Immunocytochemical localization of glutamate and gamma-aminobutyric acid in the accessory olfactory bulb of the rat.

The synaptic organization of the accessory olfactory bulb (AOB) was studied in the rat with antibodies against the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). To a large extent, the immunoreactivity patterns produced by the two antibodies were complementary. Glu-like immunoreactivity (-LI) was observed in the glomerular neuropil, in the mitral cells, and in large neurons located in the periglomerular region. Immunogold electron microscopy revealed particularly high levels of Glu-LI in the axon terminals of vomeronasal neurons. GABA-LI was present in granule and periglomerular cells and in their processes. The dendritic spines of granule cells, which were presynaptic to mitral cells, were strongly labelled by the antiserum against GABA. Labelling of serial semithin sections showed that the GABA-positive and Glu-positive neurons of the periglomerular region are generally distinct, and colocalization of Glu and GABA occurred only in a few cells. These results are consistent with electrophysiological studies indicating that the synaptic organization of the AOB is similar to that of the main olfactory bulb. In both systems, Glu is the neurotransmitter used by primary afferents and output neurons, whereas GABA is involved in the circuits underlying lateral and feed-back inhibition.

Distribution of neuropeptide Y-like immunoreactivity in the brain of the lizard Gallotia galloti.

The distribution of neuropeptide Y (NPY)-like immunoreactivity was studied in the brain of the lizard Gallotia galloti, in order to gain insight into the comparative topography of this peptide. Antisera against both NPY and its C-terminal flanking peptide (C-PON) were used, demonstrating a general coexistence of both peptides, as described in other vertebrates. Most NPY-like immunoreactive (NPY-LI) cell bodies were observed in the telencephalon, specifically in various olfactory structures, all cortices, septum, basal ganglia (except for the globus pallidus), the nucleus of the diagonal band of Broca, the amygdaloid complex, and the bed nucleus of the anterior commissure. NPY-LI cells were also seen in the preoptic and hypothalamic regions and the dorsal thalamus (mainly in the perirotundal belt), as well as in the mesencephalic tegmentum (in the ventral tegmental area, the substantia nigra, and the retrorubral area). NPY-LI fibers and terminals were widely distributed in the brain. All visual and auditory neuropiles were densely innervated. Specially dense plexuses were seen in the nucleus accumbens, the ventral pallidum, the suprachiasmatic and ventromedial hypothalamic nuclei, the nucleus medialis thalami, the left habenula, and the central nucleus of the torus semicircularis. Our analysis shows that the distribution of NPY-like immunoreactivity in the forebrain of Gallotia largely resembles that of other vertebrates, whereas differences are mainly observed in the brainstem. The widespread distribution of NPY in the lizard brain suggests several modulatory functional roles, either in local-circuit systems of the forebrain, or in various limbic, neuroendocrine, and sensory pathways.

Immunohistochemistry and neurochemistry of the habenulo-interpeduncular connection after partial developmental depletion of habenular cholinergic neurons in the rat.

The habenulo-interpeduncular system of the rat constitutes an interesting model to address quantitatively problems related to synaptogenesis and to the interactions between neuronal populations after selective alteration of these elements during development. In the present study this has been achieved by experimentally reducing, through gestational treatment with methylazoxymethanol acetate (MAM), the population of cholinergic neurons of the medial habenula which projects to the interpeduncular nucleus. Immunohistochemical analysis gave evidence that the topographical localization of the cholinergic and the substance P-containing populations in the medial habenula was not altered by MAM treatment. Furthermore, the topographical distribution of cholinergic fibers and terminals in the interpeduncular nucleus, which reflects the habenulo-interpeduncular projection as well as cholinergic projections coming from different sources, was substantially preserved. The same was also true concerning the terminal distribution of substance P in the interpeduncular nucleus. Quantitative radioassays demonstrated a sizable decrease of overall ChAT activity in both the habenulae and the interpeduncular nucleus. By comparison of 1 month-old and 3 month-old animals it appeared that this effect was partially reversed with age in the interpeduncular nucleus.

Localization of neuropeptide Y-immunoreactive cells and fibres in the brain of the Japanese quail.

In the present study, we have demonstrated, by means of the biotin-avidin method, the widespread distribution of neuropeptide Y (NPY)-immunoreactive structures throughout the whole brain of the Japanese quail (Coturnix coturnix japonica). The prosencephalic region contained the highest concentration of both NPY-containing fibres and perikarya. Immunoreactive fibres were observed throughout, particularly within the paraolfactory lobe, the lateral septum, the nucleus taeniae, the preoptic area, the periventricular hypothalamic regions, the tuberal complex, and the ventrolateral thalamus. NPY-immunoreactive cells were represented by: a) small scattered perikarya in the telencephalic portion (i.e. archistriatal, neostriatal and hyperstriatal regions, hippocampus, piriform cortex); b) medium-sized cell bodies located around the nucleus rotundus, ventrolateral, and lateral anterior thalamic nuclei; c) small clustered cells within the periventricular and medial preoptic nuclei. The brainstem showed a less diffuse innervation, although a dense network of immunopositive fibres was observed within the optic tectum, the periaqueductal region, and the Edinger-Westphal, linearis caudalis and raphes nuclei. Two populations of large NPY-containing perikarya were detected: one located in the isthmic region, the other at the boundaries of the pons with the medulla. The wide distribution of NPY-immunoreactive structures within regions that have been demonstrated to play a role in the control of vegetative, endocrine and sensory activities suggests that, in birds, this neuropeptide is involved in the regulation of several aspects of cerebral functions.

Calcitonin gene-related peptide immunoreactivity in the hypothalamo-hypophysial system of the green frog, Rana esculenta.

The distribution of calcitonin gene-related peptide (CGRP) has been investigated in the hypothalamus of the frog Rana esculenta L. by means of different immunohistochemical techniques. A few immunopositive cell bodies and several fibers have been demonstrated in the preoptic area and in the caudal hypothalamus. Some CGRP-like fibers were also recognized in the outer zone of the median eminence. Simultaneous double immunofluorescence methods showed CGRP-like immunoreactivity to be often contiguous to substance P-like positive structures, but separated from them.

Hypothalamic target cells of endogenous sexual steroids in female rat. An immunocytochemical approach utilizing antiestradiol antiserum.

The immunostaining proposed in this study identifies cells containing endogenous estrogens in the Central Nervous System, in the presence of both very high and very low serum estrogen levels. Ten adult rats, seven normal and three ovariectomized females, were used for this study. Paraffin sections of hypothalamus and ovary of each female were stained using Avidin-Biotin Complex method and antiestradiol antiserum. Two different antiestradiol antisera were tested in this work: tests for method and antisera specificity are described. The immunostaining used here shows to be specific and sensitive revealing a higher number of labeled hypothalamic areas than those revealed by other techniques.

Hypothalamus-hypophysis and testicular GnRH control of gonadal activity in the frog, Rana esculenta: seasonal GnRH profiles and annual variations of in vitro androgen output by pituitary-stimulated testes.

The binding of a gonadotrophin-releasing hormone (GnRH) long acting analog (GnRHA), D-Ser (But)6,Pro9-NEt GnRH (HOE 766), to pituitary and testicular extracts and the presence of GnRH-like material in testes and hypothalamuses were measured in the frog, Rana esculenta. Also, the cellular localization of immunoreactive GnRH was investigated in testes by immunohistochemical staining. Furthermore, lyophilized preparations of pituitary crude homogenates from animals caught monthly were tested in vitro for their ability to stimulate androgen production by December testes. Satisfactory results on specific 125I-GnRH binding were difficult to obtain in view of its low binding capacity. Moreover, binding in testicular homogenates was of the same order of magnitude (about 2%) as that found in pituitaries. In a cospecific radioimmunoassay for GnRH nonapeptide, both hypothalamic and testicular extracts gave displacement parallel to the standard curve. Immunoreactive GnRH did not significantly fluctuate in hypothalamuses, while it peaked in testes during December and July. Immunoreactive GnRH was evidenced in June and September testes employing immunohistochemical staining. In particular, the interstitial cells and the Sertoli cells were faintly stained. Testes of December animals stimulated by February pituitaries produced larger quantities of androgens as compared with testes stimulated with hypophyseal preparations from the remaining periods of the year. In conclusion, the present results are consistent with the idea that seasonal changes of the hypothalamus-hypophyseal activity play an important role in regulating the hormonal response in vertebrate testes. Moreover, we report that, in addition to rats, GnRH-like material is present in frog testes and for the first time it has been shown that such putative intratesticular material undergoes seasonal fluctuations in a vertebrate.

CRF-like immunoreactive system in the quail brain.

Nerve cells and fibres immunoreactive for synthetic ovine corticotropin releasing factor were shown to be present in several brain areas of the quail by use of immunocytochemical techniques. CRF-like immunoreactive cell bodies are mainly clustered in the hypothalamic paraventricular nucleus, in the telencephalic nucleus accumbens and in the mesencephalic periventricular grey. The external zone of the anterior median eminence contains a rich network of immunoreactive fibers. CRF-like immunopositive fibers are not restricted to circumventricular organs, but have been observed in several prosencephalic regions and in close proximity of large vasotocin-containing neurons in the supraoptic and paraventricular regions. These observations suggest that, in birds, CRF-like material might be not only involved in the hypothalamic control of anterior pituitary hormone secretion, but also in different functions likely related to neurotransmission or neuromodulation.

Habenular connections in the brain of the newt, Triturus cristatus carnifex Laurenti.

The connections of the habenular complex have been studied in the crested newt (Amphibia Urodela) by means of degeneration and HRP transport techniques. With the Fink-Heimer method, habenular efferents have been traced to the basal telencephalon, the pallium, the stria medullaris, the dorsal and ventral thalamus, the preoptic and anterior hypothalamus, the fasciculus retroflexus, the tegmentum and the interpeduncular neuropil. Anterograde transport of HRP by habenular neurons reveals fibers projections to the thalamus, the fasciculus retroflexus and the interpeduncular neuropil. After HRP injections in the habenulae, retrograde labelling of cells and fibres was observed, in the striatum, the posterior pole of the telencephalon, the thalamus, the preoptic area, the tegmentum and the raphe. The present results indicate that the habenular complex of the newt receives inputs of various sources (striatum, thalamus, hypothalamus, tegmentum) and is less directly involved in the olfactory functions.

Immunohistochemical localization of substance P-like immunoreactivity in the hypothalamus of the lizard, Podarcis sicula, R.

The distribution of substance P (SP) immunoreactivity was investigated in the hypothalamus (preoptic area included) of the lizard by single and double immunocytochemical procedures, SP-immunopositive cell bodies were seen in the paraventricular nucleus and periventricular hypothalamic gray (including the paraventricular organ) together with some more lateral elements. Extensive nerve fibers were seen in the white matter and surrounding the paraventricular and supraoptic neurons, and more caudally reaching the hypothalamic periventricular gray, suggesting a massive involvement of SP-like substance in the control of hypothalamic neuroendocrine areas.

Immunohistochemical localization of corticotropin-releasing factor (CRF)-like immunoreactivity in the hypothalamus of the newt, Triturus cristatus.

The distribution of corticotropin-releasing factor (CRF) immunoreactivity was investigated in the hypothalamus and preoptic area of the newt by single and double immunocytochemical procedures. CRF immunopositive cell bodies were seen in the preoptic area (from the anterior wall of the preoptic recess to the dorsal parts of the preoptic nucleus) and in the tuberal portions of the posterior hypothalamus. Abundant nerve fibres are seen in the outer zone of the median eminence, while the pars nervosa lacks CRF-immunoreactivity. CRF immunoreactive material is seemingly separated from neurophysins- and somatostatin immunoreactive cell bodies and fibres.

The hypothalamus of Lacerta sicula R. II. A Golgi study of the preoptic area.

An analysis of the preoptic area of the lizard, Lacerta sicula R., with the use of the Golgi method revealed that: 1) in principle, the dendritic pattern of its neurons is relatively simple; 2) the supraoptic nucleus contains large- to medium-sized bipolar or multipolar neurons together with small, usually multipolar nerve cells; 3) the preoptic periventricular gray and the paraventricular nucleus exhibit a varied neuronal typology, including large multipolar or bipolar elements, abundant CSF-contacting neurons and some tufted elements; and 4) the lateral regions display some conspicuous multipolar neurons.

Ultrastructural analysis of the posterior hypothalamus of salamander.

The anatomical organization of the tuberal area of th posterior hypothalamus in Salamandra was investigated by the Golgi methods, SEM and TEM techniques. The ependymal cells of this area are poor in cilia and often show morphological features of actively secreting or absorbing elements. Intermingled with ependymal cells, two kinds of CSF-contacting processes are seen, arising from neurons of the periventricular grey. Their contents and morphological characteristics allow them to be divided into two groups, A and B. In the neuropil different synaptic contacts are seen. The observation of unconventional synaptic contacts possibly of axo-axonic type suggests the occurrence of local circuits on the final neurosecretory pathway.

The hypothalamus of Lacerta sicula R. I. A Golgi study on the caudal hypothalamus.

The posterior (caudal) hypothalamus of the lizard, Lacerta sicula R. was investigated by means of Golgi methods. The periventricular grey is formed mainly by isodendritic bipolar and multipolar neurons, while in the lateral hypothalamus a more stellate form of neuronal elements is encountered. CSF-contacting neurons are restricted to the tuberal area and to the paraventricular organ. In the latter area they are highly differentiated and endowed with laterally branched processes. The overall pattern of the lizard hypothalamus (organization of neuropil, lateral nuclei, appearance of cell clusters, morphology of the neuronal elements) represents an intermediate stage in the phylogenetic development of the hypothalamus, being more advanced than the amphibian stage.

Substance P-related peptides in the hypothalamus of amphibia.

The occurrence of Substance P-(SP)-related peptides in the hypothalamus of three species of Amphibia (newt, clawed, toad, frog) was studied immunohistochemically employing the direct immunofluorescence method or a double-step technique (indirect immunofluorescence followed by the peroxidase-antiperoxidase complex method). SP-like immunopositive fibers are seen throughout the hypothalamus. They are especially abundant in the preoptic area and in the outer zone of the median eminence, suggesting a role of SP-related peptides in the hypothalamo-hypophysial regulation in these animals. Some SP-like neurons are seen in the posterior hypothalamus and in the preoptic area. In the newt, such SP-like immunopositive neurons occur frequently in the preoptic periventricular grey.

A Golgi study of the hypothalamus of Actinopterygii. II. The posterior hypothalamus.

The posterior hypothalami of the polypteriform, Calamoichthys, and of the teleost, Anguilla, were studied by means of the Golgi technique. In Calamoichthys, the lateral lobes are not developed and the median lobe is simple. In Anguilla, the median (tuberal) lobe shows lophodendritic, CSF-contacting cells and horizontal cells in the periventricular grey and some reticular elements directed toward the cell-poor lateral areas. In the lateral lobes the periventricular grey is formed by multipolar neurons and a diffuse population of multipolar cells of uncertain identity. The nucleus diffusus lobi lateralis is formed by scarce multipolar neurons, often placed next to the external surface of the brain. The organization of the lateral lobes in Actinopterygii is reminiscent of highly developed integrative regions.

A Golgi study of the hypothalamus of actinopterygii.

The preoptic area of a primitive actinopterygian, the polypteriform, Calamoichthys calabaricus Smithi, and of the teleost, Anguilla anguilla L., have been studied by Golgi and other neurohistological and histochemical methods. In the preoptic area two regions can be recognized: a cell rich median region and a lateral one, composed mostly of fiber systems. After Golgi staining a varied population of neurons is seen, including: (1) magnocellular neurons, endowed with a number of dendrites and an axon emerging from the main dendritic shaft; (2) lophodendritic tufted or pyriform neurons of variable morphology (often of the midget type); (3) cerebrospinal fluid contacting neurons; (4) bipolar and horizontal neurons. Of relevant interest is also the observation of commissural neurons, of a distinct precommissural necleus, and of bipolar neurons, the axons of which course toward the ventral hypopallium.

A Golgi study on the hypothalamus of amphibia. The neuronal typology.

The neuronal typology in the hypothalamus of the frog and the crested newt was studied by the Golgi technique. In the newt, piriform, multipolar or cerebrospinal fluid (CSF)-contacting neurons of relatively primitive type, according to the classification of Ramón-Moliner, are encountered in the preoptic area. Moreover, magnocellular neurons are impregnated. In the frog the preoptic area shows a more varied typology. The posterior hypothalami of the frog and the newt exhibit mainly bipolar CSF-contacting and piriform neurons. These latter are generally "tufted", but some bipolar of multipolar cells are encountered, especially in the frog. The simple anatomical organization of the amphibian hypothalamus corresponds well with the pattern of "generalized" integrative area where multimodal sensory inputs converge--including visceral information from cerebrospinal fluid by means of hypothalamic CSF-contacting sensors--to regulate the neuroendocrine outflow.