Ultrastructure of ChAT-immunoreactive synaptic terminals in the thalamic reticular nucleus of the rat.

The thalamic reticular nucleus has been shown to receive cholinergic innervation from both the nucleus basalis of Meynert in the forebrain and the pedunculopontine and laterodorsal tegmental nuclei in the brainstem (Steriade et al.: Brain Res. 408:372-376, '87; Levey et al.: Neurosci. Lett. 74:7-13, '87). Relatively dense populations of choline acetyltransferase-(ChAT) immunoreactive axons and terminallike varicosities have been shown to be distributed throughout this nucleus (Levey et al.: J. Comp. Neurol. 257:317-332, '87). In this study, the ultrastructure of ChAT-immunoreactive axons and of their synaptic terminals in the reticular nucleus was examined in the electron microscope. All ChAT-immunoreactive axonal profiles in the reticular nucleus were presynaptic; the postsynaptic elements were exclusively dendritic profiles; and no axo-axonic or axosomatic contacts from labelled axons were observed. Most ChAT-immunoreactive synaptic contacts were made by profiles less than 0.25 micron in minor diameter. Single ChAT-immunoreactive axons made synaptic contact with several dendritic profiles as the axons were followed through serial sections. These results suggest that the cholinergic innervation of the reticular nucleus will modulate the function of reticular neurons by synapsing onto the dendrites of its neurons without direct effect on the corticothalamic and thalamocortical terminals which also innervate the reticular nucleus.

Basal forebrain efferents to the medial dorsal thalamic nucleus in the rhesus monkey.

Thalamic efferent connections of the basal forebrain (BF); medial septal nucleus (MS), vertical limb of the diagonal band (VDB), horizontal limb of the diagonal band (HDB), nucleus basalis (NB), and ventral pallidum (VP) were investigated in twelve rhesus monkeys. In five animals, injections of radioactively labeled amino acids were placed in the BF. In four animals, the injections involved different divisions of the NB, HDB, and the most ventral part of the VDB. In those four cases, labeled fibers in the medial forebrain bundle were observed traveling caudally towards the hypothalamus where some turned dorsally to enter the inferior thalamic peduncle. These fibers terminated in the ventral half of the magnocellular part of the medial dorsal thalamic nucleus (MDmc). In a fifth case, the amino acid injection involved most of the MS and the VDB. Labeled fibers traveled caudally from the injection site and entered the stria medullaris. These fibers then traveled caudally before turning ventrally to terminate in the dorsal half of MDmc. To determine which of the diverse neuronal types in the BF gives rise to these thalamic projections, in two monkeys injections of horseradish peroxidase (HRP) were placed into MDmc. Labeled neurons were observed throughout the full extent of the NB, the VDB, the MS, and part of the VP. In order to determine the extent of the cholinergic input to MDmc from the BF, one of the HRP cases was processed for the simultaneous visualization of HRP, and acetylcholinesterase (AChE), the hydrolytic enzyme for acetylcholine, and a second case was processed for simultaneous visualization of HRP, and choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine. We observed that 30-50% of the HRP-labeled neurons were putatively cholinergic. In order to determine if the NB projection to MD is a collateral of the NB projection to orbital frontal cortex, one fluorescent retrograde tracer was injected into the orbital frontal cortex and one into MD. This case showed that approximately 5% of the BF neurons that project to MDmc also project to the orbital frontal cortex. These results confirm a significant subcortical projection by which the cholinergic system of the basal forebrain may influence higher cortical functions through the thalamus.

The origins of cholinergic and other subcortical afferents to the thalamus in the rat.

The origins of the cholinergic and other afferents of several thalamic nuclei were investigated in the rat by using the retrograde transport of wheat germ agglutinin conjugated-horseradish peroxidase in combination with the immunohistochemical localization of choline acetyltransferase immunoreactivity. Small injections placed into the reticular, ventral, laterodorsal, lateroposterior, posterior, mediodorsal, geniculate, and intralaminar nuclei resulted in several distinct patterns of retrograde labelling. As expected, the appropriate specific sensory and motor-related subcortical structures were retrogradely labelled after injections into the principal thalamic nuclei. In addition, other basal forebrain and brainstem structures were also labelled, with their distribution dependent on the site of injection. A large percentage of these latter projections was cholinergic. In the brainstem, the cholinergic pedunculopontine tegmental nucleus was retrogradely labelled after all thalamic injections, suggesting that it provides a widespread innervation to the thalamus. Neurons of the cholinergic laterodorsal tegmental nucleus were retrogradely labelled after injections into the anterior, laterodorsal, central medial, and mediodorsal nuclei, suggesting that it provides a projection to limbic components of the thalamus. Significant basal forebrain labelling occurred only with injections into the reticular and mediodorsal nuclei. Only injections into the reticular nucleus resulted in retrograde labelling of the cholinergic neurons in the nucleus basalis of Meynert. The results provide evidence for an organized system of thalamic afferents arising from cholinergic and noncholinergic structures in the brainstem and basal forebrain. The brainstem structures, especially the cholinergic pedunculopontine tegmental nucleus, appear to project directly to principal thalamic nuclei, thereby providing a possible anatomical substrate for mediating the well-known facilitory effects of brainstem stimulation upon thalamocortical transmission.

Nuclear distribution of glutamate, gamma-aminobutyrate and aspartate within the normal human thalamus.

Detailed mapping of amino acid neurotransmitter candidates, glutamate, gamma-aminobutyrate and aspartate within the human thalamus, was performed with a grid microdissection method using two horizontal slices of the right thalamus from four neurologically normal cases. Although the fine distribution of these amino acids varied considerably from case to case, characteristic patterns of distribution for each amino acid could be demonstrated by superimposing maps of each case. Glutamate was high around a caudal part of nucleus medialis and a rostral part of nucleus pulvinaris. Interestingly enough, gamma-aminobutyrate distributed in a similar manner to glutamate, suggesting the possible interaction between glutamate and gamma-aminobutyrate in these areas of the thalamus. The distribution of aspartate was different from the former two amino acids and highly concentrated areas were patchy and scattered in the middle part of the thalamus. The results were discussed in relation to the recently accumulated evidence obtained from animal brains for neurotransmitter roles of these amino acids in the thalamus.

Nuclear origin of thalamic afferents of the ventral striatum determines their relation to patch/matrix configurations in enkephalin-immunoreactivity in the rat.

The relation of thalamic terminal fibers in the ventral striatum with patch and matrix compartments, as defined by enkephalin-immunohistochemistry, was studied in adjacent or double-stained sections of the rat brain by combining anterograde tracing of Phaseolus vulgaris-leucoagglutinin (PHA-L) and enkephalin staining. Experiments with small PHA-L injections in the dorsal thalamus show that the paraventricular thalamic nucleus projects to the medial nucleus accumbens and rather selectively to the patch compartment in more lateral parts of the nucleus. The central medial thalamic nucleus sends its fibers to more lateral parts of the nucleus accumbens and the ventral part of the caudate-putamen, where these fibers terminate in the matrix compartment.

The distribution of dopamine-beta-hydroxylase, neuropeptide Y and galanin in locus coeruleus neurons.

The locus coeruleus (LC) is composed of noradrenaline-producing neurons that project widely throughout the neuraxis. Subpopulations of LC neuron perikarya have been shown to contain neuropeptide Y (NPY) and galanin (GAL). In the major terminal fields of LC projections, the cerebral cortex, dorsal thalamus and cerebellar cortex, there are differing plexuses of dopamine-beta-hydroxylase (DBH), NPY and GAL immunoreactive axons. DBH immunoreactive plexuses are found in all areas which conform in appearance to previous demonstrations of noradrenaline localization by fluorescence histochemistry. In contrast, there are few NPY immunoreactive axons in thalamus and cerebellum, and the cortical plexus, while similar to the DBH immunoreactive plexus, is not affected by 6-hydroxydopamine treatment. Similarly, there are few GAL immunoreactive axons in either cerebral cortex, dorsal thalamus or cerebellar cortex. Transection of ascending LC axons results in accumulation of DBH but not NPY or GAL immunoreactivity proximal to the lesion. These observations indicate that NPY and GAL are distributed differently in LC neurons from noradrenaline and DBH.

Iodinated-NPY binding sites: autoradiographic study in the rat brain.

Several authors have described the presence of iodinated neuropeptide-Y binding sites on membranes of the mammalian CNS. In the present study we show a mapping of iodinated-NPY binding sites in the rat brain using receptor autoradiography. The sections were incubated with 125I-Bolton-Hunter coupled NPY (0.5-03 nM), in the absence or presence of 1 microM cold NPY. Some autoradiograms are studied by means of an image analyzer (VDC 501 Tesak) equipped with the host computer PDP 11 Digital, in order to enhance the contrast of the labeling. A very high density of NPY receptors is present in the limbic regions (hippocampus, amygdaloid complex, septal nuclei), in the cortex, and in some thalamic nuclei, while in some hypothalamic regions (paraventricular nucleus and median eminence) we detected a lower amount of NPY receptors. At the mesencephalic level, the substantia nigra presents a very high density of NPY receptors.

Effects of drugs which influence noradrenergic activity on metenkephalin levels in specific brain areas.

The effects of pretreatment with clonidine, an alpha 2 adrenoceptor agonist, and isobutylmethylxanthine, a phosphodiesterase inhibitor, were measured on the metenkephalin content of the locus coeruleus, the amygdala and the nucleus periventricularis thalamis of rat brain. No significant effect of either drug on metenkephalin content could be detected.

A survey of MabQ113 immunoreactivity in the adult rat brain: differential staining of the lateral and medial habenular nuclei.

Monoclonal antibody mabQ113 recognizes a polypeptide antigen which, in rat cerebellum, is confined exclusively to a subset of Purkinje cells which are organized into parasagittal bands. In this report we have explored the distribution of mabQ113 immunoreactivity in some other regions of the rat brain. The most interesting result was a dramatic differential staining of the habenular complex in which mabQ113 densely and uniformly stained the lateral habenula but did not stain the medial habenula. Within the lateral habenula reaction product is localized primarily in the cellular processes of astrocytes but there is also staining of neighboring neuronal dendritic and axonal profiles. The afferent and efferent tracts of the habenular nuclei are not immunoreactive and there was no systematic difference in staining between the afferent and efferent nuclear groups of the two habenular nuclei. The pattern of mabQ113 immunoreactivity in rat brain is distinct from previously described biochemical differentiation markers of the two nuclei and thus may serve as a useful probe to study habenular anatomy, development and function.

A sex difference in the pattern of substance P-like immunoreactivity in the bed nucleus of the stria terminalis.

The caudal part of the dorsomedial bed nucleus of the stria terminalis (BNST) contained a discrete region of dense substance P (SP) staining in both male and female rats. However, the pattern of this staining differed markedly between the sexes. Males exhibited a densely stained capsule surrounding a less dense core. In females, this core was barely detectable (small and present on only 1 or 2 sections) or absent. The perimeter of this area of dense staining was traced and the area inside calculated. The mean area was found to be 2.4 times larger in males. The component of the BNST which receives this SP innervation was identified on Cresyl violet-stained sections. The area occupied by this distinct cell group was 32% larger in males. The areas of the suprachiasmatic nuclei and whole coronal sections through the BNST were measured in the same way and found not to differ between the sexes.

The effect of lesions in the locus coeruleus on the physiological responses of the cerebral blood vessels in cats.

The effects of cerebral blood flow (CBF) of lesions placed stereotactically in or near the locus coeruleus were studied in 15 lightly anesthetized cats; 5 control cats in which the electrode was placed but no lesion created, and 10 experimental cats in which a lesion was created. The response of CBF to changes in Paco2 and in mean arterial blood pressure was determined by 133Xe-washout studies 10 days after the stereotactic procedures. The sites of the lesions were studied histologically, and their effects on catecholamine concentrations in the paraventricular hypothalamic nucleus, anterior ventral nucleus of the thalamus, and parietal cortex were determined by radio-chemical assay. Control animals and those with lesions near, but not in, the locus coeruleus had normal Paco2--CBF response curves and normal catecholamine concentrations in the areas of biopsy. Bilateral destruction of the locus coeruleus was confirmed in 3 animals on histological examination and in these animals there were decreased levels of catecholamines in the areas of assay, higher resting CBFs at normocapnia, and significantly abnormal CBF--Paco2 response curves. The autoregulatory response to changes in perfusion pressure was preserved. Thus, noradrenergic neurons originating in the locus coeruleus may contribute to the control of intraparenchymal cerebral vessels and disturbance of this control may be important in the pathology of cerebral ischemia.

Neurons of the subthalamic nucleus in primates display glutamate but not GABA immunoreactivity.

Immunohistochemical studies undertaken with a highly specific antiserum raised against gamma-aminobutyric acid (GABA)-glutaraldehyde-lysyl-protein conjugate showed that cell bodies of the subthalamic nucleus in the squirrel monkey (Saimiri sciureus) were closely surrounded by several GABA-positive terminals but were not themselves immunoreactive. In contrast, after incubation with a monoclonal antibody directed against carbodiimide-fixed glutamate, virtually all cell bodies of the subthalamic nucleus displayed an intense immunoreactivity. They were surrounded by various neuronal processes that also stained for glutamate. These results suggest that the neurons of the subthalamic nucleus in primates utilize the excitatory neurotransmitter glutamate instead of the inhibitory neurotransmitter GABA.

Single thalamic dopaminergic neurons project to both the neocortex and spinal cord.

Cells in the rat subparafascicular thalamic nucleus (Spf) belonging to the diencephalic A11 cell group, were immunohistochemically stained with antibodies against tyrosine hydroxylase (TH) and dopamine itself. Employing a combination of retrograde fluorescent double-labeling and TH immunofluorescence techniques, we revealed the existence of dopaminergic Spf cells, giving rise to collateral projections to the neocortex and spinal cord.

Effect of hypophysectomy on alpha-melanotropin in discrete regions of the rat brain.

The effect of hypophysectomy on alpha-melanotropin (alpha MSH) concentrations in discrete brain regions was investigated. Hypophysectomy resulted in a 38-69% decrease in alpha MSH concentration in alpha MSH terminal regions 4 weeks after surgery. In contrast, the alpha MSH concentration in the arcuate nucleus, site of alpha MSH containing perikary, was unaffected by hypophysectomy. These results indicate that the brain alpha MSH system is distinct from, but related to that of the pituitary.

The distribution pattern of adrenocorticotropin-like immunoreactivity in the cat central nervous system.

The distribution pattern of adrenocorticotropin-like immunoreactivity (ACTH-LI) in cats using the avidin-biotin modification of an immunocytochemical method shows cell bodies containing ACTH-LI in the medial basal hypothalamus, especially in the infundibular nucleus. The fibers from these neurons extended beyond the hypothalamus, into the paraventricular nucleus of the thalamus, rostral amygdala, periaqueductal gray, locus coeruleus, parabrachial nucleus and medial nucleus of the nucleus tractus solitarius. The distribution pattern of the cell bodies and fibers containing ACTH-LI bears several similarities to that seen in rats. The pattern differs from that of rats in the fact that the termination in the amygdala is more extensive and that ACTH-LI was not observed in cell bodies in any location other than the medial basal hypothalamus.

Immunohistochemical demonstration of serotonin nerve fibers in the subthalamic nucleus of the rat, cat and monkey.

Distribution of serotonin-immunoreactive nerve fibers (5-HT fibers) in the subthalamic nucleus (STN) of the rat, cat and monkey was examined with a modified peroxidase-anti-peroxidase method using 5-HT antiserum without pretreatment. Fine varicose 5-HT fibers formed a fine network in the STN. Overall density of 5-HT fibers in the STN was highest in the monkey, and lowest in the rat. In the rat and cat, these fibers were distributed almost diffusely in the STN, whereas in the monkey they were particularly abundant in the ventral and medial parts of the STN. Many thick 5-HT fibers ran through the monkey STN.

Vasopressin modulates the activity of catecholamine containing neurons in specific brain regions.

Following the i.c.v. administration of antivasopressin serum the alpha-MPT-induced disappearance of noradrenaline was decreased in the dorsal septal nucleus, parafascicular nucleus and the rostral part of the nucleus tractus solitarii, whereas that of dopamine was lowered in the caudate nucleus and in the A2 region of the medulla oblongata. In general the effects are opposite to those previously found following the i.c.v. administration of vasopressin. The results support the hypothesis that vasopressin modulates catecholamine neurotransmission in specific brain regions of the rat.

Putative amino acid neurotransmitters and the nucleus dorsomedialis thalamus-prefrontal cortex pathway in the rat.

Endogenous levels of putative amino acid neurotransmitters (glycine, glutamic acid, aspartic acid, and GABA) in medial and sulcal prefrontal cortex of the rat were analyzed using gas liquid chromatography. No changes were found in the levels of these amino acids in medial and sulcal prefrontal cortex after lesion of the nucleus dorsomedialis of the thalamus suggesting, therefore, that the NDMT-prefrontal cortex pathway is not mediated by these amino acids.

An electrochemical approach to sleep metabolism: a pO2 paradoxical sleep system.

Oxygen cathodes chronically implanted in the cat brain recorded changes of local oxygen concentration during paradoxical sleep. Phasic high amplitude pO2 changes were consistently observed in some regions and were characterized by a dramatic increase in the amplitude of the oscillations. The regions displaying these responses included part of the reticular formation, hypothalamus, amygdala and cerebellum which we refer to as the "pO2 paradoxical sleep system." This pO2 pattern was not observed in white matter, in the neocortex or in specific thalamic nuclei. It is postulated that the phasic response is due to a local increase of neuronal activity requiring increased oxygen availability and augmented protein synthesis during paradoxical sleep and may form part of a system related to "plastic" phenomena.