Immunohistochemical studies of localization and co-localization of glutamate, aspartate and GABA in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat

Localization and possible co-localization of glutamate, aspartate and GABA immunoreactivities was examined in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat by double antigen immunohistochemistry using diaminobenzidine and benzidine dihydrochloride in one series and double immunofluorescence labelling with rhodamine and fluorescein in a second series of animals. In three of these regions, retrosplenial granular cortex, anterior thalamic nuclei, and mammillary nuclei, glutamate immunoreactivity was co-localized with aspartate immunoreactivity in a majority of the projection neurons (pyramidal neurons, predominantly in layers V and VI in retrosplenial granular cortex; rounded polygonal multipolar neurons throughout the rostrocaudal extent of the anterior thalamic and mammillary nuclei). None of the cells showing glutamate and/or aspartate immunoreactivity in these regions also displayed GABA immunoreactivity, which was present in non-pyramidal cells in the retrosplenial granular cortex (chiefly in layers I–III) and in small numbers of cells within the anterior thalamic nuclei. In the thalamic reticular nucleus, in contrast, most neurons were immunoreactive for GABA and in the majority of these neurons glutamate (and/or aspartate) immunoreactivity was co-localized with GABA.     

Collateral axons of cholinergic pontine neurones projecting to midline, mediodorsal and parafascicular thalamic nuclei in the rat

The organization of collateral axons projecting from neurones in the pontine laterodorsal tegmental nucleus (LDTg) has been examined using combinations of retrograde neuronal tracers with immunocytochemical markers for the acetylcholine-synthesizing enzyme choline acetyltranferase (CHAT), focussing on projections to the midline, mediodorsal and parafascicular thalamic nuclei and the ventral tegmental area. 25–59% of LDTg neurones projecting to the mediodorsal nucleus provided collaterals to the midline nuclei. Virtually all (87–96%) of these double retrogradely labelled neurones appeared cholinergic. 9–18% of LDTg neurones projecting to the parafascicular nuclei also provided a collateral to the midline nuclei and 50–78% of these double retrogradely labelled neurones stained for CHAT. 26–29% of the single LDTg neurones which projected collaterals to both the mediodorsal and midline nuclei, were found to project a third collateral to the ventral tegmental area. These anatomical findings, taken together with functional evidence, suggest that cholinergic terminals arising from LDTg are involved in coordinating thalamic mechanisms of brain state control; and in regulating dopaminergic pathways, both directly and via the thalamus.     

The organization of thalamic neurons projecting to the premotor cortex and the caudate nucleus in the cat studied by a fluorescent retrograde double labeling technique

Thalamic neurons projecting to both the head of the caudate nucleus and the premotor cortex in the cat were studied by the retrograde fluorescent double labeling technique. After injections of Evans blue into the caudate nucleus, and diamidino-phenylindol into the premotor cortex, a small number of double labeled neurons appeared in the ventral anterior, ventral lateral, anteromedial, rhomboid, central dorsal, central lateral, central medial, paracentral and parafascicular nuclei, in addition to numerous single-labeled neurons. This indicates that some neurons in the thalamic nuclei send bifurcating axons to both the head of the caudate nucleus and the premotor cortex. The caudatal projections of these thalamic neurons are organized in a topical manner.     

The thalamic reticular nucleus of the adult rat: experimental anatomical studies

Summary The thalamic reticular nucleus (TRN) is a sheet-like nucleus partially enclosing the dorsolateral and anterior aspects of the thalamus and traversed by the thalamo-cortical and cortico-thalamic fibre systems. This paper describes the cellular and synaptic organization of the TRN in adult albino rats on the basis of LM and EM studies of normal animals and experimental animals with injections of horseradish peroxidase (HRP) and/or lesions in various parts of the brain. Particular attention was paid to the dorso-caudal part of the TRN, which establishes connections with visual centres.LM-HRP preparations show that the neurons of TRN project only to ipsilateral dorsal thalamus; no labelled cell bodies were found in TRN after injections into the cortex or any part of the brain stem caudal to the thalamus. Small injections into dorsal thalamus result in a small cluster of labelled neurons and an associated patch of terminal label in TRN. The dorso-caudal part of the nucleus projects to the dorsal lateral geniculate nucleus, the ventro-caudal part to the medial geniculate nucleus and a large part of the nucleus anterior to the areas associated with the geniculate nuclei projects to the ventrobasal nucleus. No evidence was found for a widespread distribution of reticulo-thalamic axons and the connections between TRN and the dorsal lateral geniculate nucleus and between TRN and the ventrobasal nucleus show a fine-grain topographical organization with more rostral and dorsal parts of TRN projecting to more rostral and dorsal parts of the dorsal lateral geniculate and ventrobasal nuclei.The neurons of TRN are variable in size (range of somal diametersc. 10–20 m), shape (cell bodies are most commonly ellipsoidal) and dendritic morphology (bitufted and bipolar arrangements most common), but no basis for subdividing them into more than one class was found with any of the techniques used. The cell body and dendrites are commonly aligned parallel to the surface of TRN and at right angles to the traversing fibre bundles. The dendrites do not branch extensively and are only moderately spinous. Long, hair-like spines corresponding to those described by Scheibel & Scheibel (1966) were not found: nor were dendritic bundles found to be as prominent in EM material as reported by these authors in LM-Golgi material. Plasma membranes of dendrites in small bundles and of contiguous somata were commonly in direct contact over large areas, but gap junctions between them were not seen.The neuropil of TRN is simple with three major axon terminal types.D-type terminals (about 56% of all terminals in visual TRN) have closely packed spherical synaptic vesicles (42 nm diameter);L-type terminals (about 31%) are paler, slightly larger and have less densely packed synaptic vesicles (46 nm diameter); both terminal types make Gray type 1 synaptic contacts on dendritic spines and dendritic shafts and rarely also on cell bodies and axon hillocks.F-type terminals (about 8%) contain flattened synaptic vesicles in a dark matrix and make Gray type 2 contacts with dendrites, cell bodies and axon hillocks. In visual TRN, D-type terminals (but not all) degenerate after ablation of ipsilateral visual cortex and L-type terminals (but not all) degenerate after lesion of ipsilateral dorsal lateral geniculate nucleus; the density of degenerating terminals is higher after cortical than after geniculate lesions. Indirect evidence suggests that F-type terminals may be (or may include) collaterals of reticulo-thalamic projection cells, but no evidence was found for a widespread or dense plexus of such collaterals.After injection of HRP into the dorsal lateral geniculate nucleus, labelled axon terminals in visual TRN (many clearly L-type) were found in synaptic contact with retrogradely labelled dendrites of reticulo-geniculate projection cells. When HRP injection was combined with ablation of ipsilateral visual cortex, degenerating axon terminals (most of them identifiable as D-type) were also found in synaptic contact with retrogradely-labelled dendrites of reticulo-geniculate projection cells.Thus, neurons of visual TRN in the rat receive monosynaptic, presumptively excitatory input from collaterals of cortico-geniculate and geniculo-cortical axons, and project in a topographically-organized manner to the ipsilateral dorsal lateral geniculate nucleus (where they make Gray type 2 GABAergic and presumptively inhibitory synaptic contacts chiefly with the dendrites of geniculo-cortical projection cells). A similar pattern of organization is seen in other parts of the TRN and these data are compatible with the view that the TRN (and the perigeniculate nucleus of the cat thalamus, which is similar in several respects to visual TRN) forms part of a negative feed-back system by which the activity of thalamo-cortical projection neurons is regulated.     

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.     

The second, intralaminar thalamo-cortical projection system

Summary In the marmoset (Callithrix jacchus), HRP and ³H-apo-HRP were injected into various cortical regions and the positions of labelled neurons in the non-specific, intralaminar thalamic nuclei (N. centralis and centre médian) were investigated. Although neuron populations projecting to the different cortical regions overlap widely, a coarse topology exists inasmuch as intralaminar neurons projecting to the posterior cortex were located more rostrally and those projecting to the anterior cortex were located more caudally in the intralaminar complex. With injections into nearby cortical regions of the parieto-temporal association cortex with HRP and ³H-apo-HRP, respectively, no double labelled cells were found in the intralaminar nuclei, although the fields of labelled cells completely overlapped. Also in the specific projection nuclei no double labelled cells were encountered. About 10–20% of the thalamo-cortical projection cells are located in the intralaminar nuclei. Some functional aspects of this second thalamo-cortical projection system are discussed.     

Cholinergic and non-cholinergic projections from the canine pontomesencephalic tegmentum (Ch5 area) to the caudal intralaminar thalamic nuclei

Summary The distribution and morphology of cholinergic and non-cholinergic neurons projecting to the caudal intralaminar thalamic nuclei from the Ch5 area in the dog were examined using a technique combining horseradish peroxidase (HRP) retrograde labeling with choline acetyltransferase (ChAT) immunocytochemistry. After processing for ChAT, cholinergic neurons were found primarily within the nucleus tegmenti pedunculopontinus (PPN) and the central tegmental tract (ctt). ChAT positive neurons were also located in the nucleus cuneiformis and among the fibers of the lateral lemniscus and medial longitudinal fasciculus. On the basis of immunocytochemical and cytoarchitectonic data, PPN was divided into two distinct cell groups — a compact cell group located dorsolateral to the brachium conjunctivum and a diffuse cell group intermingled among the fibers of the brachium conjunctivum. Tissue processed for WGA-HRP and ChAT following injections of lectin-conjugated horseradish peroxidase into either the centrum medianum (CM) or parafascicular (Pf) nucleus resulted in double labeled cholinergic projection neurons in both PPN and ctt. Injections which involved CM and the caudal part of the central lateral thalamic nucleus (CL) resulted in more retrogradely labeled neurons than did those injections involving Pf. Injections of CM and CL also resulted in more double labeled cells in the dorsolateral compact portion of PPN than did injections confined to Pf. In all cases a small number of cholinergic neurons located in the contralateral PPN were retrogradely labeled as well. A substantial number of retrogradely labeled neurons were not ChAT positive, and in some cases, comprised up to 27% of the total population of projection neurons. Measurements of cell soma areas indicated that cells comprising the general cholinergic population were mostly medium (300–600 m²) or large (>600 m²) in size. The majority of cholinergic projection neurons fell within the medium size category while the non-cholinergic projection neurons were significantly smaller than their cholinergic counterparts. The results of this study suggest that in the dog, Ch5 cholinergic neurons which project to the caudal intralaminar thalamic nuclei are medium in size and are located primarily within PPN and ctt. In addition, a parallel projection to the caudal intralaminar nuclei exists which originates from smaller, non-cholinergic neurons in these same regions. Based on the results of this study, it appears that cholinergic projections to intralaminar thalamic nuclei which in turn project to the neostriatum may be one of the pathways over which PPN can affect basal ganglia activity.     

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.     

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.     

Spinally projecting neurons in the dorsal column nuclei: Distribution,dendritic trees and axonal projections

The distribution, dendritic trees and axonal courses of spinally projecting cells in the dorsal column nuclei were studied after labelling by retrograde HRP transport. The region of densest distribution was at the base of the two nuclei and in the area between them, extending for about 2 mm caudally from the obex. Only very few cells were found inside the cell cluster regions of the nuclei, where their dendrites had a free stellate form. The great majority, lying between, deep, or rostral to the cluster regions, also had a stellate form, except where they impinged on the boundaries of the cluster regions or on other nuclear borders; the spread of dendrites was dramatically restricted at such boundaries, often leading to a fusiform appearance in transverse sections which however was not evident in the parasagittal plane. No justification was therefore found for subdividing the population on morphological grounds. Axons of these cells descended ipsilaterally in either the medial part of the dorsolateral fascicle or in the adjacent lateral part of the cuneate fascicle, at cervical levels, and probably in about equal numbers. Most axons destined for the DLF followed a deep caudolateral trajectory, while many destined for the DC had a more dorsal or lateral course. Collateral branches were seen within the nuclei but could not be followed far. The fact that few if any cells lying in the region of maximum distribution of the spinally projecting cells were labelled following injections of HRP into the thalamic ventroposterior nucleus emphasizes that they form a distinctive entity within this medullary nuclear complex, and that any axon branches they give into the contralateral brainstem must have some other destination than the VPL. Two other groups of neurons were labelled by HRP implants into the dorsal columns - one in the ventrolateral medullary reticular formation, and the other in the nucleus of the solitary tract.     

Unique presynaptic and postsynaptic roles of Group II metabotropic glutamate receptors in the modulation of thalamic network activity

The thalamic reticular nucleus (TRN) is a sheet of GABAergic neurons that project to other TRN neurons and to associated thalamocortical relay nuclei. The TRN receives glutamatergic synaptic inputs from cortex as well as reciprocal inputs from the collaterals of thalamocortical neurons. In addition to ionotropic glutamate receptors, metabotropic glutamate receptors (mGluRs) are present in the TRN circuitry. Using whole cell voltage clamp recordings, we pharmacologically characterized unique pre- and postsynaptic functions for Group II mGluRs (mGluR 2 and mGluR 3) within the TRN circuitry in ferrets. mGluR 2 was found on presynaptic cortical axon terminals in the TRN, where it reduced glutamate release, while mGluR 3 acted postsynaptically on TRN cells to increase membrane conductance. Using miniature inhibitory postsynaptic current analysis, we also found that picrotoxin-sensitive intra-TRN GABA-mediated neurotransmission was not affected by administration of a Group II mGluR agonist, indicating that neither mGluR 2 nor 3 acts on presynaptic GABA-containing terminals within the TRN. Because strong corticothalamic activation is implicated in abnormal thalamic rhythms, we used extracellular recordings in the lateral geniculate nucleus to study the effect of Group II mGluR agonists upon these slow oscillations. We induced approximately 3 Hz spike-and-wave discharge activity through corticothalamic stimulation, and found that such activity was reduced in the presence of the Group II mGluR agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268). These data indicate that Group II mGluR reduce the impact of corticothalamic excitation, and that they may be a useful target in the reduction of absence-like rhythms.     

Dual chemoarchitectonic lamination of the visual sector of the thalamic reticular nucleus

The chemoanatomical organization of the visual sector of the cat's thalamic reticular nucleus (TRN)—that is at the dorsal lateral geniculate nucleus (dLGN) and at the pulvinar nucleus (Pul)—was investigated with two novel cytoarchitectonic markers. The Wisteria floribunda agglutinin (WFA) binding reaction visualized the extracellular perineuronal net (PN) and the SMI 32 immunoreaction stained intracellular neurofilaments. Two distinct layers of the TRN could be detected, particularly by WFA- but also by SMI 32-staining. The outer tier outlined a canopy of labeling placed a bit detached from the diencephalon dorsolaterally, while the inner TRN tier is very tightly attached to the thalamic lamina limitans externa. The labeled neurons showed typically fusiform morphology with dendrites orienting in the plane of TRN. Additionally, these chemoarchitectural reactions identified a chain of structures in the ventral diencephalon connected to the TRN tiers. One stained string is formed by the subthalamic nucleus bound laterally to the peripeduncular nucleus extending further dorsolateral into the outer TRN tier. The other chain laced up the field of Forel, the zona incerta, the ventral LGN, the perigeniculate nucleus (PGN) and the previously-overlooked peripulvinar nucleus (PPulN) and so formed the inner TRN tier. In the third most distanced TRN tier, in the perireticular nucleus, a very few WFA-binding presenting neuron were found. In addition to the PN possessing TRN neurons, WFA-reactive presumable interneurons were also labeled within the visual thalamus. Following tracer injections into the feline Pul, two stripes of cells were retrogradely labeled in the neighboring visual TRN sector. The location of these reticular neurons coincided precisely with the chemoanatomically identified inner and outer TRN tiers. On the analogy of the PGN-TRN duality at the dLGN, the chemoanatomical and tract tracing findings strongly suggest a similar dual organization in the pulvinoprojecting TRN portion.     

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).     

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.     

Glutamate decarboxylase-immunoreactive neurons and terminals in the periaqueductal gray of the rat

The periaqueductal gray of 5 rats was processed for immunocytochemistry using an antiserum to glutamate decarboxylase. In both colchicine-pretreated (4 rats) and untreated (1 rat) animals, glutamate decarboxylase-positive cell bodies were present in all periaqueductal gray subdivisions, especially in the dorsal and ventrolateral subdivision. The perikaryal cross-sectional area of labelled neurons was smaller than that of periaqueductal gray projecting neurons retrogradely labelled with horseradish peroxidase in separate experiments. The morphology of glutamate decarboxylase-containing neurons resembled that of small polygonal, triangular and fusiform cells described in previous Golgi studies. Glutamate decarboxylase immunoreactivity was also observed in a large number of terminal-like structures, most of which were distributed close to the somata and dendrites of both glutamate decarboxylate-positive and -negative neurons. At all rostrocaudal levels the highest concentration of these elements was observed around the aqueduct. These results suggest that two sub-populations of neurons are present in the periaqueductal gray of rats, one consisting of small-sized glutamate decarboxylase-positive neurons (intrinsic neurons) and the other of large-sized glutamate decarboxylase-negative neurons (projecting neurons). Intrinsic circuits could be present between glutamate decarboxylase-positive and -negative neurons and between glutamate decarboxylase-positive neurons.     

Organization of serotoninergic projections from the raphé nuclei to the anterior thalamic nuclei in the rat: a combined retrograde tracing and 5-HT immunohistochemical study

We combined retrograde transport of horseradish peroxidase (HRP) with 5-hydroxytryptamine (5-HT) immunohistochemistry to study serotoninergic projections to the anterior thalamic nuclei (ATN) of the rat. Small iontophoretic injections of HRP into the anterodorsal thalamic nucleus resulted in double-labelled neurons predominantly in the ventromedial and also in the ventrolateral part of the ipsilateral dorsal raphé (DR). A smaller number of double-labelled neurons was also found in the dorsomedial part of the nucleus, predominantly ipsilaterally, and in the median raphé nucleus (MnR), close to the midline. After injection into the medial subdivision of the anteroventral thalamic nucleus, the pattern of labelling in DR and MnR was similar to that detected following injections into the anterodorsal thalamic nucleus. However, injection into the posterior subdivision of the anteroventral thalamic nucleus resulted in bilateral retrograde labelling of a few 5-HT-containing neurons in the dorsolateral part of the DR. Labelling in the ventromedial, ventrolateral and dorsomedial regions of DR and MnR was similar to that detected after injections into the medial subdivision of the anteroventral thalamic nucleus. After all injections into the ATN, double-labelled cells were found throughout the rostrocaudal extent of MnR and throughout the rostral two-thirds of DR. The caudal extension of DR was devoid of double-labelled cells. Although double-labelled cells were observed bilaterally in the dorsolateral part of the DR, the projection from DR to ATN was predominantly ipsilateral. These results show that there is an internal organization within DR such that subnuclei of the DR can be defined on the basis of their efferent projections to specific subdivisions of the ATN.     

GABA and glycine as putative transmitters in subcortical pathways to the pontine nuclei. A combined immunocytochemical and retrograde tracing study in the cat with some observations in the rat.

Using the retrograde tracers horseradish peroxidase-wheatgerm agglutinin and gold particles conjugated to wheatgerm agglutinin apo-horseradish peroxidase in combination with an antiserum against glutaraldehyde-fixed GABA, it was examined whether the pontine nuclei of the cat receive projections from GABA-like immunoreactive neurons in the brainstem, diencephalon, or deep cerebellar nuclei, contributing to the GABA-like immunoreactive fibre plexus previously demonstrated in the pontine nuclei [Brodal et al. (1988) Neuroscience 25, 27-45]. Following tracer injections that covered both the pontine nuclei and the reticular tegmental nucleus in two cats, it was found that 125 out of 1166 (10.7%) and 29 out of 294 (9.9%) retrogradely labelled neurons in the cerebellar nuclei were GABA-like immunoreactive. In the same two experiments only six out of 2029 (0.3%) and 10 out of 1398 (0.7%) retrogradely labelled neurons in the brainstem and diencephalon were GABA-like immunoreactive. Among the regions in the brainstem and diencephalon known to project to the pontine nuclei, double-labelled cells were seen in the reticular formation, the periaqueductal gray, and the nucleus praepositus hypoglossi, but not in the zona incerta or the anterior pretectal nucleus, regions that have been shown to contain glutamate decarboxylase-like immunoreactive neurons projecting to the pontine nuclei in the rat [Border et al. (1986) Brain Res. Bull. 17, 169-179]. In order to test whether this is due to species differences, the same experimental approach was used in the rat, and it was found that 54 out of 3249 (1.7%) retrogradely labelled neurons in the brainstem and diencephalon were double-labelled. Notably, in the zona incerta 2% of the retrogradely labelled cells were also GABA-like immunoreactive, and in the reticular formation there was a higher proportion of double-labelled cells than was found in the cat. Additional sources were identified, that may contribute to the GABA-like immunoreactive fibre plexus in the pontine nuclei of the rat. This, in conjunction with the previous finding that the pontine nuclei of the rat contain only very few putative GABAergic neurons [Border and Mihailoff (1985) Expl Brain Res. 59, 600-614; Brodal et al. (1988) Neuroscience 25, 27-45], lead to the suggestion that the GABA-like immunoreactive fibre plexus in the pontine nuclei of the rat is predominantly of extrinsic origin, possibly representing a mosaic of the terminal fields of several subcorticopontine projections.(ABSTRACT TRUNCATED AT 400 WORDS)     

Segregation of lemniscal inputs and motor cortex outputs in cat ventral thalamic nuclei: application of a novel technique

Summary A double labeling method that permits accurate delineation of the terminals of medial lemniscal fibers was used to determine whether thalamic neurons projecting to motor cortex in the cat are in a position to be contacted by such terminals. Thalamic neurons in the VL nucleus were retrogradely labeled by injections of fluorogold placed in the cytoarchitectonically defined area 4, while lemniscal axons and their terminal boutons were anterogradely labeled, in a Golgi-like manner, from injections of Fast Blue placed under physiological control in different parts of the contralateral dorsal column nuclei. In additional experiments, spinothalamic fibers were similarly labeled by injections of Fast Blue in the spinal cord. The results reveal that there is no significant overlap in the distributions of lemniscal terminals and motor cortex-projecting neurons and that no somata or proximal dendrites of motor cortex-projecting neurons are in a position to receive lemniscal terminals. Spinothalamic terminals, on the other hand, end in clusters around motor cortex-projecting neurons in the VL nucleus as well as in other nuclei and are a more likely route for short latency somatosensory inputs to the motor cortex.Abbreviations AD anterodorsal nucleus - AM anteromedial nucleus - AP area postrema - AV anteroventral nucleus - C cuneate nucleus - CeM central medial nucleus - CL central lateral nucleus - CM centre médian nucleus - EC external cuneate nucleus - G gracile nucleus - L limitans nucleus - LD lateral dorsal nucleus - LP lateral posterior nucleus - MGM magnocellular medial geniculate nucleus - MD mediodorsal nucleus - MTT mamillothalamic tract - MV medioventral nucleus - Pc paracentral nucleus - Pf parafascicular nucleus - Po posterior nuclei - R reticular nucleus - RF fasciculus retroflexus - S solitary nucleus - SG suprageniculate nucleus - T spinal trigeminal nucleus - VA ventral anterior nucleus - VIN vestibular nuclei - VL ventral lateral nucleus - VMb basal ventral medial nucleus - VMp principal ventral medial nucleus - VPL ventral posterior lateral nucleus - VPM ventral posterior medial nucleus - ZI zona incerta - 1,2,3a,3b,4 fields of cerebral cortex - C4, C5, C6 spinal cord segments - 5SP,5ST spinal trigeminal nucleus and tract - 10, 12 vagal and hypoglossal nuclei     

GABAergic projections from the thalamic reticular nucleus to the anteroventral and anterodorsal thalamic nuclei of the rat

We have studied GABAergic projections from the thalamic reticular nucleus to the anterior thalamic nuclei of the rat by combining retrograde labelling with horseradish peroxidase and GABA-immunohistochentistry. Small iontophoretic injections of the tracer into subnuclei of the anterior thalamic nuclear complex resulted in retrograde labelling of cells in the rostrodorsal pole of the ipsilateral thalamic reticular nucleus. All of these cells were also GABA-positive. The projections were topographically organized. Neurons located in the most dorsal part of the rostral reticular nucleus projected to the dorsal half of both the posterior subdivision and the medial subdivision of the anteroventral thalamic nucleus, and to the rostral portion of the anterodorsal thalamic nucleus. Immediately ventral to this group of neurons, but still within the dorsal portion of the reticular nucleus, a second group of neurons, extending from the dorsolateral to the dorsomedial edge of the nucleus, projected to the ventral parts of the posterior and medial subdivisions of the anteroventral nucleus. Following injection of tracer into the dorsal part of the rostral anteroventral nucleus, retrograde labelled GABA-containing cell bodies were also found in the ipsilateral anterodorsal nucleus.     

Collateral projections of trigeminal ganglion neurons to both the principal sensory trigeminal and the spinal trigeminal nuclei in the rat

Employing a combination of fluorescent retro grade double labelling and immunofluorescence histo chemistry for substance P (SP) and calcitonin gene-relat ed peptide (CGRP), we examined collateral projections from single neurons in the trigeminal ganglion (TG) of the rat to both the principal sensory trigeminal nucleus (Vp) and the oral, interpolar or caudal subnuclei of the spinal trigeminal nucleus (Vo, Vi or Vc). In the rats that were unilaterally injected with fast blue (FB) into the Vp and with diamidino yellow (DY) into the Vo, Vi or Vc, neurons labelled with FB and/or DY were observed in the TG ipsilateral to the injections. Of the labelled TG neurons, about 2% were double labelled with both trac ers in the rats that were injected with FB into the Vp and with DY into the Vo or Vi, and about 10% were double labelled in the rats that were injected with FB into the Vp and with DY into the Vc. The results indicate that TG neurons sending their axons to the Vp project, by way of axon collaterals, to the Vc more frequently than to the Vo or Vi.Some of the TG neurons double labelled with FB and DY exhibited SP-or CGRP-like immunoreactivity (LI): Of the TG neurons that were double labelled with FB injected into the Vp and with DY injected into the Vo, Vi or Vc, about 38%, 49% and 42%, respectively, displayed SP-LI, and about 54%, 58% and 59%, respectively, showed CGRP-LI. Some of the SP-or CGRP-LI TG neurons that were double labelled with FB and DY were assumed to mediate pain signals to both the Vp and the spinal trigeminal nucleus (Vo, Vi and/or Vc) by way of axon collaterals.Yun-Qing Li is on leave from the Department of Anatomy, The Fourth Military Medical University, Xian, People's Republic of China