Localization of met-enkephalin-like immunoreactivity within pain-related nuclei of cervical spinal cord, brainstem and midbrain in the cat

Met-enkephalin immunoreactivity was investigated with an indirect immunoperoxidase technique in the cervical spinal cord, brainstem and midbrain of the cat, paying special attention to pain-related nuclei. Different technical conditions were used to reveal preferentially met-enkephalin-containing fibres and terminals or perikarya. Immunoreactive fibres and terminals were revealed optimally in sections from control animals incubated with detergent (Triton X-100). Immunoreactive perikarya were revealed in colchicine treated animals. Comparison between different routes of administration showed that local injections of colchicine are needed to reveal optimally immunoreactive perikarya in nuclei located far from the ventricles. Met-enkephalin-containing fibres and terminals are widely distributed in the posterior brain and spinal cord. The densest network of immunoreactive fibres are observed in the superficial layers of the cervical spinal cord and the caudal trigeminal nucleus, in the nucleus of the solitary tract, the nucleus of the facial nerve, the nucleus of the prepositus hypoglossi, the nucleus raphe pallidus, the medial vestibular nucleus, the interpedoncular nucleus and the substantia nigra. A moderate staining of fibres is observed in various nuclei including the ventral horn of the spinal cord and caudal trigeminal nucleus, the brainstem and midbrain reticular formation, the inferior olivary complex, the nucleus of the descending trigeminal tract and the periaqueductal grey. Met-enkephalin-containing perikarya are present in all the nuclei cited before, except in the inferior olivary complex. The densest aggregation of enkephalin-like perikarya is observed in the nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, nucleus reticularis gigantocellularis pars α and nucleus reticularis lateralis. The general distribution of enkephalin-containing structures in the cervical spinal cord, brainstem and midbrain of the cat appears very similar to that of the rat except in the substantia nigra where met-enkephalin cell bodies are found in the cat but not in the rat. In particular the pain-related nuclei present a similar distribution of the peptide in the two species; however, met-enkephalin-containing cell bodies are much more numerous in the cat than in the rat (notably in the reticular formation). Similar types of metenkephalin innervation occur in the dorsal and intermediate grey of the spinal cord and of the caudal trigeminal nucleus supporting further that the functional organizations of these regions are closely related.     

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.     

Spinovestibular projections in the rat,with particular reference to projections from the central cervical nucleus to the lateral vestibular nucleus

Projections from the spinal cord to the vestibular nuclei were examined following injections of Phaseolus vulgaris–leucoagglutinin, cholera toxin subunit B, or biotinylated dextran at various levels of the spinal cord in the rat. Labeled terminals were abundant after injections of the tracers into the C2 and C3 segments containing the central cervical nucleus. Labeled terminals were seen in the descending vestibular nucleus and the parvocellular, magnocellular, and caudal parts of the medial vestibular nucleus throughout its rostrocaudal extent. Labeled terminals were most numerous in the lateral vestibular nucleus throughout its rostrocaudal extent. The projections from the central cervical nucleus to the vestibular nuclei were exclusively contralateral to the cells of origin because the axons of the central cervical nucleus neurons cross in the spinal cord. Following tracer injections in the cervical enlargement, many labeled terminals were seen in the magnocellular part of the medial vestibular nucleus, but a few were seen in the lateral and the descending vestibular nucleus. Injections into more caudal segments resulted in sporadic terminal labeling in the magnocellular part of the medial vestibular nucleus, the descending vestibular nucleus, and the caudal part of the lateral vestibular nucleus. The results indicate that primary neck afferent input relayed at the central cervical nucleus is mediated directly to the contralateral vestibular nuclei. It is suggested that this projection serves as an important linkage from the upper cervical segments to the lateral vestibulospinal tract in the tonic neck reflex. © 1995 Wiley-Liss, Inc.     

Immunohistochemical localization of gamma-aminobutyric acid- and aspartate-containing neurons in the guinea pig vestibular nuclei

The immunohistochemical distributions of gamma-aminobutyric acid (GABA)- and aspartate-containing neurons were studied in the guinea pig vestibular nuclei using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered throughout all regions of the vestibular nuclei. The largest number of these cells was found in the medial nucleus. Intraventricular injection of colchicine markedly increased GABA-like immunoreactivity in these cell bodies. GABA-containing terminals were distributed throughout all 4 subdivisions of the nuclei, with the richest localization found around the floor of the fourth ventricle. Various sized aspartate-containing neurons were noted in the vestibular nuclei and small cells were present in the superior, medial and lateral nucleus. Medium-sized cells were observed throughout the vestibular nuclei. Giant cells in the lateral nucleus also contained aspartate and were surrounded by GABA-like immunoreactive terminals, thereby suggesting the modulation of aspartate-containing neurons by GABAergic fibers from Purkinje cells.     

Central projections of cervical primary afferent fibers in the guinea pig: an HRP and WGA/HRP tracer study

The central course and the projections of the first and the second cervical dorsal root ganglia and of suboccipital muscle primary afferent fibers in the guinea pig were studied by means of anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA/HRP) or aqueous solution of horseradish peroxidase (HRP). Injections of WGA/HRP into the second cervical dorsal root ganglion produced labeling in the dorsal and ventral horns. Within the spinal cord, the largest amount of HRP reaction product was found within the lateral third of the substantia gelatinosa and within the central cervical nucleus. The main area of termination in the medulla was the external cuneate nucleus. However, HRP reaction product was also found within the medial and inferior vestibular nuclei, cell group x, the perihypoglossal nuclei, the nucleus of the solitary tract, and the nucleus of the spinal trigeminal tract. Descending fibers could be detected as caudal as spinal segment T5. Injections of WGA/HRP into the first cervical dorsal root ganglion produced heavy terminal label within the central cervical nucleus but not within the substantia gelatinosa. Again, the external cuneate nucleus was the main area of termination within the medulla. Label could not be observed within the vestibular nuclear complex or within the spinal trigeminal nucleus. Injections of aqueous HRP into the suboccipital muscles produced heavy transganglionic label within the central cervical nucleus, whereas the substantia gelatinosa totally lacked terminal label. Ascending proprioceptive fibers reached the external cuneate nucleus and group x. Scanty projections could be detected within the vestibular nuclei as well as within the perihypoglossal nuclei except for the nucleus prepositus hypoglossi. Label was absent in the spinal trigeminal nucleus.     

Somatostatin in the caudal spinal cord: An immunohistochemical study of the spinal centers involved in the innervation of pelvic organs

The distribution of somatostatin in the rat spinal cord was studied immunohistochemically with particular reference to the localization in the caudal centers that innervate the pelvic organs. For detailed studies of the laminar distribution of somatostatin the combination of immunohistochemistry and acetylcholinesterase enzyme histochemistry was employed. Deafferentation experiments were carried out to shed light on the origin of the somatostatin-containing axons. These experiments showed that the bulk of the spinal somatostatin has a spinal origin. The structures showing somatostatin immunoreactivity formed a distinct and detailed pattern. The marginal layer and particularly the substantia gelatinosa contained a dense immunoreactivity in terminallike structures. Such structures were also found in the reticular nucleus, along the medial border of the dorsal horn, and in the nucleus of the dorsolateral funiculus. In all of these regions somatostatin-positive cell bodies were also observed. In the intermediate gray matter stained terminals were present around the central canal in a varying number. The most prominent stainability was found in the lumbosacral transition zone. Many terminals were also observed in the sacral parasympathetic intermediolateral nucleus. In contrast, very few appeared in the sympathetic nuclei. Immunoreactive somata were present in the surroundings of the central canal at all levels. Moreover, positive neurons were found in the intermediolateral nucleus of the sacral cord. By combined retrograde tracing and immunohistochemistry the existence of somatostatin-containing parasympathetic visceromotoneurons was ascertained. Corresponding to this, somatostatin-positive terminals were seen in the major pelvic ganglion. The ventral horn generally contained few terminals, and the density was particularly low in the motoneuron neuropil. However, a dense somatostatin network was found in the sixth lumbar segment in relation to the neurons in Onuf's nucleus X complex, the nucleus that innervates the small pelvic muscles including the striated sphincters. It is concluded that somatostatin, besides being involved in the processing of sensory input, serves an important motor task, that of taking part in the complex control of the pelvic organs and their associated striated muscles.     

Labelling neurons with fluorescent dyes administered via intravenous, subcutaneous or intraperitoneal route

Fast blue, true blue and fluorogold injected into neonatal and subadult rats via subcutaneous or intraperitoneal route labelled certain forebrain nuclei and the central and peripheral neurons whose axons form the spinal and most cranial nerves. The A1 and A5 noradrenergic neurons, nucleus of the tractus solitarius and some neurons in the nucleus raphe medianus, nucleus reticularis gigantocellularis were also labelled. In addition, occasional fluorescent neurons were seen in the vestibular nucleus, locus coeruleus, fastigial nucleus and Purkinje cell layer of the cerebellum. Some nuclei labelled in neonatal rats hardly showed any labelled neurons in subadult rats. After injection the subarachnoid space, blood vessels in the spinal cord, brain and peripheral ganglia, the circumventricular organs and the choroid plexuses were labelled much faster than the neurons. A similar pattern of labelling was seen in subadult rats receiving intravenous injection of fluorogold. Evans blue and rhodamine injected intravenously failed to label any neurons in the brain or spinal cord.     

Immunohistochemical localization of γ-aminobutyric acid- and aspartate-containing neurons in the guinea pig vestibular nuclei

The immunohistochemical distributions of γ-aminobutyric acid (GABA)- and aspartate-containing neurons were studied in the guinea pig vestibular nuclei using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered throughout all regions of the vesticular nuclei. The largest number of these cells was found in the medial nucleus. Intraventricular injection of colchicine markedly increased GABA-like immunoreactivity in these cell bodies. GABA-containing terminals were distributed throughout all 4 subdivisions of the nuclei, with the richest localization found around the floor of the fourth ventricle. Various sized aspartate-containing neurons were noted in the vestibular nuclei and small cells were present in the superior, medial and lateral nucleus. Medium-sized cells were observed throughout the vestubular nuclei. Giant cells in the lateral nucleus also contained aspartate and were surrounded by GABA-like immunoreactive terminals, thereby suggesting the modulation of aspartate-containing neurons by GABAergic fibers from Purkinje cells.     

Glycine receptor distribution in mouse CNS: autoradiographic localization of [3H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino-acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [3H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.     

Studies of suspected neurotransmitters in the vestibuloocular pathways.

Isolated fresh cat trochlear and oculomotor nuclei, which contain the axon terminals of inhibitory neurons whose cell bodies are in the superior vestibular nucleus (SVN), actively synthesize and store [3H]GABA, [14C]acetylcholine, [3H]dopamine and [3H]tyramine from labeled precursors of these compounds. Twelve to 14 days following lesions of the ipsilateral superior vestibular nucleus or its efferent pathway to the oculomotor and trochlear nuclei, at a time when there is extensive degeneration of superior vestibular nucleus axon terminals in these nuclei, the synthesis and storage of GABA in the ipsilateral trochlear nucleus is markedly reduced compared to that in the contralateral trochlear nucleus; the synthesis of acetylcholine, dopamine and tyramine is not measurably affected. The oculomotor nuclei, which unlike the trochlear nuclei receive a heavy bilateral projection from the SVN, show no asymmetric decrease after SVN lesions in their ability to synthesize any of the compounds tested. The data support the identity of GABA as an inhibitory transmitter in the superior vestibular nucleus-trochlear nucleus pathway.     

Immunohistochemical localization of phospholipase D1 in rat central nervous system

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the distribution of PLD isozyme, PLD1 in the rat brain and spinal cord using an immunological approach. Western blot analysis showed the presence of PLD1 protein in all tissues studied, with significantly higher levels in the brainstem and spinal cord, which was correlated with the results obtained from PLD activity assay. Prominent and specific signals of PLD1 were observed in many functionally diverse brain areas, including the olfactory bulb, medial septum-diagonal band complex, cerebral cortex, brainstem, cerebellum, and spinal cord. In the brainstem, the red nucleus, substantia nigra, interpeduncular nucleus, cranial motor nuclei (trigeminal motor, abducent, facial, and hypoglossal), sensory cranial nerve nuclei (spinal trigeminal, vestibular, and cochlear), as well as nuclei of the reticular formation, all showed intense immunoreactivity. Purkinje cells and deep cerebellar nuclei of the cerebellum were also labeled intensely. However, no significant labeling was found in the thalamus, epithalamus, and basal ganglia. Although many of the PLD1 immunoreactive cells were neurons, PLD1 was also expressed in glial cells such as presumed astrocytes and tanycytes. These findings suggest that PLD1 may play an important role in the central nervous system of the adult rat.     

Glycine receptor distribution in mouse CNS: autoradiographic localization of [H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [³H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.     

Possible morphological substrates for GABA-mediated presynaptic inhibition in the lamprey spinal cord

Gamma-aminobutyric acid (GABA) neurons intrinsic to the lamprey spinal cord are known to modulate synaptic transmission from interneurons active during locomotion and from mechanosensory dorsal cells. Many of these physiological effects are presynaptic. To establish the morphological substrates for these axo-axonic interactions, an ultrastructural analysis was performed with an antiserum to fixed GABA. The GABA immunoreactivity (ir) was detected by postembedding peroxidase-antiperoxidase and immunogold techniques. GABA-ir terminals were found to make appositions with unlabelled axons located in the dorsal columns and in the ventrolateral aspect of the spinal cord. In the ventrolateral part of the cord, similar appositions between different GABA-ir terminals were also observed. The immunolabelled terminals contained spherical to pleomorphic synaptic vesicles, and also glycogen granules and dense core vesicles. In some cases, the fine structure of the contacts between immunogold-labelled terminals and unlabelled axons suggested a synaptic relationship. Such a relation was found in a relatively small proportion (2–3%) of the appositions studied. These specializations were always observed in close relation to an output synapse of the postsynaptic axon. It is suggested that the axo-axonal contacts described may provide an effective modulation of the synaptic transmission from axons in the lamprey spinal cord. © 1993 Wiley-Liss, Inc.     

Afferent pathways to the region of the vestibular nuclei that participates in cardiovascular and respiratory control

Prior experiments have shown that a region of the medial and inferior vestibular nuclei contributes to cardiovascular and respiratory regulation. In addition to labyrinthine inputs, the majority of neurons in this region of the vestibular nuclei receive signals from the skin, muscle, and viscera, although the pathways conveying these nonlabyrinthine inputs to the vestibular nucleus neurons are unknown. To gain further insight into the afferent pathways to this functionally distinct subdivision of the vestibular complex, we combined monosynaptic mapping with viral transneuronal tracing in the ferret. First order afferent projections were defined by retrograde transport of the beta-subunit of cholera toxin (CTbeta), and the extended polysynaptic circuitry was defined in the same animals by injection of a recombinant of pseudorabies virus Bartha (PRV) into the contralateral vestibular nuclei. Neurons containing CTbeta or infected by retrograde transneuronal transport and replication of PRV were distributed throughout the spinal cord, but were 10 times more prevalent in the cervical cord than the lumbar cord. The labeled spinal neurons were most commonly observed in Rexed's laminae IV-VI and the dorsal portions of laminae VII-VIII. Both the CTbeta and PRV injections also resulted in labeling of neurons in all four vestibular nuclei, the prepositus hypoglossi, the reticular formation, the inferior olivary nucleus, the medullary raphe nuclei, the spinal and principal trigeminal nuclei, the facial nucleus, and the lateral reticular nucleus. Following survival times >/=3 days, PRV-infected neurons were additionally present in nucleus solitarius and the gracile and cuneate nuclei. These data show that an anatomical substrate is present for somatosensory and visceral inputs to influence the activity of cells in the autonomic region of the vestibular nuclei and suggest that these signals are primarily transmitted through brainstem relay neurons.     

Observations on the afferent and efferent organization of the vagus nerve and the innervation of the stomach in the squirrel monkey

Horseradish peroxidase was injected into the cervical vagus nerve or stomach wall of adult squirrel monkeys. Following cervical vagus nerve injections, labelled afferent fibres were present in the tractus solitarius and labelled fibres and terminals were present in medial and lateral parts of the nucleus of the tractus solitarius (NTS) ipsilaterally. Afferent labelling was also seen in the ipsilateral commissural nucleus and in the area postrema. Labelling was present contralaterally in caudal levels of the medial parts of the NTS, in the commissural nucleus, and in the area postrema. Afferent projections to the ipsilateral pars interpolaris of the spinal trigeminal nucleus and to the substantia gelatinosa of the C1 segment of the spinal cord were also labelled. Following injections of HRP into the anterior and posterior stomach walls, the tractus solitarius was labelled bilaterally. Afferent labelling was concentrated bilaterally in the dorsal parts of the medial division of the NTS, i.e., in the subnucleus gelatinosus, and in the commissural nucleus. The regions of NTS immediately adjacent to the tractus solitarius were largely unlabelled. Injections of HRP into the cervical vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal nucleus of the vagus (DMX) and in the nucleus ambiguus (NA). In addition a few neurones were labelled in the intermediate zone between these two nuclei. Retrogradely labelled neurons were also present in the nucleus dorsomedialis in the rostral cervical spinal cord and in the spinal nucleus of the accessory nerve. Injections of HRP into the left cricothyroid muscle in two cases resulted in heavy retrograde labelling of large neurons in the left NA. Following stomach wall injections of HRP retrograde labelling of neurons was seen throughout the rostrocaudal and mediolateral extent of the DMX; there was no apparent topographical organization of the projection. In these cases, a group of labelled smaller neurons was found lying ventrolateral to the main part of the NA through its rostral levels. This study in a primate indicates that a large vagal afferent projection originates in the stomach wall and terminates primarily in the subnucleus gelatinosus of the NTS and in the commissural nucleus with a distribution similar to that described previously in studies in several subprimate mammalian species. The present results and those of other studies suggest some degree of segregation of visceral input within different subnuclei of the NTS.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative topography of projections from the mesodiencephalic junction to the inferior olive, vestibular nuclei, and upper cervical cord in the cat

Distributions of neurons located in the central rostral mesencephalon and caudal diencephalon that project to the upper cervical spinal cord, vestibular nuclei, or inferior olive were studied in the cat by using retrograde axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Afferent sources to all of these targets were observed in the interstitial nucleus of Cajal (INC), the region surrounding the fasciculus retroflexus (PF), and the nucleus of the fields of Forel (NFF). Three-dimensional reconstruction revealed differences in densities of cells projecting from these common areas. Spinal projecting cells were present in slightly greater numbers in the caudal two-thirds of the INC, whereas those projecting to the vestibular complex were more numerous in the rostral two-thirds of this nucleus. A relatively smaller number of olivary projecting cells were dispersed throughout the INC. Olivary afferent sources outnumber those with spinally directed or vestibularly directed axons in the PF region. In the fields of Forel, cells projecting to the vestibular nuclei or inferior olive were concentrated medially, whereas cells projecting to the spinal cord appeared both medially and laterally. Each type of afferent source was also seen in the nucleus of the posterior commissure and the posterior ventral lateral hypothalamic area. Unique sources of afferents to the inferior olive were observed in the parvicellular red nucleus (ipsilateral to the injections) and the anterior and posterior pretectal nuclei. A large number of labeled neurons was seen in the nucleus of Darkschewitsch after injections of tracer into the inferior olive, but this projection did not appear to be unique, as small numbers of labeled cells were also seen after injections into the cervical spinal cord. The Edinger-Westphal nucleus and the adjacent somatic oculomotor nucleus contained cells which projected separately to the spinal cord or the vestibular complex, and the superior colliculus contained cells which projected separately to the contralateral spinal cord or the contralateral inferior olive. In this study, it was also noted that neurons in the medial terminal nucleus of the accessory optic tract projected to the ipsilateral inferior olive or to the contralateral vestibular complex. These differences in locations and densities of cells projecting to the cervical spinal cord, vestibular complex, and inferior olive may underlie functional specializations in these areas in relation to vertical eye and head movement control and to neural systems controlling postural adjustments accompanying limb movements.     

Observations on the brainstem-spinal descending systems of normal and reeler mutant mice by the retrograde HRP method

Brainstem neurons which project to the lumbar spinal level were identified in both reeler mutant mice and normal controls (Balb/c mice) by the retrograde horseradish peroxidase (HRP) technique. In normal controls after HRP injection into the lumbar cord, retrogradely labelled neurons were observed in (1) the lateral vestibular nucleus, (2) the pontine and medullary reticular formations including the nucleus centralis caudalis pontis, nucleus gigantocellularis, nucleus paragigantocellularis, nucleus raphe magnus et pallidus, and nucleus centralis medullae oblongatae pars ventralis et dorsalis, and (3) the dorsal column nuclei, i.e., the nucleus gracilis and nucleus cuneatus medialis. In reeler mutant mice, labelled neurons were again seen in the nuclei referred to above, and their cellular type and distribution patterns within the corresponding nuclei were similar to those of the normal controls. These observations suggest that (1) the brainstem nuclei of reeler mutant mice which project to the lumbar spinal cord are cytoarchitecturally normal, (2) the reeler genetic locus (rl) does not affect the nonlaminated structures in the brainstem, at least those referred to above, and (3) the motor dysfunctions observed in the reeler, such as action tremor, dystonic posture, and reeling ataxic gait, are not attributable to the brainstem-spinal descending systems.     

Parvalbumin immunoreactivity in the thalamus of guinea pig: Light and electron microscopic correlation with gamma-aminobutyric acid immunoreactivity

The relationship of the calcium binding protein parvalbumin (PV) with gamma-aminobutyric acidergic (GABAergic) neurons differs within different thalamic nuclei and animal species. In this study, the distribution of PV and GABA throughout the thalamus of the guinea pig was investigated at the light microscopic level by using immunoperoxidase methods. Intense PV labelling was found in all the GABAergic neurons of the reticular nucleus and in scattered GABAergic neurons in the anteroventral nucleus, whereas GABAergic interneurons in the ventrobasal and lateral geniculate nuclei were not PV labelled. At the electron microscopic level, preembedding immunuperoxidase for PV was combined with postembedding immunogold for GABA. In the ventrobasal nucleus, four types of profiles were recognized: 1) terminals with flattened vesicles and forming symmetric synapses, which were labelled with both PV and GABA and could therefore be identified as afferents from the reticular nucleus; 2) boutons morphologically similar to presynaptic dendrites of interneurons, labelled only with GABA; 3) large terminals with round vesicles and asymmetric synapses, labelled only with PV, which contacted GABAergic presynaptic dendrites in glomerular arrangements and resembled ascending excitatory afferents; and 4) terminals unlabelled by either antiserum. In the ventrobasal nucleus of the guinea pig a double immunocytochemical labelling permits therefore the differentiation of two populations of GABAergic vesicle-containing profiles, i. e., the terminals originating from reticular nucleus (that are double labelled) and the presynaptic dendrites originating from interneurons (that are GABA-labelled only). The possibility to differentiate GABAergic inputs from the reticular nucleus and from interneurons can shed light to the functional interpretation of synaptic circuits in thalamic sensory nuclei. © 1994 Wiley-Liss, Inc.     

Postsynaptic Targets of Purkinje Cell Terminals in the Cerebellar and Vestibular Nuclei of the Rat

The cerebellar and vestibular nuclei consist of a heterogeneous group of inhibitory and excitatory neurons. A major proportion of the inhibitory neurons provides a GABAergic feedback to the inferior olive, while the excitatory neurons exert more direct effects on motor control via non-olivary structures. At present it is not clear whether Purkinje cells innervate all types of neurons in the cerebellar and vestibular nuclei or whether an individual Purkinje cell axon can innervate different types of neurons. In the present study, we studied the postsynaptic targets of Purkinje cell axons in the rat using a combination of pre-embedding immunolabelling of the Purkinje cell terminals by L7, a Purkinje cell-specific marker, and postembedding GABA and glycine immunocytochemistry. In the cerebellar nuclei, vestibular nuclei and nucleus prepositus hypoglossi Purkinje cell terminals were found apposed to GABAergic and glycinergic neurons as well as to larger non-GABAergic, non-glycinergic neurons. In the cerebellar and vestibular nuclei individual Purkinje cell terminals innervated both the inhibitory and excitatory neurons. Both types of neurons were contacted not only by GABAergic Purkinje cell terminals but also by GABA-containing terminals that were not labelled for L7 and by non-GABAergic, non-glycinergic terminals that formed excitatory synapses. Glycine-containing terminals were relatively scarce (<2% of the GABA-containing terminals) and frequently contacted the larger non-GABAergic, non-glycinergic neurons. To summarize, Purkinje cell axons evoke their effects through different types of neurons present in the cerebellar and vestibular nuclear complex. The observation that individual Purkinje cells can innervate both excitatory and inhibitory neurons suggests that the excitatory cerebellar output system and the inhibitory feedback to the inferior olive are controlled simultaneously.     

gamma-Aminobutyric acid transporters in the cat periaqueductal gray: a light and electron microscopic immunocytochemical study

The gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) mediate GABA uptake into presynaptic axon terminals and glial processes, thus contributing to the regulation of the magnitude and duration of the action of GABA at the synaptic cleft. The aim of the present study was to investigate the expression of three high-affinity GABA transporters (GAT-1, GAT-2, and GAT-3) in the periaqueductal gray matter (PAG) of adult cats by using immunocytochemistry with affinity-purified antibodies. Light microscopic observations revealed GAT-1 immunoreactivity in punctate structures, particularly dense in the lateral portion of the dorsolateral PAG column. Weak GAT-2-immunopositive puncta were homogeneously distributed in the PAG. GAT-3 immunoreactivity was detected in each column of the PAG but was more intense in the dorsolateral PAG column and around the aqueduct. Electron microscopic studies showed GAT-1 immunoreactivity in distal astroglial processes, in unmyelinated and small myelinated axons, and in axon terminals making symmetric synapses on both PAG neurons and dendrites. GAT-2 immunoreactivity was present mostly in the form of patches of different sizes in the cytoplasm of neuronal elements like the perikarya and dendrites of PAG neurons, in myelinated and unmyelinated axons, and in the axon terminals forming both symmetric and asymmetric synapses. Labeling was also observed in nonneuronal elements. Astrocytic cell bodies and their distal processes as well as the ependymal cells lining the wall of the aqueduct showed patches of GAT-2 immunoreactivity. Electron microscopic observation revealed GAT-3 immunoreactivity exclusively in distal astrocytic processes adjacent to the somata of PAG neurons and in axon terminals making both symmetric and asymmetric synapses. The present results suggest that three types of termination systems of GABAergic transmission are present in the cat periaqueductal gray matter.