Projections of brainstem core cholinergic and non-cholinergic neurons of cat to intralaminar and reticular thalamic nuclei

We combined the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry to study the projections of cholinergic and non-cholinergic neurons of the upper brainstem core to rostral and caudal intralaminar thalamic nuclei, reticular thalamic complex and zona incerta in the cat. After wheat germ agglutinin-horseradish peroxidase injections in the rostral pole of the reticular thalamic nucleus, the distribution and amount of retrogradely labeled brainstem neurons were similar to those found after tracer injection in thalamic relay nuclei (see preceding paper). After wheat germ agglutinin-horseradish peroxidase injections in the caudal intralaminar centrum medianum-parafascicular complex, rostral intralaminar central lateral-paracentral wing, and zona incerta, the numbers of retrogradely labeled brainstem neurons were more than three times higher than those found after injections in thalamic relay nuclei. The larger numbers of horseradish peroxidase-positive brainstem reticular neurons after tracer injections in intralaminar or zona incerta injections results from a more substantial proportion of labeled neurons in the central tegmental field at rostral midbrain (perirubral) levels and in the ventromedial part of the pontine reticular formation, ipsi- and contralaterally to the injection site. Of all retrogradely labeled neurons in the caudal midbrain core at the level of the cholinergic peribrachial area and laterodorsal tegmental nucleus, 45-50% were also choline acetyltransferase-positive after the injections into central lateral-paracentral and reticular nuclei, while only 25% were also choline acetyltransferase-positive after the injection into the centrum medianum-parafascicular complex. These findings are discussed in the light of physiological evidence of brainstem cholinergic mechanisms involved in the blockade of synchronized oscillations and in activation processes of thalamocortical systems.     

GABA-containing neurons in the pontine nuclei of rat, cat and monkey. An immunocytochemical study

Putative GABAergic elements in the pontine nuclei have been studied in the rat, cat and two old world monkeys (Macaca mulatta and Papio papio) using an antiserum against GABA-glutaraldehyde-protein conjugates and the peroxidase-antiperoxidase method. In addition, an antiserum against glutamate decarboxylase has been used in the cat. For comparison, Golgi impregnated material from cat and macaque has been studied. In all species there is a moderately dense plexus of fibres with GABA-like immunoreactivity with only minor regional differences between different parts of the pontine nuclei. The number of cell bodies showing GABA-like immunoreactivity is, however, strikingly different. Thus, in the rat there are very few such neurons. In the cat, they make up about 1% of the total cell population, while the corresponding number in the two primate species is about 5%. The number is consistently somewhat higher in rostral than in caudal parts of the pontine nuclei. Numbers in the cat are essentially the same with the glutamate decarboxylase antiserum as with the GABA antiserum. The size of GABA-like immunoreactivity positive somata is very similar in cat, macaque and baboon, averaging about 160 micron2 in cross-sectional area. The average cross-sectional area of the total neuronal population as measured in adjacent thionin-stained sections is about 280 micron2. However, the range of sizes for GABA-like immunoreactivity positive cells is wide, so that size alone is not a good criterion for their identification. Although their dendritic morphology is varied, a significant proportion of GABA-like immunoreactivity positive cells have very long and straight dendrites. A few examples were found in the primate species of GABA-like immunoreactivity positive cells with processes tentatively identified as axons. Such processes could be seen to divide several times. No such branching processes could be identified, however, in Golgi impregnated material from the same species. In order to determine whether GABA-like immunoreactivity positive cells project to the cerebellum, retrograde tracing of horseradish peroxidase-wheat germ agglutinin was combined with immunocytochemistry. No double labelled cells could be found in the pontine nuclei. Comparison of size distribution of retrogradely labelled pontocerebellar and GABA-like immunoreactivity positive cell bodies showed a high degree of overlap, although the average size of projection neurons and GABA-like immunoreactivity positive ones is clearly different.(ABSTRACT TRUNCATED AT 400 WORDS)     

Topographic localization of neuromedin U-like structures in the rat brain: an immunohistochemical study

The distribution of neuromedin Us, uterus-stimulating and hypertensive peptides newly identified in porcine spinal cord, was examined in the rat brain by the indirect immunofluorescent method. Neuromedin U-like immunoreactive structures were found to be unevenly distributed in the neuronal system. Neuromedin U-like immunoreactive neurons were present in the cranial motor nuclei, reticular nuclei, nucleus vestibularis lateralis, trigeminal sensory nuclei, colliculus superior and inferior, lemniscus lateralis, nucleus pontis, nucleus ruber, zona incerta, substantia innominata, horizontal limb of the diagonal band and cerebral cortex. The immunoreactive fibres were found in the above areas, particularly near the labelled cells, forming a fibre plexus with various intensities of immunoreactivity. In addition, dense plexuses were also seen in the nucleus reticularis thalami, nucleus ventralis posteromedialis, nucleus ventralis posterolateralis, nucleus tegmentalis dorsalis and ventralis, vertical limb of the diagonal band, nucleus olivaris superior, and nucleus pontis. In the first six structures, no labelled neurons were present and in the remaining structures, a few scattered neurons were noted. This indicates that these fibres are probably of extrinsic origin.     

The pathways connecting the hippocampal formation, the thalamic reuniens nucleus and the thalamic reticular nucleus in the rat

Most dorsal thalamic nuclei send axons to specific areas of the neocortex and to specific sectors of the thalamic reticular nucleus; the neocortex then sends reciprocal connections back to the same thalamic nucleus, directly as well indirectly through a relay in the thalamic reticular nucleus. This can be regarded as a 'canonical' circuit of the sensory thalamus. For the pathways that link the thalamus and the hippocampal formation, only a few comparable connections have been described. The reuniens nucleus of the thalamus sends some of its major cortical efferents to the hippocampal formation. The present study shows that cells of the hippocampal formation as well as cells in the reuniens nucleus are retrogradely labelled following injections of horseradish peroxidase or fluoro-gold into the rostral part of the thalamic reticular nucleus in the rat. Within the hippocampal formation, labelled neurons were localized in the subiculum, predominantly on the ipsilateral side, with fewer neurons labelled contralaterally. Labelled neurons were seen in the hippocampal formation and nucleus reuniens only after injections made in the rostral thalamic reticular nucleus (1.6-1.8 mm caudal to bregma). In addition, the present study confirmed the presence of afferent connections to the rostral thalamic reticular nucleus from cortical (cingulate, orbital and infralimbic, retrosplenial and frontal), midline thalamic (paraventricular, anteromedial, centromedial and mediodorsal thalamic nuclei) and brainstem structures (substantia nigra pars reticularis, ventral tegmental area, periaqueductal grey, superior vestibular and pontine reticular nuclei). These results demonstrate a potential for the thalamo-hippocampal circuitry to influence the functional roles of the thalamic reticular nucleus, and show that thalamo-hippocampal connections resemble the circuitry that links the sensory thalamus and neocortex.     

Substance P-containing neurons in the pontomesencephalic tegmentum of the human brain.

We have employed immunohistochemical and computerized morphometric procedures to study substance P-containing neurons in the tegmentum of adult humans. An estimated 192,500 +/- 40,500 substance P-containing neurons were found in three main cytoarchitectural regions: the mesencephalic reticular formation, the central gray, and the pontine reticular formation. The morphology of the immunoreactive neurons varied according to the region in which they were found. On the basis of size alone two types of substance P-containing neurons, large and small, were readily distinguishable by eye and measurement. Within each of the three main regions it was possible to distinguish distinct subgroups using cell size, morphology and position. Large neurons were concentrated in the caudal midbrain (pedunculopontine tegmental nuclei), in the oral pontine reticular nucleus and in the lateral dorsal tegmental nucleus. In contrast, small neurons were concentrated in the rostral mesencephalic reticular formation (cuniform nuclei). Both small and large neurons were found in the midbrain and pontine raphe nuclei. In addition, small neurons were concentrated in discrete midline regions (the periaqueductal gray, the tegmental nuclei of the pontine central gray, and the interpeduncular nucleus). The findings suggest that the majority of neurons in the brainstem tegmental nuclei previously identified as cholinergic also contain substance P in humans.     

Localization of amino acids, neuropeptides and cholinergic neurotransmitter markers in identified projections from the mesencephalic tegmentum to the mammillary nuclei of the rat

Retrograde labelling has been combined with immunohistochemistry to localize neurons containing GABA, glutamate, choline acetyltransferase, leu-enkephalin, neurotensin and substance P-like immunoreactivity in the projection pathways from the midbrain tegmental nuclei to the mammillary nuclei in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the medial mammillary nucleus resulted in retrogradely labelled neurons in the ventral tegmental nucleus of Gudden, whereas injections into the lateral mammillary nucleus resulted in large numbers of retrogradely labelled neurons in the ipsilateral dorsal tegmental nucleus of Gudden and in the laterodorsal tegmental nucleus. In the ventral tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also immunolabelled for GABA and approximately ten to 18 WGA-HRP-labelled neurons per section were immunoreactive for leu-enkephalin. In addition, small numbers of WGA-HRP-labelled neurons in the principal subnucleus of the ventral tegmental nucleus were immunoreactive for Glu whereas small numbers of retrogradely labelled neurons in the compact subnucleus of the central superior nucleus displayed neurotensin-like immunoreactivity. In the ventral subnucleus of the dorsal tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also GABA-immunoreactive and approximately ten to 14 WGA-HRP labelled neurons per section were immunoreactive for leu-enkephalin. The ventral subnucleus of the dorsal tegmental nucleus also contained small numbers of retrogradely labelled neurons that displayed either glutamate or substance P-like immunoreactivity. In addition, moderate to small numbers of WGA-HRP-labelled neurons (five to 20 per section) in the laterodorsal tegmental nucleus were immunoreactive for choline acetyltransferase. These results are compatible with the possibility that tegmentomammillary projection neurons use several different neurochemicals as neurotransmitter(s) and/or neuromodulator(s).     

Afferent projections to the periaqueductal gray in the rabbit

The afferents to the periaqueductal gray in the rabbit have been described following hydraulic pressure injection of horseradish peroxidase at various sites throughout this structure. Every third section was reacted with tetramethylbenzidine, for the localization of afferent neurons. At the site of the deposit alternate sections were reacted with tetramethylbenzidine, Hanker-Yates reagent, or diaminobenzidine, for comparative assessment of the injection site. A large number of retrogradely labelled cells, assessed by bright- and dark-field microscopy, were observed in a wide range of areas throughout the brain. Major labelled areas within the telencephalon were cortical areas 5, 20, 21, 32 and 40. Within the diencephalon, the hypothalamus contained quantitatively by far the largest number of labelled cells. Of these nuclei, the dorsal pre-mammillary nucleus contained the largest number of labelled cells. Considerable labelling was also found within medial and lateral preoptic nuclei, anterior hypothalamic area, and ventromedial hypothalamic nucleus. Another diencephalic region containing a significant number of retrogradely labelled neurons was the zona incerta. At midbrain, pontine and medullary levels, additional labelled regions were: the substantia nigra, cuneiform nucleus, parabigeminal nucleus, raphe magnus, and reticular areas. Heavy labelling was seen within the periaqueductal gray itself, rostral and caudal to deposits placed within each subdivision. In addition, a large number of other areas labelled throughout the brain (Tables 2A-D). Not only were some differences noted in the pattern of labelled cells with deposits placed rostrally or caudally within periaqueductal gray, but certain topographical differences with respect to the degree of labelling within nuclei were also seen with injection sites ventral, lateral or dorsal to the aqueduct. In addition, a further difference was noted, in that over one third of the areas labelled with deposits in just one or other of the "divisions" within periaqueductal gray. The results therefore suggest that the periaqueductal gray might be divisible to some extent on the basis of connectivity with intrinsic subdivisions of the complex. It is hoped that, with time, it might prove possible to resolve any such differential input in functional terms. The wide variety of afferent input to the periaqueductal gray, and its strategic location, would seem to place it in a unique position for integrating and modifying a diversity of motor, autonomic, hormonal, sensory and limbic influences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Does the reticular thalamic nucleus project to the midbrain?

In this study, we investigated whether the reticular thalamic nucleus has a projection to major centres of the midbrain in rats, rabbits and cats. Various tracers (biotinylated dextran, cholera toxin B subunit, fluorescent latex beads) were injected either into the midbrain tectum (deep layers of the superior colliculus) or tegmentum (midbrain reticular and pedunculopontine nuclei). In other experiments, different coloured latex beads (red and green) were injected into the deep layers of the superior colliculus and into the midbrain reticular nucleus of the same animal (rabbits). Our major finding is that in rats, rabbits and cats, there are no retrogradely labelled cells in the reticular thalamic nucleus after tracer injections into the abovementioned midbrain centres. In rabbits and cats, however, there are retrogradely labelled cells lying close to the ventromedial edge of the reticular thalamic nucleus after such injections. We show, by means of immunocytochemical double-labelling, that these retrogradely labelled cells do not lie in the reticular thalamic nucleus as suggested by previous studies, but in the inner small-celled region, a group of small cells that forms part of the zona incerta. Although there appears to be no clear topography of projection of the inner small-celled region, our tracer double-labelling experiments show that separate cells in the inner small-celled region project to individual centres of the midbrain (i.e., there are very few double-labelled cells after double injections). In rats, unlike in rabbits and cats, there is no clearly defined inner small-celled region and there are no retrogradely labelled cells seen along the ventromedial edge of the reticular thalamic nucleus. Our results suggest that in rats, rabbits and cats, there is no projection of the reticular thalamic nucleus to major centres of the midbrain, suggesting that the nucleus may not have a very strong influence on midbrain function, as it does on dorsal thalamic function.     

Neurotransmitter characteristics of neurons projecting to the supramammillary nucleus of the rat

Retrograde labelling was combined with immunohistochemistry to localize neurons containing choline acetyltransferase, gamma-aminobutyric acid (GABA), glutamate, serotonin, somatostatin, Leu-enkephalin, neurotensin, and substance P-immunoreactivity in neurons projecting to the supramammillary nucleus in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the supramammillary nucleus resulted in retrogradely labelled neurons in the medial septal nucleus, the nuclei of the diagonal band of Broca, the infralimbic cortex, the medial and lateral preoptic nucleus, the subiculum, the laterodorsal tegmental nucleus, the compact subnucleus of the central superior nucleus and the dorsal raphe nucleus. In the medial septal nucleus and in the nuclei of the diagonal band of Broca, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for choline acetyltransferase and small numbers of WGA-HRP-labelled neurons were immunoreactive for GABA, glutamate, neurotensin or substance P. In the medial preoptic nucleus, 85-90% of retrogradely labelled neurons (25-30 per section) were immunoreactive for somatostatin and a few WGA-HRP-labelled neurons displayed neurotensin-immunoreactivity. In the rostroventral part of the subiculum, small numbers of retrogradely labelled neurons were also immunoreactive for neurotensin or for glutamate. In the laterodorsal tegmental nucleus, 90% of WGA-HRP-labelled neurons (20-25 per section) were immunoreactive for choline acetyltransferase and small numbers of retrogradely labelled neurons also displayed substance P immunoreactivity. In the compact subnucleus of the central superior nucleus, 50-60% of retrogradely labelled neurons (15-20 per section) were also immunolabelled for GABA and approximately 30-40% of WGA-HRP-labelled neurons (10-12 per section) were immunoreactive for Leu-enkephalin. The compact subnucleus of the central superior nucleus also contained small numbers of retrogradely labelled neurons that displayed neurotensin immunoreactivity. In the dorsal raphe nucleus, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for serotonin and small numbers of retrogradely labelled neurons displayed neurotensin or glutamate immunoreactivity. These results suggest that the multiple neurochemicals contained in ascending and descending projections to the SuM participate in complex interactions in the transmission process of SuM neurons.     

An autoradiographic analysis of ascending projections from the medullary reticular formation in the rat

Ascending projections from the several nuclei of the medullary reticular formation were examined using the autoradiographic method. The majority of fibers labeled after injections of [3H]leucine into nucleus gigantocellularis ascended within Forel's tractus fasciculorum tegmenti which is located ventrolateral to the medial longitudinal fasciculus. Nucleus gigantocellularis injections produced heavy labeling in the pontomesencephalic reticular formation, the intermediate layers of the superior colliculus, the pontine and midbrain central gray, the anterior pretectal nucleus, the ventral midbrain tegmentum including the retrorubral area, the centromedian-parafascicular complex, the fields of Forel/zona incerta, the rostral intralaminar nuclei and the lateral hypothalamic area. Nucleus gigantocellularis projections to the rostral forebrain were sparse. Labeled fibers from nucleus reticularis ventralis, like those from nucleus gigantocellularis, ascended largely in the tracts of Forel and distributed to the pontomedullary reticular core, the facial and trigeminal motor nuclei, the pontine nuclei and the dorsolateral pontine tegmentum including the locus coeruleus and the parabrachial complex. Although projections from nucleus reticularis ventralis diminished significantly rostral to the pons, labeling was still demonstrable in several mesodiencephalic nuclei including the cuneiform-pedunculopontine area, the mesencephalic gray, the superior colliculus, the anterior pretectal nucleus, the zona incerta and the paraventricular and intralaminar thalamic nuclei. The main bundle of fibers labeled by nucleus gigantocellularis-pars alpha injections ascended ventromedially through the brainstem, just dorsal to the pyramidal tracts, and joined Forel's tegmental tract in the midbrain. With the brainstem, labeled fibers distributed to the pontomedullary reticular formation, the locus coeruleus, the raphe pontis, the pontine nuclei, and the dorsolateral tegmental nucleus and adjacent regions of the pontine gray. At mesodiencephalic levels, labeling was present in the rostral raphe nuclei (dorsal, median and linearis), the mesencephalic gray, the deep and intermediate layers of the superior colliculus, the medial and anterior pretectal nuclei, the ventral tegmental area, zona incerta as well as the mediodorsal and reticular nuclei of the thalamus. Injections of the parvocellular reticular nucleus labeled axons which coursed through the lateral medullary tegmentum to heavily innervate lateral regions of the medullary and caudal pontine reticular formation, cranial motor nuclei (hypoglossal, facial and trigeminal) and the parabrachial complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

The site of origin of calcitonin gene-related peptide-like immunoreactive afferents to the inferior olivary complex of the mouse.

The intent of the present study is to define the brainstem nuclei which give rise to CGRP-immunolabeled afferents to the inferior olivary complex of the mouse. A technique which combines retrograde transport of fluorescent microspheres with immunohistochemistry was used to address this question. In the present study, intensely labeled CGRP neurons were localized within several cranial nerve nuclei including the hypoglossal, facial, oculomotor, motor nucleus of the trigeminal nerve and nucleus ambiguus, as well as in the parabrachial nucleus, locus coeruleus and medullary and pontine reticular formation. In addition, lightly labeled CGRP neurons were identified within the deep cerebellar nuclei, the inferior olivary complex, lateral reticular nucleus, medial and lateral vestibular nuclei, nucleus Darkschewitsch, interstitial nucleus of Cajal, the central gray area adjacent to the third ventricle, and the zona incerta. The origin of the projection to the inferior olivary complex primarily arises from the deep cerebellar nuclei, the locus coeruleus, and the central gray matter of the mesodiencephalic area. In addition, a small CGRP input is derived from the superior and lateral vestibular nuclei as well as the zona incerta. In conclusion, we have identified several extrinsic sources of CGRP to the inferior olivary complex and have localized it within afferents that have been shown to have either excitatory (mesodiencephalic nuclei) or inhibitory (cerebellar nuclei) effects on olivary circuits. The presence of CGRP in these functionally diverse brainstem and cerebellar afferents suggests that the peptide may act as a co-transmitter to modulate the activity of olivary neurons.     

The cerebellar projection from the reticular formation of the brain stem in the rabbit

Summary By retrograde transport of horseradish peroxidase the reticulocerebellar projections were examined in twenty-six rabbits.After injections in the cerebellum retrogradely labeled neurons were more numerous in the caudal reticular formation (ventral and gigantocellular reticular nuclei) than in its rostral part (caudal and oral pontine reticular nuclei). The labeled cells were of all sizes, large, medium-sized and small. Giant cells were labeled only after injections in the posterior lobe vermis.After injections in the anterior lobe, the posterior vermis, the fastigial nucleus and the flocculus, retrogradely labeled neurons were found bilaterally in the ventral reticular nucleus, the gigantocellular reticular nucleus and the caudal pontine reticular nucleus. Some cases with posterior vermal and fastigial injections in addition showed labeled neurons bilaterally in the oral pontine reticular nucleus. There were no major side differences. The cases with injections in the anterior part of the paramedian lobule gave rise to only a few labeled cells in the gigantocellular reticular nucleus.Negative findings were consistently made in the mesencephalic reticular formation.     

Thalamic distribution of zinc-rich terminal fields and neurons of origin in the rat

Several cortico-cortical and limbic-related circuits are enriched in zinc, which is considered as an important modulator of glutamatergic transmission. While heavy metals have been detected in the thalamus, the specific presence of zinc has not been examined in this region. We have used two highly sensitive variations of the Timm method to study the zinc-rich innervation in the rat thalamus, which was compared to the distribution of acetylcholinesterase activity. The origin of some of these zinc-rich projections was also investigated by means of retrograde transport after intracerebral infusions of sodium selenium (Na2SeO3). The overall zinc staining in the thalamus was much lower than in the neocortex, striatum or basal forebrain; however, densely stained terminal fields were observed in the dorsal tip of the reticular thalamic nucleus, the anterodorsal and lateral dorsal thalamic nuclei and the zona incerta. In addition, moderately stained zinc-rich terminal fields were found in the rostral intralaminar nuclei, nucleus reuniens and lateral habenula. Intracerebral infusions of Na2SeO3 in the lateral dorsal nucleus resulted in retrogradely labeled neurons that were located in the postsubiculum, and also in the pre- and parasubiculum. These results are the first to establish the existence of a zinc-rich subicular-thalamic projection. Similar infusions in either the intralaminar nuclei or the zona incerta resulted in labeling of neurons in several brainstem structures related to the reticular formation. Our results provide morphological evidence for zinc modulation of glutamatergic inputs to highly selective thalamic nuclei, arising differentially from either cortical limbic areas or from brainstem ascending activation systems.     

Glutamate immunoreactivity in the rat basilar pons: light and electron microscopy reveals labeled boutons and cells of origin of afferent projections.

Immunohistochemical methods that employed a polyclonal antiserum directed against a glutamate-hemocyanin conjugate were utilized to examine the rat basilar pontine nuclei at both light and electron microscopic levels in order to identify putative glutamatergic neural elements. A large number of cells ranging in size from 11 to 32 microns in diameter and present in all subdivisions and at all rostrocaudal levels of the basilar pons exhibited intense glutamate immunoreactivity. Immunoreactive punctate structures, confirmed by electron microscopy to be axon terminals, were homogeneously distributed throughout the pontine neuropil, although a somewhat greater accumulation was apparent medially at mid-levels of the basilar pons and laterally at more caudal levels. Immunolabeled axons were also present throughout the pontine nuclei. In order to demonstrate possible extrinsic sources of glutamate-immunoreactive axon terminals within the pontine gray, injections of wheat germ agglutinin-horseradish peroxidase were made directly into the basilar pons. Tissue was then evaluated for the presence of retrogradely transported wheat germ agglutinin-horseradish peroxidase and the same tissue sections processed for glutamate immunocytochemistry. Following this combined protocol, neuronal somata exhibiting both wheat germ agglutinin-horseradish peroxidase and glutamate immunoperoxidase reaction products were observed within layer Vb of the cerebral cortex, zona incerta, the dentate nucleus of the cerebellum, nucleus paragigantocellularis of the medullary reticular formation, and the dorsal column nuclei. Such double-labeled cells were considered to represent glutamatergic neurons that provide axonal projections to the basilar pons. Ultrastructural studies of the pontine nuclei confirmed the presence of glutamate immunogold labeling in dendrites, neuronal somata, axons, and axon terminals. Immunoreactive boutons contained round vesicles and primarily formed asymmetric synapses at various postsynaptic loci which included glutamate-immunolabeled dendritic profiles and somata. These results suggest that glutamatergic basilar pontine neurons form one segment of a multisynaptic pathway involving glutamatergic afferents to the basilar pons, glutamatergic pontocerebellar projection neurons, and the glutamatergic granule cells of the cerebellar cortex.     

Organization of afferent connections to the lateral and interpositus cerebellar nuclei from the brainstem relay nuclei: a horseradish peroxidase study in the cat

Afferent projections to the lateral (dentate) and interpositus cerebellar nuclei from the brainstem relay nuclei were studied in cats using the horseradish peroxidase (HRP) method. In the first series of experiments, HRP was injected into the brachium pontis. Mossy fiber terminals were anterogradely labeled, predominantly in the lateral (hemispherical) part, moderately in the intermediate part, and slightly in the vermal part of the cerebellum. Besides these terminals in the cerebellar cortex, axon terminals labeled anterogradely were also found in the cerebellar nuclei. The labeled terminals appeared almost exclusively in the lateral nucleus and rarely in the interpositus nucleus. Cells labeled retrogradely were found both in the pontine nuclei and the tegmental reticular nucleus, but not in other brainstem nuclei. In the second series of experiments, HRP was injected into the lateral and interpositus nuclei, and retrograde labeling was examined in the brainstem relay nuclei. After HRP injection into the lateral nucleus, the number of labeled cells was significantly large in the pontine nuclei, but fairly small in the reticular or vestibular nuclei. The number of labeled cells was generally large in the inferior olive, mainly in the principal olive. After HRP injection into the interpositus nucleus, the number of labeled cells was moderate in the reticular or vestibular nuclei, but small in the pontine nuclei. The number of labeled cells in the inferior olive was also large, being distributed mainly in the accessory olives. These results indicate that the pontine nuclei and the principal olive provide major afferent inputs to the lateral nucleus, whereas the reticular nuclei, the vestibular nuclei and the accessory olives are the major afferent sources to the interpositus nucleus.     

Electron microscopic data on the neurons of nuclei subpretectalis and posterior-ventralis thalami. A combined immunohistochemical study

 The nucleus rotundus receives GABA-like immunoreactive fibres from the nuclei subpretectalis and postero-ventralis thalami. This result was confirmed by Phaseolus vulgaris leucoagglutinin (PhA-L) anterograde tracer and with electron microscopic (EM) γ-aminobutiric acid (GABA)-immunogold staining. The detailed electron microscopic analysis of the structure of the neurons in these nuclei revealed that the neurons in the nucleus subpretectalis displayed GABA-like immunoreactivity. In the postero-ventral thalamic nucleus a group of neurons was GABA-positive. The surface of the neurons was covered both with numerous GABA-negative and GABA-like immunoreactive terminals that established asymmetrical and symmetrical synapses, respectively, with the GABA-positive neurons. The GABA-like immunonegative terminals are supposed to be the axon terminals of the collaterals of tecto-rotundal fibres in the subpretectal nucleus and the collateral terminal branches of contralateral tecto-rotundal fibres in the postero-ventralis thalami. In both nuclei, the GABA-like immunoreactive terminals may be developed by the collaterals of local neurons that establish symmetrical synapses. In the Phaseolus lectin-stained preparations these terminals may be labelled. The morphological characteristics of the neurons in the subpretectal and partly, in the postero-ventral nuclei are similar to those of interneurons (local circuit neurons) and the numerous asymmetrical and symmetrical axo-somatic synapses, respectively. But these neurons locate outside of their target nucleus, and exert their modulatory effect on rotundo-ectostriatal transmission. Also, a contralateral influence is present in the nucleus rotundus that may interact in the cooperation of the eyes. The neurons of the subpretectal and postero-ventral nuclei, similarly to the neurons of isthmic nuclei, are a special group of modulatory neurons with effects at a distance. Accepted: 28 July 1998     

Distributions and colocalization of neuropeptide Y and somatostatin in the goldfish brain.

The distributions of single- and double-labelled neuropeptide Y- (NPY-) and somatostatin-immunoreactive (SOM-IR) perikarya and processes were determined in the goldfish brain using immunoperoxidase and immunofluorescence techniques, respectively. In double-labelled material, it was evident that although these two peptides showed markedly similar distributions, they were colocalized in very few instances. A high degree of colocalization of NPY and SOM was noted in the neurons of the ventrolateral telencephalon (VI), the entopenduncular nucleus (NE) and, to a lesser extent, in the dorsocentral nucleus of the telencephalon (Dc). In Vl and NE, neurons showing NPY-IR displayed SOM-IR and vice versa. The only other instance of colocalization was that noted in the brainstem, where SOM and NPY were colocalized in the large cell bodies of the medial column of the vagal motor complex. Single-labelled SOM- and NPY-IR neurons shared a very similar distribution in various nuclei in the diencephalon and in the optic tectum. Colocalization was also noted within fibers throughout many nuclei of the telencephalon and within fibers innervating the swim bladder, one of the peripheral organs to which neurons of the medial column of the vagal motor complex project. Processes in the torus semicircularis and vagal lobe showed single-labelled immunoreactivity for both SOM and NPY in distinct laminar patterns. Large single-labelled SOM-IR terminals appeared to form pericellular baskets in the eminentia granularis of the cerebellum. Single-labelled NPY- or SOM-IR fibers were also found in the secondary gustatory nucleus and tract, the facial lobe, descending trigeminal tract, reticular formation and spinal cord. As in mammalian species, select groups of neurons in teleosts colocalize the neuropeptides SOM and NPY.     

Immunocytochemical localization of D-amino acid oxidase in rat brain

d-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.     

The organization of preoptic-medullary circuits in the male rat: evidence for interconnectivity of neural structures involved in reproductive behavior, antinociception and cardiovascular regulation.

The present studies used anatomical tract-tracing techniques to delineate the organization of pathways linking the medial preoptic area and the ventral medulla, two key regions involved in neuroendocrine, autonomic and sensory regulation. Wheatgerm agglutinin-horseradish peroxidase injections into the ventromedial medulla retrogradely labeled a large number of neurons in the medial preoptic area, including both the median and medial preoptic nuclei. The termination pattern of preoptic projections to the medulla was mapped using the anterograde tracers Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine. Tracer injections into the preoptic area produced a dense plexus of labeled fibers and terminals in the ventromedial and ventrolateral pons and medulla. Within the caudal pons/rostral medulla, medial preoptic projections terminated heavily in the nucleus raphe magnus; strong anterograde labeling was also present in the pontine reticular field. At mid-medullary levels, labeled fibers focally targeted the nucleus paragigantocellularis, in addition to the heavy fiber labeling present in the midline raphe nuclei. By contrast, very little labeling was observed in the caudal third of the medulla. Experiments were also conducted to map the distribution of ventral pontine and medullary neurons that project to the medial preoptic area. Wheatgerm agglutinin-horseradish peroxidase injections in the preoptic area retrogradely labeled a significant population of neurons in the ventromedial and ventrolateral medulla. Ascending projections from the medulla to the preoptic area were organized along rostral-caudal, medial-lateral gradients. In the caudal pons/rostral medulla, retrogradely labeled cells were aggregated along the midline raphe nuclei; no retrograde labeling was present laterally at this level. By contrast, in the caudal half of the medulla, cells retrogradely labeled from the medial preoptic area were concentrated as a discrete zone dorsal to the lateral reticular nucleus; labeled cells were not present in the ventromedial medulla at this level. The present findings suggest that the medial preoptic area and ventral midline raphe nuclei share reciprocal connections that are organized in a highly symmetrical fashion. By contrast, preoptic-lateral medullary pathways are not reciprocal. These preoptic-brainstem circuits may participate in antinociceptive, autonomic and reproductive behaviors.