 60–70% by all three doses of dimethoate. Statistically significant elevation of GABA levels was observed followin gt he administration of dimethoate 180.0 mg/kg bw (121.5% of control, p < 0.05), while the Bmax values for the GABAA receptors of the hippocampal synaptic membranes were 50.6% and 51.6% of control values in the 83.7 and 180.0 mg/kg bw dimethoate groups, respectively (p < 0.05). Statistically significant decreases in the Kd values of 19.8, 51.7, and 53.4% vs. controls were observed in a dose-dependent manner (p < 0.05). In conclusion, it is suggested that the GABAergic system is maybe involved in the neurotoxicity of organophosphates as well as the cholinergic mechanisms.

Vagal afferent control of opioidergic effects in rat brainstem circuits

We demonstrated recently that increasing the levels of cAMP allows opioids to modulate GABAergic synaptic transmission between the nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). Using a combination of electrophysiological, immunohistochemical and biochemical approaches, we provide evidence that vagal afferent fibres dampen cAMP levels within the vagal brainstem circuits via tonic activation of group II metabotropic glutamate receptors (mGluRs). Whole-cell patch-clamp recordings were made from identified neurons of the rat DMV. Following chronic vagal deafferentation, the opioid agonist methionine-enkephalin (ME) inhibited the amplitude of evoked IPSC (eIPSC) in 32 of 33 neurons, without exogenous enhancement of cAMP levels. The ME-induced inhibition was prevented by the group II mGluR-selective agonist APDC. Following perfusion with the group II mGluR-selective antagonist EGLU, ME inhibited eIPSC amplitude in brainstem slices of control rats. Immunohistochemical experiments revealed that, following vagal deafferentation, mu-opioid receptors were colocalized on GABAergic profiles apposing DMV neurons; the number of colocalized profiles was significantly decreased by pretreatment with APDC. Radioimmunoassay and Western blot analysis showed that cAMP and phosphorylated cyclic AMP response element binding protein (pCREB) levels in the dorsal vagal complex were increased following vagal deafferentation. Our data show that by tonically dampening the levels of cAMP within the GABAergic synaptic contacts, activated group II mGluRs prevent the modulation of this synapse by endogenous opioids. These data suggest that the plasticity, hence the response, of central circuits controlling the vagal motor outflow to visceral organs is modulated and finely tuned by vagal afferent fibres.

Comparison of the ultrastructure of cortical and retinal terminals in the rat superior colliculus

We compared the ultrastructure and synaptic targets of terminals of cortical or retinal origin in the stratum griseum superficiale and stratum opticum of the rat superior colliculus. Following injections of biotinylated dextran amine into cortical area 17, corticotectal axons were labeled by anterograde transport. Corticotectal axons were of relatively small caliber with infrequent small varicosities. At the ultrastructural level, corticotectal terminals were observed to be small profiles (0.44 +/- 0.27 microm(2)) that contained densely packed round vesicles. In tissue stained for gamma amino butyric acid (GABA) using postembedding immunocytochemical techniques, corticotectal terminals were found to contact small (0.51 +/- 0.69 microm(2)) non-GABAergic dendrites and spines (93%) and a few small GABAergic dendrites (7%). In the same tissue, retinotectal terminals, identified by their distinctive pale mitochondria, were observed to be larger than corticotectal terminals (3.34 +/- 1.79 microm(2)). In comparison to corticotectal terminals, retinotectal terminals contacted larger (1.59 +/- 1.70 microm(2)) non-GABAergic dendrites and spines (73%) and a larger proportion of GABAergic profiles (27%) of relatively large size (2.17 +/- 1.49 microm(2)), most of which were vesicle-filled (71%). Our results suggest that cortical and retinal terminals target different dendritic compartments within the neuropil of the superficial layers of the superior colliculus.

Ultrastructural analysis of projections to the pulvinar nucleus of the cat. II: Pretectum

The pretectum (PT) can supply the pulvinar nucleus (PUL), and concomitantly the cortex, with visual motion information through its dense projections to the PUL. We examined the morphology and synaptic targets of pretecto-pulvinar (PT-PUL) terminals labeled by anterograde transport in the cat. By using postembedding immunocytochemical staining for gamma-aminobutyric acid (GABA), we additionally determined whether PT-PUL terminals or their postsynaptic targets were GABAergic. We found that the main projection from the PT to the PUL is an ipsilateral, non-GABAergic projection (72.4%) that primarily contacts thalamocortical cell dendrites (87.6%), and also the dendritic terminals of interneurons (F2 profiles; 12.4%). The PT additionally provides GABAergic innervation to the PUL (27.6% of the ipsilateral projection), which chiefly contacts relay cell dendrites (84.6%) but also GABAergic profiles (15.4%). These GABAergic pretectal terminals are smaller, beaded fibers that likely branch to bilaterally innervate the PUL and dLGN, and possibly other targets. We also examined the neurochemical nature of PT-PUL cells labeled by retrograde transport and found that most are non-GABAergic cells (79%) and devoid of calbindin. Taking existing physiological and our present morphological data into account, we suggest that, in addition to the parietal cortex, the non-GABAergic PT-PUL projection may also strongly influence PUL activity. The GABAergic pretectal fibers, however, may provide a more widespread influence on thalamic activity.

Immunoreactivity for the group III receptor subtype mGluR4a in the visual layers of the rat superior colliculus

Several studies indicate that metabotropic glutamate receptors (mGluRs) participate in the transmission of visual stimuli in optic layers of the superior colliculus (SC). We examined the cellular and subcellular distribution of the group III mGluR4a in superficial layers of the rat SC by means of a specific antiserum and a preembedding immunogold method for electron microscopy. Deposits of mGluR4a immunoparticles were mostly observed on presynaptic membranes of large synaptic terminals, which made asymmetrical synapses and contained abundant spherical, clear synaptic vesicles and numerous electron translucent mitochondria. These characteristic ultrastructural features correspond to retinocollicular synaptic terminals. Also, chains of synaptic retinal terminals along dendrites were labeled for mGluR4a. About 70% of morphologically identified retinal terminals were mGluR4a immunopositive. Furthermore, mGluR4a immunoreactivity in SC greatly disappeared following retinal ablation. About 28% of cortical terminals identified by anterograde tracing showed mGluR4a labeling, whereas only 2% of collicular GABAergic profiles were labeled for mGluR4a. These results reveal that retinal terminals are the major contributors to the mGluR4a immunoreactivity observed in the superior collicular circuitry.

Comparison of the fine structure of cortical and collicular terminals in the rat medial geniculate body

Neurons throughout the rat medial geniculate body, including the dorsal and ventral divisions, display a variety of responses to auditory stimuli. To investigate possible structural determinants of this variability, measurements of axon terminal profile area and postsynaptic dendrite diameter were made on inferior colliculus and corticothalamic terminal profiles in the medial geniculate body identified by anterograde tracer labeling following injections into the inferior colliculus or cortex. Over 90% of the synapses observed were axodendritic, with few axosomatic synapses. Small (<0.5 μm 2) and large (>1.0 μm 2) collicular profiles were found throughout the medial geniculate, but were smaller on average in the dorsal division (0.49±0.49 μm 2) than in the ventral division (0.70±0.64 μm 2). Almost all corticothalamic profiles were small and ended on small-caliber dendrites (0.57±0.25 μm diameter) throughout the medial geniculate. A few very large (>2.0 μm 2) corticothalamic profiles were found in the dorsal division and in the marginal zone of the medial geniculate. GABA immunostaining demonstrated the presence of GABAergic profiles arising from cells in the inferior colliculus. These profiles were compared with GABAergic profiles not labeled with anterograde tracer, which were presumed to be unlabeled inferior colliculus profiles or thalamic reticular nucleus profiles. The distributions of dendritic diameters postsynaptic to collicular, cortical and unlabeled GABAergic profiles were compared with dendritic diameters of intracellularly labeled medial geniculate neurons from rat brain slices. Our results demonstrate a corticothalamic projection to medial geniculate body that is similar to other sensory corticothalamic projections. However, the heterogeneous distributions of excitatory inferior collicular terminal sizes and postsynaptic dendritic diameters, along with the presence of a GABAergic inferior collicular projection to dendrites in the medial geniculate body, suggest a colliculogeniculate projection that is more complex than the ascending projections to other sensory thalamic nuclei. These findings may be useful in understanding some of the differences in the response characteristics of medial geniculate neurons in vivo.

Location of muscarinic type 2 receptors within the synaptic circuitry of the cat visual thalamus

A cholinergic projection from the parabrachial region (PBR) of the brainstem to the visual thalamus has been studied in great detail during the past 20 years. A number of physiological studies have demonstrated that this projection causes a dramatic change in thalamic activity during the transition from sleep to wakefulness. Additionally, the PBR may mediate more subtle changes in thalamic activity as attentional levels fluctuate during the waking state. The synaptic circuitry underlying these events has been identified in the cat thalamus. However, there is currently no anatomical information regarding the distribution of cholinergic receptors in relation to this circuitry. To begin to understand how the PBR projection modulates thalamic activity, we used immunocytochemical techniques to examine the distribution of muscarinic type 2 (M2) receptors in the visual thalamus of the cat. The distribution of M2 receptors correlates well with previous reports of the distribution of cholinergic terminals in the visual thalamus. At the light microscopic level, dense M2 staining was seen in the neuropil of the dorsal lateral geniculate nucleus (dLGN) and pulvinar nucleus and in somata and proximal dendrites of cells in the thalamic reticular nucleus (TRN). In the dLGN and pulvinar nucleus, we quantitatively analyzed the distribution of M2 receptors using electron microscopy. Postembedding immunocytochemistry for gamma aminobutyric acid (GABA) was used to determine whether M2 receptors are present on interneurons or thalamocortical cells. In particular, we examined the distribution of M2 receptors with respect to the known sites of PBR terminations. The dendrites of both thalamocortical cells and interneurons were stained for the M2 receptors in both the glomerular and extraglomerular neuropil. However, the densest staining was found in glomerular GABAergic profiles that displayed the morphology associated with interneuron dendritic terminals (F2 profiles). Our data suggest that M2 receptors play an important role both in blocking thalamic spindle oscillations and in increasing the efficacy of signal transmission during increased attentional states. J. Comp. Neurol. 410:431–443, 1999. © 1999 Wiley-Liss, Inc.

Location of muscarinic type 2 receptors within the synaptic circuitry of the cat visual thalamus

A cholinergic projection from the parabrachial region (PBR) of the brainstem to the visual thalamus has been studied in great detail during the past 20 years. A number of physiological studies have demonstrated that this projection causes a dramatic change in thalamic activity during the transition from sleep to wakefulness. Additionally, the PBR may mediate more subtle changes in thalamic activity as attentional levels fluctuate during the waking state. The synaptic circuitry underlying these events has been identified in the cat thalamus. However, there is currently no anatomical information regarding the distribution of cholinergic receptors in relation to this circuitry. To begin to understand how the PBR projection modulates thalamic activity, we used immunocytochemical techniques to examine the distribution of muscarinic type 2 (M2) receptors in the visual thalamus of the cat. The distribution of M2 receptors correlates well with previous reports of the distribution of cholinergic terminals in the visual thalamus. At the light microscopic level, dense M2 staining was seen in the neuropil of the dorsal lateral geniculate nucleus (dLGN) and pulvinar nucleus and in somata and proximal dendrites of cells in the thalamic reticular nucleus (TRN). In the dLGN and pulvinar nucleus, we quantitatively analyzed the distribution of M2 receptors using electron microscopy. Postembedding immunocytochemistry for gamma aminobutyric acid (GABA) was used to determine whether M2 receptors are present on interneurons or thalamocortical cells. In particular, we examined the distribution of M2 receptors with respect to the known sites of PBR terminations. The dendrites of both thalamocortical cells and interneurons were stained for the M2 receptors in both the glomerular and extraglomerular neuropil. However, the densest staining was found in glomerular GABAergic profiles that displayed the morphology associated with interneuron dendritic terminals (F2 profiles). Our data suggest that M2 receptors play an important role both in blocking thalamic spindle oscillations and in increasing the efficacy of signal transmission during increased attentional states.

Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts

Abnormal pain-related behaviour that accompanies peripheral nerve injury may be the result of altered spinal neuronal function. The long-term loss of inhibitory function by GABA neurons in particular may be a mechanism by which abnormal neural hyperactivity occurs, leading to exaggerated sensory processing following nerve injury. In order to assess this, changes in spinal GABA immunoreactivity at several time points following constriction nerve injury was quantified in parallel with behavioural assessments of abnormal sensory responses to noxious and innocuous stimuli. In addition, the effects of spinal adrenal medullary transplants were determined since previous findings have demonstrated alleviation of behavioural pain symptoms by such transplants. In response to unilateral sciatic nerve injury, GABAergic profiles normally found in lumbar dorsal horn laminae I–III, significantly decreased. The decrease was apparent three days following ligation, particularly on the side ipsilateral to the nerve injury. By two weeks, no GABAergic profiles could be seen, with the deficit appearing in the spinal dorsal horn both ipsilateral and contralateral to the unilateral peripheral nerve injury. Marked decreases in GABA-immunoreactive profiles persisted for at least up to five weeks post-injury, with partial restoration occurring by seven weeks. However, even at seven weeks, losses in GABA-immunoreactive profiles persisted in the dorsal horn ipsilateral to peripheral nerve injury. These findings were comparable in animals receiving control striated muscle transplants. In contrast, adrenal medullary transplants markedly reduced the loss in GABA-immunoreactive profiles at all time-points examined. In addition, GABA-immunoreactive profile levels were normalized near that of intact animals by five to seven weeks following nerve injury in animals with adrenal medullary transplants. Parallel improvements in sensory responses to innocuous and noxious stimuli were also observed in these animals.The results of this study indicate that peripheral nerve injury can result in severe losses in spinal inhibitory mechanisms, possibly leading to exaggerated sensory processes in persistent pain states. In addition, adrenal medullary transplants may provide a neuroprotective function in promoting recovery and improving long-term survival of GABAergic neurons in the spinal dorsal horn which have been damaged by excitotoxic injury.

Synaptic reorganisation in the rat striatum after dopaminergic deafferentation: an ultrastructural study using glutamate decarboxylase immunocytochemistry.

The ultrastructure of GABAergic and non-GABAergic synapses in the adult rat neostriatum was examined 6-8 months after unilateral removal of the nigrostriatal dopaminergic pathway by 6-hydroxydopamine injection into the medial forebrain bundle. GABAergic profiles were identified by preembedding glutamate decarboxylase (GAD) immunocytochemistry performed on parasagittal vibratome sections. In three representative fields of the striatum, the nature and number of boutons and their postsynaptic partners were determined and the differences between the striata ipsi- and contralateral to the lesion analyzed. The percentage of GAD-immunoreactive boutons was increased from 23% on the intact side to 28% on the lesioned side. In addition, the GABAergic boutons underwent significantly more multiple contacts with several independent postsynaptic profiles, preferentially with dendritic spines. This could reflect a lesion-induced sprouting of local GABAergic axon terminals. On the other hand, although the vast majority of GABAergic boutons underwent synaptic contacts with dendrites (77% vs. 80%), the number of boutons per dendrite or per dendritic circumference remained unchanged. Thus, the higher frequency of GABAergic boutons may simply reflect the loss of the dopaminergic nerve endings, without a heterosynaptic replacement by GABAergic boutons. The deafferentation also induced structural changes of the postsynaptic profiles. Some dendritic spines had a shortened neck; others were completely integrated in the dendrite which now contained a spine apparatus and was contacted by boutons with the features of axospinous synapses. The spine retraction resulted in a quantitative decrease in the number of spines. Analysis of the synaptic curvature revealed that only spines with a flat contact zone were lost. Concurrently, the number of dendrites was increased, of the GAD-containing in particular, suggesting that the denrites of GABAergic interneurons tend to elongate and/or arborize. Taken together, the results of the present study show that the dopaminergic denervation caused a remodeling of the postsynaptic neurons. The relative increase of the number of GABAergic boutons and their synaptic contacts suggests that an altered wiring of the intrinsic GABAergic system contributes to the changes in the striatal output activity.

Ultrastructural organization of GABA in the rabbit superior colliculus revealed by quantitative postembedding immunocytochemistry.

We have studied the organization of gamma-aminobutyric acid (GABA)ergic profiles in the superior colliculus of the rabbit to determine whether the synaptic types found in cat and monkey also exist in a mammalian species whose visual system has a different organization. Ultrastructure of GABAergic profiles was examined by use of a polyclonal antibody to GABA and quantitative postembedding immunocytochemistry. Three distinct types of vesicle-containing profiles were labeled by the GABA antibody in the rabbit superior colliculus. One type was a putative presynaptic dendrite (PSD profile) that received synaptic input from other profiles and contained pleomorphic synaptic vesicles scattered throughout the profile. These PSD profiles frequently received retinal input and formed dendrodendritic synapses. A second type of profile was a large caliber dendrite, often horizontal in orientation (H profile), that had one or more discrete clusters of pleomorphic synaptic vesicles at sites of synaptic contact with conventional dendrites. These H profiles received few synaptic contacts. A third profile type was a putative axon terminal (F profile) with smaller, more flattened synaptic vesicles that densely and uniformly filled the profile. Quantitative analysis of gold particle density revealed that F profiles had a significantly higher gold particle density (14.3/microns 2) than did PSD or H profiles (10.4 and 10.2/microns 2), suggesting that GABAergic profile types contain different concentrations of GABA. The vesicle density of these profile types also differed, but no obvious relationship between vesicle and particle distributions was observed. We conclude that the profiles labeled by GABA in rabbit superior colliculus are similar to those in cat and monkey and must represent a phylogenetically conserved organization common to many mammals, and that particle density analysis of postembedding immunocytochemistry can distinguish different GABAergic profile types.

Inhibitory synaptic input to identified rubrospinal neurons in Macaca fascicularis: an electron microscopic study using a combined immuno-GABA-gold technique and the retrograde transport of WGA-HRP.

Rubrospinal neurons of the magnocellular division of the red nucleus of Macaca fascicularis were retrogradely labeled following spinal cord microinjections of wheat germ agglutinin-horseradish peroxidase, as demonstrated by the chromagen tetramethylbenzidine, identifying the mesencephalic cells of origin of this descending motor pathway. The tissue was processed for electron microscopy and subsequently tested on the electron microscope grid for immunoreactivity of gamma aminobutyric acid (GABA) in presumed local circuit neuronal somata, in dendrites, and in axonal terminals. Results demonstrate the presence of retrogradely labeled rubrospinal neurons of medium and large diameters (30-90 microns) and immunoreactive neurons of small size (less than 20 microns in diameter) within the nucleus. In addition, there are substantial numbers of GABAergic, presumably inhibitory, synaptic structures contacting somata and primary, medium, and small sized dendrites, as well as spineheads of rubrospinal neurons. The immunoreactive presynaptic profiles exhibit two different morphological appearances: one axonal and the other dendritic. Axonal terminals contain densely packed pleomorphic to flattened vesicles and form primarily symmetrical synapses with somata and all regions of the dendritic arbor. GABAergic profiles resembling presynaptic dendrites (PSDs) are also present. These profiles possess scattered flattened to pleomorphic synaptic vesicles in a translucent cytoplasm and are often postsynaptic to axonal terminals of unknown origin, or to GABAergic profiles. GABAergic local circuit neurons (LCNs), the neurites of which remain within the confines of the nucleus, appear to be contacted primarily by cortical and cerebellar afferents. These LCNs may or may not possess axons and thus may represent both the source of the GABAergic axonal terminals as well as that of the PSDs. Inhibitory afferents from other sources, such as the mesencephalic reticular formation, may also account for GABAergic terminals involved in this inhibition. We propose that the level of excitability of rubrospinal neurons and their subsequent activation of spinal motor neurons and interneurons is significantly regulated by the local circuit GABAergic inhibitory interneuronal population of the nucleus proper and probably by axons entering the nucleus from an extranuclear source.

Catecholaminergic, GABAergic, and hippocamposeptal innervation of gabaergic “somatospiny” neurons in the rat lateral septal area

This study deals with the neurochemical characterization of the rat lateral septal area (LSA) somatospiny neurons and their innervation by hippocamposeptal, catecholaminergic, and GABAergic fibers. Electron microscopic single and double immunostaining methods were used to label catecholaminergic fibers and GABAergic cells and boutons. Axon terminals originating in the hippocampus were labeled by acute anterograde axon degeneration induced by fimbria-fornix transection 36 hours before sacrifice. Three types of experiments were performed. The convergent catecholaminergic and hippocamposeptal innervation of LSA somatospiny neurons was studied by combining immunostaining for tyrosine hydroxylase (TH) with fimbria-fornix transection. GABAergic neurons and their hippocamposeptal afferents were identified and characterized in colchicine pretreated animals immunostained for glutamic acid decarboxylase (GAD) combined with fimbria-fornix transection. The third experiment aimed at simultaneously visualizing the relationships between catecholaminergic boutons, hippocamposeptal excitatory amino acid containing axon terminals and GABAergic profiles by double immunostaining for TH (the PAP technique) and GAD (the immunogold method) combined with fimbria-fornix transection. The results are summarized as follows: 1) The same LSA somatospiny neurons receive synaptic inputs from the hippocampus and TH immunoreactive fibers which form pericellular baskets around these cells. 2) LSA somatospiny neurons are GABAergic and are postsynaptic targets of GABAergic boutons with unknown origin and hippocamposeptal axon terminals. 3) The double immunostaining experiment, finally, provided direct evidence that the same GABAergic somatospiny neurons are postsynaptic targets of both catecholaminergic and hippocamposeptal afferents. The synaptic interconnections described in this study provide anatomical basis for a better understanding of the action of catecholamines, excitatory amino acids, and GABA on the activity of LSA neurons.

The morphogenesis of glutamic acid decarboxylase in the neostriatum of the cat: neuronal and ultrastructural localization

Correlative light and electron microscopic immunohistochemical methods were adapted for a descriptive analysis of the normal time course and pattern of expression and intraneuronal localization of the enzyme glutamic acid decarboxylase (GAD) in the neostriatum (Ns) of fetal, postnatal and adult cats. The differentiation of this synthesizing enzyme demonstrated the establishment of γ-aminobutyric acid (GABA) transmitter identity in these neurons and their connections. The structural modifications of these GABAergic profiles revealed the morphogenesis of important inhibitory synaptic inputs in the Ns. The expression of GAD began during late fetal development and proceeded in a diagonal gradient from the first-formed ventrolateral putamen to the last-formed dorsomedial caudate nucleus. The frequency of GAD-positive elements increased with age particularly during the early postnatal period. After the initial expression of GAD, 3 interrelated processes contributed to its differentiation: (1) enzyme accumulation; (2) enzyme association with membranous organelles and (3) progressive elaboration of neuronal infrastructure. Synaptogenesis was both coincident and subsequent to GAD differentiation. Two principal types of GABAergic structures, cell bodies and axonal ‘terminals’, were evident from the initiation of GAD expression. The GABAergic cell bodies were polymorphic by and after the day of birth and consisted of ubiquitous medium sized cells (often having somatic and/or dendritic spines) and rare large sized cells (apparently aspiny and confined to a limited region of the Ns). The GABAergic axonal terminals changed from growth cone and prototerminal forms to mature bouton en passage and bouton terminaux forms establishing axosomatic and axodendritic contracts, having symmetric synaptic specializations and providing inputs to both medium- and large-sized GABAergic target neurons.