Localization of Inhibin Alpha-Subunit Immunoreactivity in the Rat Adrenal Cortex

Inhibin, a water soluble, non-steroidal glycoprotein hormone that inhibits follicle-stimulating hormone secretion, is produced by the gonads and several non-gonadal tissues (placenta, pituitary and brain). This study describes the presence of inhibin α-chain-immunoreactive cells in the adrenal cortex of intact adult male rats and the effects of hypophysectomy and adrenocorticotropin treatment on the expression of inhibin in that gland. In intact rats, the majority of the immunoreactive cells were found scattered in the zona glomerulosa and reticularis. The zona fasciculata contained only a few cells immunopositive for the inhibin α-chain. No immunoreactive cells were found in the medulla. Hypophysectomy resulted in a dramatic reduction in α-subunit immunoreactivity with only scattered cells observed in the zona glomerulosa and reticularis (0 to 2 cells/section). No immunoreactive cells were found in the zona fasciculata or the medulla. Following supraphysiologic adrenocorticotropin administration to hypophysectomized animals, the number of inhibin α-chain-immunoreactive cells significantly increased in each zone of the adrenal cortex. The most prominent changes were seen in the zona reticularis. Interestingly, in the adrenocorticotropin-treated animals scattered immunopositive cells were also present in the medulla. Our results suggest that inhibin-related peptide(s) exist within the adrenal gland and that the expression of these peptides is regulated by adrenocorticotropin. Inhibin-related peptide(s) may, therefore, play a paracrine and/or endocrine role in the adrenal function.     

Long-term effects of neuropeptide-Y on the rat adrenal cortex

Adult female rats, intact or dexamethasone suppressed, were i.p. injected twice a day for 4 consecutive days with 0.5 ug neuropeptide-Y (NPY). In intact rats, NPY lowered serum ACTH and aldosterone levels, and had no effects either on serum and adrenal corticosterone or the morphology of the adrenal cortex. In dexamethasone-treated rats, NPY again decreased serum aldosterone concentration. Moreover, it caused a small but significant drop both in the volume of zona fasciculata cells and the adrenal content of corticosterone. These findings seem to suggest an inhibitory effect of NPY on the function of rat zona glomerulosa and perhaps zona fasciculata.     

Occurrence of myelin-associated glycoprotein (MAG)-like immunoreactivity in some nervous, endocrine, and immune-related cells of the rat

The occurrence and distribution of myelin-associated glycoprotein (MAG)-like immunoreactivity was investigated in the rat using a polyclonal antibody to MAG purified from rat brain. In the nervous system, MAG immunoreactivity was found in the periaxonal portion of the myelinated fibers and in a small number of oligodendroglia in the cortex, hippocampus, and the spinal cord. The sheath of Schwann cells in unmyelinated fibers and satellite cells in the spinal ganglia were also immunoreactive for MAG. In the endocrine system, the noradrenaline-containing cells in the adrenal medulla and some endocrine cells in the duodenum showed MAG immunoreactivity. In the immune system, numerous reticular cells with slender cytoplasmic processes, which formed a dense network, were immunopositive for MAG within the germinal center in the lymph nodes and spleen. In the thymus, a number of epithelial reticular cells within the medulla showed variation in staining intensity. These findings provide new information on the wide distribution of MAG immunoreactivity in the nervous, endocrine, and immune systems, and may contribute to the further understanding of the biological roles of this protein.     

Glial fibrillary acidic protein immunoreactivity in adrenocortical and Leydig cells of the Syrian golden hamster (Mesocricetus auratus).

In an immunocytochemical investigation of the expression of glial fibrillary acidic protein (GFAP) in non-nervous system tissues ten anti-GFAP antibodies were used on a range of normal adult organs from different species. All four polyclonal and six monoclonal antibodies revealed the expression of GFAP in cells of the zona fasciculata and reticularis of the adrenal cortex and Leydig cells of the Syrian hamster. The Chinese hamster, mole, rat, mouse, guinea pig, rabbit, pig, duck and man were negative. Co-expression of immunoreactivity for GFAP and vimentin was observed in adrenocortical and Leydig cells of the Syrian hamster but there were differences in the staining patterns of these intermediate filament proteins. Expression of GFAP in adrenal cortex of Syrian hamster is confirmed by immunoblot and limited proteolysis analysis which reveal a light form which is immunochemically indistinguishable from its counterpart in the central nervous system. The results presented here suggest a new model for the study of the possible role of GFAP expression in cells known to be sites of steroid synthesis.     

Distribution and cellular localization of adrenoleukodystrophy protein in human tissues: implications for X-linked adrenoleukodystrophy

Defects of adrenoleukodystrophy protein (ALDP) lead to X-linked adrenoleukodystrophy (X-ALD), a disorder mainly affecting the nervous system white matter and the adrenal cortex. In the present study, we examine the expression of ALDP in various human tissues and cell lines by multiple-tissue RNA expression array analysis, Western blot analysis, and immunohistochemistry. ALDP-encoding mRNA is most abundant in tissues with high energy requirements such as heart, muscle, liver, and the renal and endocrine systems. ALDP selectively occurs in specific cell types of brain (hypothalamus and basal nucleus of Meynert), kidney (distal tubules), skin (eccrine gland, hair follicles, and fibroblasts), colon (ganglion cells and epithelium), adrenal gland (zona reticularis and fasciculata), and testis (Sertoli and Leydig cells). In pituitary gland, ALDP is confined to adrenocorticotropin-producing cells and is significantly reduced in individuals receiving long term cortisol treatment. This might indicate a functional link between ALDP and proopiomelanocortin-derived peptide hormones.     

Long-term stimulatory effect of neuropeptide-Y on the growth and steroidogenic capacity of rat adrenal zona glomerulosa

A prolonged infusion with neuropeptide-Y (NPY) caused a notable hypertrophy of the adrenal zona glomerulosa and its parenchymal cells in rats whose hypothalamo-hypophyseal axis and renin-angiotensin system were pharmacologically interrupted. Zona glomerulosa hypertrophy was associated with a significant rise in both basal and stimulated plasma levels of aldosterone. Zona fasciculata cells and the blood concentration of corticosterone were not affected. These findings suggest that NPY is specifically involved in the stimulation of the growth and steroidogenic capacity of rat adrenal zona glomerulosa.     

Epidermal nerve fiber density in sensory ganglionopathies: clinical and neurophysiologic correlations.

We assessed the involvement of somatic unmyelinated fibers in sensory ganglionopathies by skin biopsy and quantitative sensory testing (QST). Sixteen patients with ganglionopathy, 16 with axonal neuropathy, and 15 normal controls underwent skin biopsy at the proximal thigh and the distal leg. Intraepidermal nerve fibers (IENF) were immunostained by antiprotein gene product 9.5, and their linear density was quantified under light microscopy. Confocal microscopy studies with double staining of nerve fibers and basement membrane were also performed. Healthy subjects and neuropathy patients showed the typical proximodistal gradient of IENF density; in neuropathies, values were significantly lower at the distal site of the leg, confirming the length-dependent loss of cutaneous innervation. Conversely, ganglionopathy patients with hyperalgesic symptoms did not show any change of IENF density between the proximal thigh and the distal leg. The distinct pattern of epidermal denervation seen in sensory ganglionopathy reflected the degeneration of somatic unmyelinated fibers in a fashion that was not length-dependent, which was consistent with both clinical and neurophysiologic observations and supported the diagnosis.     

Serotoninergic innervation of the ferret cerebral cortex. II. Postnatal development.

We have investigated the serotoninergic innervation of the ferret cortex from the day of birth to adulthood with immunohistochemical techniques. Due to the premature birth of ferrets, this period spans the entire generation of cells located within the upper cortical layers and their subsequent migration to their final positions. Already at birth, serotoninergic fibers innervate the developing cortex. This innervation is most dense within the marginal zone, the subplate region, and the lower portion of the cortical plate. As long as cell migration continues, serotoninergic fibers enter the expanding portions of the cortex. Only the region just below the marginal zone where newly arriving cells are added to the cortical plate is not innervated by the ingrowing fibers. When the bulk of cell migration ceases, during the third postnatal week, this gap disappears and the fibers gradually form a continuous innervation from the pia to the ventricle. As the cortex matures, the serotoninergic fibers become successively confined to the upper layers, to generate the adult pattern. In the adult ferret cortex, the highest innervation density is found within layers 1, 2, and 3, with a much sparser innervation within the lower layers (Voigt and de Lima, J. Comp. Neurol. 314:403-414, 1991). The dense innervation in the deep cortical layers is only transient, virtually disappearing toward adulthood. These results suggest that serotoninergic axons innervate cortical layers as soon as newly arriving cells reach their final positions within the cortex. This early innervation lends support to the idea that serotonin may play a role during development of the cerebral cortex.     

Nervale Einflußnahme des Hypothalamus auf die Funktion der Nebennierenrinde

Zusammenfassung Gezielte unilaterale Hypothalamusläsionen im Gebiet des Nucleus ventromedialis führen zur Ausbildung eines karyometrisch faßbaren signifikanten Seitendimorphismus im Bereich der Zona fasciculata der Nebennierenrinde. Dieser Effekt setzt offensichtlich eine vorerst anatomisch nicht definierbare nervale Regelstrecke zwischen Hypothalamus und Nebennierenrinde als zusätzliche Einrichtung neben dem humoralen Weg im Dienste der Funktionsregulation voraus.

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Summary Unilateral lesions of the hypothalamic ventromedial nuclei produce a significant difference in nuclear size of cells of the zona fasciculata as both adrenals in adult male rats are compared. This effect obviously requires an at present anatomically not definable nerval connection between hypothalamus and adrenal cortex that may represent an additional pathway next to the humoral one for the regulation of adrenal function.

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Neuropeptide FF distribution in the human and rat forebrain: a comparative immunohistochemical study

Neuropeptide FF (NPFF) is an octapeptide implicated in a variety of physiological functions, including nociception, cardiovascular responses, and neuroendocrine regulation. The NPFF gene and its mRNA are highly conserved across species. A comparative study of NPFF distribution in the human and rat forebrain was carried out by using single NPFF and double NPFF + vasopressin (VP) immunohistochemistry. NPFF is extensively localized within neurochemical circuits of human and rat forebrain. Semiquantitative analysis revealed that the densities of NPFF cells and fibers in many forebrain nuclei in the human correlate well with those observed for the same structures in the rat. High numbers of NPFF positive neurons in the dorsomedial hypothalamic nucleus and a dense plexus of NPFF fibers surrounding the fornix within the bed nucleus of the stria terminalis were identified in the human and rat forebrain. Within the hypothalamus of both species, dense NPFF innervation was observed in the perinuclear zone of the supraoptic nucleus (SO) just dorsolateral to the VP-positive neurons. Extensive NPFF innervation of ventricular ependyma and brain microvasculature were common for both species. At the same time, obvious differences in NPFF localization between the two species were also apparent. For example, in contrast to the rat SO, no NPFF- or NPFF- + VP-immunostained cells were observed in the human SO. Knowledge of NPFF neuroanatomical localization in the human brain and the relationship of these observations to those in the rat brain may provide insight into the role of this peptide in central cardiovascular and neuroendocrine regulation.     

Differential cholinergic innervation within functional subdivisions of the human cerebral cortex: a choline acetyltransferase study.

The distribution of cholinergic fibers in the human brain was investigated with choline acetyltransferase immunocytochemistry in 35 cytoarchitectonic subdivisions of the cerebral cortex. All cortical areas and all cell layers contained cholinergic axons. These fibers displayed numerous varicosities and, on occasion, complex preterminal profiles arranged in the form of dense clusters. The density of cholinergic axons tended to be higher in the more superficial layers of the cerebral cortex. Several distinct patterns of lamination were identified. There were also major differences in the overall density of cholinergic axons from one cytoarchitectonic area to another. The cholinergic innervation of primary sensory, unimodal, and heteromodal association areas was lighter than that of paralimbic and limbic areas. Within unimodal association areas, the density of cholinergic axons and varicosities was significantly lower in the upstream (parasensory) sectors than in the downstream sectors. Within paralimbic regions, the non-isocortical sectors had a higher density of cholinergic innervation than the isocortical sectors. The highest density of cholinergic axons was encountered in core limbic structures such as the hippocampus and amygdala. These observations show that the cholinergic innervation of the human cerebral cortex displays regional variations that closely follow the organization of information processing systems.     

Cholinergic neurons of the feline pontomesencephalon. II. Ascending anatomical projections

Immunoreactivity for choline acetyltransferase (ChAT) was analyzed in unoperated cats and in cats in which stereotaxic lesions were made in the pedunculopontine and laterodorsal tegmental nuclei. The fine reaction product revealed moderate to dense ChAT-immunoreactive fiber plexuses throughout the telencephalon, diencephalon, and midbrain. A pontomesencephalic origin of cholinergic innervation to virtually every nucleus of the diencephalon, as well as to various midbrain and basal telencephalic sites was indicated in the cats with lesions, in which the optical density of ChAT-immunoreactivity was significantly decreased as compared to controls. Pontomesencephalic lesions produced no changes, however, in the density of ChAT staining in the cerebral cortex, basolateral amygdala, or caudate nucleus. In addition to ChAT-positive terminal fiber arborizations which were widely distributed, cholinergic fibers-of-passage were traced in the unoperated and operated feline brains. The general course of ChAT fibers cut in cross-section was followed in successive transverse levels, and although pathways originating from the pedunculopontine nucleus demonstrated orientations in every direction, many demonstrated a rostral course. A particularly dense aggregate of ascending ChAT-positive fibers was localized in the dorsolateral sector of the pedunculopontine area which could be followed at more rostral levels into the central tegmental fields and the compact part of the substantia nigra. From the central tegmental fields, numerous ChAT-immunopositive fibers cut in cross-section continued to course rostrally in the intralaminar, reticular and lateroposterior nuclei of the thalamus, and a distinct bundle of ChAT fibers coursing dorsolaterally was observed medial to the optic tract ascending to the lateral geniculate. ChAT fibers with dorsolateral orientations were additionally observed in the zona incerta, ventral anterior thalamus, and ansa lenticularis on route to the reticular thalamus, the globus pallidus, and the substantia innominata. Pathways consisting of fibers traced from ChAT-containing cells in the laterodorsal tegmental nucleus could be traced to medial structures such as the periaqueductal gray, ventral tegmental area and dorsal raphe. Medially placed ChAT fibers were additionally followed through the ventral tegmental area, the midline thalamus, and the hypothalamus, up to the medial and lateral septal nuclei. The trajectories of the ascending cholinergic pathways from the pontomesencephalon are discussed in relation to locally generated electrophysiological responses in the cat.     

Botulinum-induced alteration of nerve-muscle interactions in the human orbicularis oculi following treatment for blepharospasm

To assess longstanding alterations in human muscle innervation induced by botulinum toxin, we studied motor axons in the orbicularis oculi of nine patients previously injected with botulinum toxin for treatment of benign essential blepharospasm (BEB). Compared with untreated BEB and normal orbicularis oculi, muscle exposed to botulinum toxin developed persistent and cumulative alterations of innervation, including (1) thin, unmyelinated axonal collaterals that contact muscle end plates, (2) an increased number of muscle fibers innervated by individual terminal motor axons, (3) a profusion of unmyelinated axonal sprouts that end blindly, (4) an increased range of end plate sizes, and (5) multiple end plates on individual muscle fibers. The findings suggest that axonal sprouts which develop after botulinum-toxin-induced functional denervation can form new end plates. A single muscle fiber may then be innervated at separate sites by more than one axon.     

Dopaminergic innervation of the rat prefrontal cortex: a fluorescence histochemical study.

After the destruction of the noradrenergic ascending pathways, the localization of frontal cortical fields receiving fibers from the dopaminergic mesocortical system has been studied in rats, using a glyoxylic-paraformaldehyde method. The dopaminergic innervation was distributed in two main areas. The area of highest density was a medial field which spread in the medial cortex anterior and dorsal to the genu of the corpus callosum. It did not reach the shoulder region except in the foremost part of the frontal lobe where dopaminergic fibers were scattered in the whole cortex, including the molecular layer. A deep sulcal field was situated between the dorsal bank of the rhinal sulcus and the lateral cortex above it. In addition, a moderately dense band of dopaminergic fibers was observed between the corpus callosum and the anterior commissura, beside the accumbens nucleus. Similar data were obtained with dopamine uptake experiments on reserpine-treated but otherwise normal animals. The frontal areas receiving dopaminergic innervation coincide strikingly with the 'prefrontal cortex' as defined by neuroanatomical studies, which is assumed to be more or less equivalent to the prefrontal cortex of primates and derives direct projections from the amygdala. The functional implications of these findings are discussed.     

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.     

Diversity of relative intensities of acetylcholinesterase staining within the laminae of the visual cortex of four mammals

The histological staining patterns of acetylcholinesterase fibers in the visual cortex of the hamster, bank vole, yellow necked wood mouse, and rabbit are described. Between the species there are variations in staining intensities of the laminae in the visual cortex. In the hamster and bank vole layers IV, V, and VI are intensely stained. The yellow necked wood mouse demonstrates little difference in staining intensity between the layers of the visual cortex. The rabbit evidences a dense precipitate in the lower portion of layer IV and also in layer V. Staining intensity patterns suggest that the cholinergic afferents to the visual cortex may develop preferential innervation to laminae which receive low sensory stimulation during the formative period.     

Serotoninergic innervation of the dorsal column nuclei and its relation to cytoarchitectonic subdivisions: An immunohistochemical study in cats and monkeys (Aotus trivirgatus)

The serotoninergic innervation of the dorsal column nuclei (DCN) was investigated in cats and owl monkeys (Aotus trivirgatus) with immunohistochemical methods. A dense network of serotonin-immunoreactive fibers was present in the reticular regions of DCN in cats, and in the pars triangularis of the cuneate nucleus and the peripheral and caudal regions of the gracile nucleus in owl monkeys. The cat's cluster regions and the monkey's rotund regions were more sparsely innervated. Electron microscopic examination showed that the labeled fibers were thin and unmyelinated. Vesicle-containing, terminal-like structures were small. They were in contact with dendrites, other terminals and cell bodies, but synapses were rare. The results demonstrate that the serotoninergic projection to the DCN in both cats and owl monkeys is heterogeneously distributed in a pattern that is faithfully related to the cytoarchitectonic subdivisions of the DCN. The densely innervated reticular regions in the DCN of cats and the corresponding regions in monkeys are predominantly involved in the processing of sensory information to the cerebellum, either directly, or indirectly through projections to the inferior olive, pontine gray, tectum, pretectum, red nucleus, or zona incerta. Thus, the present findings suggest that the serotoninergic innervation of the DCN is primarily related to the DCN's involvement in motor functions. © 1993 Wiley-Liss, Inc.     

Immunocytochemical evidence of well-developed dopaminergic and noradrenergic innervations in the frontal cerebral cortex of human fetuses at midgestation

The catecholaminergic (CA) innervation of the frontal lobe was visualized in 20- to 24-week-old human fetuses with immunocytochemical techniques, by use of antibodies raised against three synthetic enzymes of the CA pathway, tyrosine-hydroxylase (TH), dopamine-β-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). DBH-like immunoreactivity (IR) was probably labeling the noradrenergic (NA) fibers and terminals in the cerebral cortex since no PNMT-IR fibers were detected. In double-labeling TH-DBH experiments, 92–95% of the DBH-IR afferents were not labeled with TH antibodies, indicating that TH-like immunoreactivity (TH-IR) was found primarily in dopaminergic (DA) fibers. Although cortical layering had not yet occurred at this stage, the widespread CA innervation observed in the different areas and layers of the fetal frontal cortex was comparable to that previously described in the adult (Gaspar, Berger, Febvret, Vigny, and Henry: J. Comp. Neurol. 279:249–271, '89). At midgestation, the distribution of CA innervation was region and laminar specific: (1) The densest dopaminergic innervation in the cerebral cortex was located caudal to the genu of the corpus callosum: TH-IR fibers were abundant throughout all layers, from the medial telencephalon (future cingulate) to the dorsal areas (presumed motor cortices) and the lateral insular areas; (2) TH-IR fibers were less dense in the rostral prefrontal cortical anlage; (3) DBH-IR noradrenergic afferents were less numerous than the dopaminergic ones in all the cortical areas studied; (4) in all areas, the highest amount of TH and DBH-IR terminals was found in the upper subplate and in the lower part of the cortical plate, followed by the molecular layer and the intermediate zone. The deep subplate exhibited a lower number of positive fibers but contained TH-IR cell bodies. The presence of dense CA innervation in the immature cortical anlage of the human frontal lobe does not exclude a reorganization of DA and NA innervations within the different cortical layers and areas during the protracted pre- and postnatal period of development. © 1993 Wiley-Liss, Inc.     

Dopamine- and adenosine-3',5'-monophosphate (cAMP)-regulated phosphoprotein of 32 kDa (DARPP-32) in the adrenal gland: immunohistochemical localization.

The cellular localization of a dopamine- and adenosine-3',5'-monophosphate (cAMP)-regulated phosphoprotein of an apparent molecular weight of 32,000 (DARPP-32) was investigated in mouse, rat, rabbit, guinea pig, cat, monkey (Macaca fascicularis and Marmoset) and human adrenal gland by means of indirect immunofluorescence histochemistry. DARPP-32-like immunoreactivity (-LI) was demonstrated in chromaffin cells in the adrenal medulla of rabbit, guinea-pig, cat, monkey and human, but not in mouse or rat. In the Marmoset monkey, DARPP-32-LI was also observed in the zona glomerulosa of the adrenal cortex. It has been shown that dopamine and dopaminergic agonists inhibit catecholamine release from chromaffin cells and aldosterone secretion from cells in the adrenal cortex. The present results suggest that DARPP-32, an intracellular third messenger for dopamine, may be part of the signal transduction mechanism for dopamine acting on the adrenal gland.