H. W. M. Steinbusch

The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat.

The distribution of serotonin-immunoreactive varicosities in the paraventricular (PVH) and supraoptic (SO) nuclei was charted in normal immunohistochemical material and the probable cells of origin of these projections were identified using a combined retrograde transport-immunohistochemical method. The density of serotonergic fibers in the PVH and the SO is quite low relative to that in the immediately surrounding neuropil, in striking contrast to noradrenergic inputs to the nuclei. Immunoreactive fibers are concentrated in specific parts of the parvocellular division of the PVH, whereas in the magnocellular division of the nucleus, and in the SO, they are found mostly in regions where oxytocinergic cells predominate. These projections appear to arise from 3 distinct serotonergic cell groups (B7, B8 and B9) in the midbrain.

On the stimulation of soluble and particulate guanylate cyclase in the rat brain and the involvement of nitric oxide as studied by cGMP immunocytochemistry

The localization of the particulate and soluble guanylate cyclase in the rat brain was studied using cGMP-immunocytochemistry. The cGMP was fixed to tissue protein using a formaldehyde fixative, and an antibody against cGMP was used which was raised against a cGMP-formaldehyde-thyroglobulin conjugate. We used the atrial natriuretic factor (ANF) as a model compound to stimulate the particulate enzyme and sodium nitroprusside (SNP) to stimulate the soluble enzyme. Sequential immunostaining for cGMP and glial fibrillary acidic protein (GFAP) showed that the great majority of the ANF-responsive, cGMP-producing cells were astrocytes. These ANF-responsive cells were found in discrete parts of the CNS; not all astrocytes in these regions were responsive to ANF. SNP stimulated cGMP in abundantly present neuronal fibres throughout the CNS; few neuronal cell bodies showed increased cGMP production after SNP. Moreover, SNP also raised cGMP in astrocytes, however, not all astrocytes showed the response to SNP. These results suggest that cells might be present in the CNS which contain both the soluble and the particulate guanylate cyclase. It was demonstrated that in the immature cerebellum, the cGMP was raised in glial structures in response to N-methyl-D-aspartate (NMDA), ANF, SNP, and kainic acid. The response to NMDA and kainic acid was sensitive to inhibition of the nitric oxide synthesis from L-arginine by NG-methyl-L-arginine. Surprisingly the response to ANF localized in the molecular layer and the granular layer was also sensitive to inhibition by NG-methyl-L-arginine, whereas the response to ANF in the deep nuclei was not. A small depolarization induced by 10 to 20 mmol/l K+ induced an increase in cGMP in chopped hippocampus tissue which showed a biphasic temporal characteristic. The initial, fast (30 sec), peak was shown to be localized in varicose fibres throughout the hippocampus, whereas the slower response (10 min) was localized in astrocytes. These studies demonstrate that the different enzymes which synthesize cGMP are differently localized. However, there is also a time dependency in the activation of the guanylate cyclases, which becomes apparent in different structures at different times. The possible role of cGMP as a regulator of ion homeostase is discussed.

cGMP-Producing, Atrial Natriuretic Factor-Responding Cells in the Rat Brain.

Using an in vitro incubation method, we stimulated cGMP production in rat brain slices by rat ANF‐(103–126). The localization of the cells responding to this ANF stimulation with an increase in cGMP production was studied by cGMP immunocytochemistry. ANF‐responding cells were found in specific loci throughout the central nervous system of the rat. Regions containing the highest number of these cells were: the olfactory bulb, the lateral septum, the bed nucleus of the accessory olfactory tract, the mediobasal amygdala, the central grey area, the medial vestibular nucleus, and the nucleus of the solitary tract. Scattered ANF‐responding, cGMP‐immunoreactive cells were found in the hippocampus, the cingulate cortex, the ventral pallidum, the medial preoptic area, and the endopeduncular nucleus. ANF‐responding cells in these areas had the same morphology, that is, multipolar with numerous processes. The nature of these ANF‐responding cells was studied by sequential staining with an antiserum against glial fibrillary acidic protein (GFAP). In the hippocampus it was demonstrated that all ANF‐responding cells are astroglial cells. However, not all astroglial cells in this area showed a cGMP response, demonstrating a regional heterogeneity. ANF‐responding cells, having the appearance of neuronal cell bodies, could be found in the subfornical organ, and the hypothalamic paraventricular nucleus. Fibres producing cGMP immunoreactivity in response to ANF were found in the median preoptic nucleus, the medial preoptic area, and the dorsal hypothalamus.

Localization of cGMP in the cerebellum of the adult rat: an immunohistochemical study

The localization of cGMP in the cerebellum of the adult rat after fixation with formaldehyde was studied with an antibody raised against a cGMP-formaldehyde-thyroglobulin conjugate. Three different protocols were used: (1) in vitro incubation of 300 microns cerebellar slices followed by fixation and cryostat sectioning; (2) in vitro incubation of 100 microns cerebellar slices followed by fixation with no further sectioning; (3) perfusion fixation of the anesthetized rat followed by vibratome sectioning. All 3 protocols gave essentially the same results: cGMP-immunoreactivity was found predominantly in Bergmann fibers in the molecular layer, in Bergmann cell bodies in the Purkinje cell layer (but not in Purkinje cells), and in astroglial cells in the granular layer.

Aging of the serotonergic system in the rat forebrain: An immunocytochemical and neurochemical study

Age-related changes in both morphological and neurochemical parameters of indol- and catecholaminergic system in the rat brain were examined. A qualitative histochemical survey of the occurrence of aberrant serotonergic fibers in the aged rat brain suggests region-specificity in the process of degeneration. Forebrain areas, such as the caudate-putamen complex, globus pallidus, prefrontal and frontoparietal cortices were consistently affected, whereas serotonergic fibers were only infrequently affected in other areas like septal and amygdaloid nuclei. Neurochemical data similarly revealed regional differences. 5-Hydroxytryptamine levels were increased in the frontoparietal cortex, hippocampus, hypothalamus and the mesencephalic raphe region but remained unchanged in the caudate-putamen complex. 5-Hydroxyindolacetic acid levels were also enhanced in all these areas. Examination of brains of 12-, 18- and 24-month-old rats revealed that aberrant serotonergic fibers were already present at the age of 12 months and their incidence increase with age. There was no difference in the number of serotonergic cells in the dorsal raphe nucleus of young and aged rats. Aberrant tyrosine hydroxylase-immunoreactive fibers were observed only infrequently. Their occurrence showed no overlap with the areas containing aberrant serotonergic fibers. Neurochemical estimates of the levels of catecholamines in young versus aged rat brain areas similarly revealed regional and neurotransmitter specific differences to occur during the process of aging.

Histaminergic projections from the premammillary and posterior hypothalamic region to the caudate-putamen complex in the rat

Immunofluorescence, using antibodies to histamine and to histidine decarboxylase, was combined with retrograde axonal tracing by injecting Granular Blue into the caudate-putamen complex. Evidence is presented for the existence of histaminergic as well as non-histaminergic projections from the posterior hypothalamus and the premammillary region to the caudate-putamen complex. The majority of the histaminergic neurons projecting to this brain region are localized in the nuclei caudalis magnocellularis and caudalis magnocellularis postmammillaris. Roughly 20-25% of the histaminergic neurons in these cell groups innervate the caudate-putamen complex.

Distribution of serotonin- and dopamine-immunoreactivity in the brain of the teleost Clarias gariepinus.

The distribution of serotonergic and dopaminergic cell bodies and varicose fibres in the brain of the teleost Clarias gariepinus was studied immunohistochemically using antisera against formaldehyde-conjugated serotonin and dopamine. Many serotonergic and dopaminergic fibres innervated the areas dorsalis telencephali pars medialis and pars lateralis dorsalis, as well as the area ventralis telencephali pars ventralis. In the diencephalon, a large number of serotonergic and some dopaminergic fibres were found in the preoptic nucleus, innervating the cells of this nucleus. In addition, serotonergic and dopaminergic fibres were observed in the pituitary stalk and in all regions of the pituitary gland. Moreover, the diencephalon contained the highest number of serotonin- or dopamine-immunoreactive cell bodies. These cells were confined to the same periventricular nuclei as the nucleus ventromedialis thalami, the nucleus posterior periventricularis, the nucleus lateralis tuberis, the nuclei recessus lateralis and recessus posterioris. Most cells of these nuclei were in contact with the cerebrospinal fluid of the third ventricle. The brainstem contained serotonergic cell bodies in the raphe nuclei and a few serotonergic and dopaminergic fibres. The torus semicircularis was densely innervated by serotonergic fibres and, to a lesser extent, dopaminergic fibres. In the midbrain of Clarias gariepinus, no dopaminergic homologue of the substantia nigra was observed. The results are discussed both in a comparative and a physiological context. In this regard, special attention has been paid to the contribution of hypothalamic monoamines in the regulation of gonadotropin secretion as an essential step in the neuro-endocrine control of reproduction.

Mapping of CRF-immunoreactive nerve fibers in the medulla oblongata and spinal cord of the rat

By use of an antiserum raised against Corticotropin Releasing Factor (CRF 1-41), nerve fibers can be stained in the medulla oblongata and spinal cord of rats. A dense plexus of CRF-immunoreactive (CRFi) nerve fibers is present in the nucleus tractus spinalis nervi trigemini from which fiber bundles enter the tractus spinalis nervi trigemini. Large numbers of CRFi fibers are present in the substantia gelatinosa of the spinal cord, while the tractus solitarius, the nucleus tractus solitarius and the nucleus ambiguus contain a low number of CRFi fibers. In rats treated with colchicine, CRFi cell bodies are found in the hypothalamus and occasionally in the nucleus tractus solitarius and the nucleus olivaris inferior. Posterolateral deafferentation of the hypothalamus did not result in a disappearance of the CRFi fibers in the medulla oblongata and spinal cord 7 days after surgery. These results indicate that CRFi fibers present in the spinal cord and medulla oblongata are part of a novel peptidergic neuronal system, which is different from the hypothalamo-infundibular CRF system.

Aberrant morphology of serotonergic fibers in the forebrain of the aged rat

The morphological aspects and density of the serotonergic innervation in the forebrain of young (2 months) and aged (28-32 months) rats was studied employing immunocytochemistry with an antibody to serotonin. In the aged rats aberrant morphology of many of the preserved fibers was observed. The aberrant fibers were characterized by swollen varicosities and swollen intervaricose connections. They formed small networks. These findings were mainly restricted to the frontoparietal cortex and caudate putamen. In the same regions we observed a decrease in serotonergic innervation. There was no overall relation between aberrant morphology and decrease of serotonin-innervation as we observed a decrease in fiber density without morphological abnormalities in the hippocampus. It is suggested that the aberrant morphology may reflect the local degeneration of serotonergic forebrain afferents during the process of aging.

Similarities between aberrant serotonergic fibers in the aged and 5,7-DHT denervated young adult rat brain.

SummaryRecent morphological observations have suggested neurotransmitter specific degeneration of amongst others, the serotonergic system in the aged rat brain. However, morphological studies can only give a static picture of the events that take place over a period of several months. In the present study we used an experimental model in which degeneration of the serotonergic system in the young adult rat brain was produced on a short time scale. Morphological changes were studied 2 h and 1 or 14 days after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT). Nonspecific damage and severe depletion of serotonergic fibers was observed in the immediate surroundings of the injection site, representing the effects of high local concentrations of 5,7-DHT. Sometime after injection swollen varicosities and dilated non-varicose fibers were observed. Fourteen days after the 5,7-DHT treatment cluster-like fibers appeared. It is argued that these swollen and crumpled fiber knots are slowly degenerating fibers. A comparison is made with the abnormal serotonergic fibers in the aged rat brain and it is concluded that these aged abnormal fibers represent axonal degeneration of the serotonergic system in the senescent rat brain.