Neuroendocrine mechanisms for reproductive senescence in the female rat: gonadotropin-releasing hormone neurons.

Reproductive aging in female rats is characterized by profound alterations in the neuroendocrine axis. The preovulatory luteinizing hormone (LH) surge is attenuated, and preovulatory expression of the immediate early gene fos in gonadotropin-releasing hormone (GnRH) neurons is substantially reduced in middle-aged compared with young rats. We tested the hypothesis that alterations in GnRH gene expression may be correlated with the attenuation of the LH surge and may be a possible mechanism involved in neuroendocrine senescent changes. Sprague-Dawley rats ages 4 to 5 mo (young), 12-14 mo (middle-aged), or 25 to 26 mo (old) were killed at 10:00 AM or 3:00 PM on proestrus, the day of the LH surge, or diestrus I in cycling rats, and on persistent estrus or persistent diestrus in acyclic rats. RNase protection assays of GnRH mRNA and GnRH primary transcript were performed. GnRH mRNA levels increased significantly with age, whereas GnRH primary transcript levels, an index of GnRH gene transcription, decreased in old compared to young and middle-aged rats. This latter result suggests that an age-related change in GnRH mRNA levels occurs independently of a change in gene transcription, indicating a potential posttranscriptional mechanism. On proestrus, GnRH mRNA levels increased significantly from 10:00 AM to 3:00 PM in young rats. This was in contrast to proestrous middle-aged rats, in which this afternoon increase in GnRH mRNA levels was not observed. Thus, the normal afternoon increase in GnRH mRNA levels on proestrus is disrupted by middle age and may represent a substrate for the attenuation of the preovulatory GnRH/LH surge that occurs in rats of this age, prior to reproductive failure.

Connections of the precommissural nucleus.

The connections of the precomissural nucleus (PRC) have been examined with anterograde and retrograde axonal tracing methods in the rat. Experiments with cholera toxin B subunit (CTb) indicate that the PRC shares a number of common afferent sources with the dorsolateral periaqueductal gray (PAG). Thus, we have shown that the nucleus receives substantial inputs from the prefrontal cortex, specific domains of the rostral part of the lateral septal nucleus, rostral zona incerta, perifornical region, anterior hypothalamic nucleus, ventromedial hypothalamic nucleus, dorsal premammillary nucleus, medial regions of the intermediate and deep layers of the superior colliculus, and cuneiform nucleus. Moreover, the PRC also receives inputs from several PAG regions and from neural sites involved in the control of attentive or motivational state, including the laterodorsal tegemental nucleus and the ventral tegmental area. The efferent projections of the PRC were analyzed by using the Phaseolus vulgaris-leucoagglutinin (PHA-L) method. Notably, the PRC presents a projection pattern that resembles in many ways the pattern described previously for the rostral dorsolateral PAG in addition to projections to a number of targets that also are innervated by neighboring pretectal nuclei, including the rostrodorsomedial part of the lateral dorsal thalamic nucleus, the ventral part of the lateral geniculate complex, the medial pretectal nucleus, the nucleus of the posterior commissure, and the ventrolateral part of the subcuneiform reticular nucleus. Overall, the results suggest that the PRC might be viewed as a rostral component of the PAG, and the possible functional significance of the nucleus is discussed in terms of its connections.

Morphology of neurons in the anterior hypothalamic area and supraoptic hypothalamic nucleus of the adult human brain.

Morphological features of neuronal cell types in the anterior hypothalamic area (AHA) and supraoptic hypothalamic nucleus (SON) of the adult human brain were analysed in Golgi impregnated preparations. Four neuronal cell types were described for the first time in these human nuclei. Type I neurons were found in both the AHA and SON, while the other three cell types (types II-IV) were found only in the SON. Type I neurons had elongated, triangular or multipolar somata, which emitted 2-5 sparsely branching primary dendrites with a moderate number of fine spines. Also many of type I neurons in the AHA had thin dendritic side-branches. Type II neurons had round or fusiform somata, and two occasionally branching primary dendrites. Type III neurons were multipolar neurons with 3-5 densely spined and sparsely branching dendrites. Their axons had collaterals. Type IV neurons had very small ovoid somata with one smooth and unbranched primary dendrite. The neurons in the human AHA and SON were similar to those observed in the same areas in other mammalian species, except for the very small neurons in the SON and the thin dendritic side-branches of type I neurons in the AHA, that had not been previously described.

Characterization of progesterone-3-[125I-BSA] binding sites in the medial preoptic area and anterior hypothalamus.

In this study we utilized radiolabeled progesterone (P) conjugated to bovine serum albumin (BSA) at position 3 (P-3-[125I-BSA]) to examine steroid receptors in membrane fractions from the medial preoptic area-anterior hypothalamus (MPOA-AH) of ovariectomized (OVXed) rats. In the MPOA-AH binding of P-3-[125I-BSA] was linear across a tissue concentration range of 0.005 to 0.02 mg protein/0.1 ml of membrane suspension. Kinetic experiments revealed an association t(1/2) of 51.4 min and a dissociation t(1/2) of 122.5 min for P-3-[125I-BSA] at 0 degrees C. Analysis of data from competition binding experiments using P-3-BSA revealed high- and low-affinity binding sites in the MPOA-AH. Involvement of MPOA-AH binding sites with a G-protein was suggested by a reduction of P-3-[125I-BSA] binding in the presence of the non-hydrolyzable GTP analog GTPgammaS but not ATPgammaS. In addition, if homogenates from the MPOA-AH were preincubated with 10(-5) M of the G-protein antagonist cholera toxin for 30 min at 37 degrees C, competition binding data indicated only high-affinity binding sites. Once daily injections of OVXed rats with 4 mg P for 12 days significantly increased the density of P-3-[125I-BSA] binding sites in the MPOA-AH. This treatment did not affect P-3-[125I-BSA] binding in the dorsal tectum, medial basal hypothalamus, ventral tegmental area or the thymus.

Stress-induced changes in messenger RNA levels of N-methyl-D-aspartate and AMPA receptor subunits in selected regions of the rat hippocampus and hypothalamus.

The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.

An ultrastructural study of somal appositions in the suprachiasmatic nucleus and anterior hypothalamus of the rat.

Somatic appositions of glial and neuronal elements in the suprachiasmatic nucleus (SCN) and the anterior hypothalamus (AH) were evaluated. Neuronal somas in the SCN exhibited more coverage by astrocytes than did those in the AH. Conversely, somas in the AH showed more coverage by neuronal elements than those in the SCN. All measurements of somal appositions were independent of circadian influences.

Fos-like expression and nuclear size in osmotically stimulated supraoptic nucleus neurons.

This study has analysed by immunocytochemistry the pattern of expression of Fos-related proteins, as well as variations in nuclear size, after the osmotically induced activation of supraoptic nucleus neurons of the rat. In control rats most supraoptic nucleus neurons were Fos-like negative. After acute and chronic dehydration by salt-loading, the number of Fos-like positive neurons increased dramatically. The level of Fos-like immunoreactivity was higher in chronically stimulated rats, and also the neurons of the ventral region of the supraoptic nucleus were more intensely stained than those of the dorsal region. The karyometric analysis was made on electron micrographs. The mean nuclear profile area showed a significant increase in dehydrated rats with respect to the controls (73 +/- 16 microns 2 in those dehydrated for six days vs 54 +/- 13 in controls, mean +/- S.D.). However, no significant differences in this parameter were found when one-day and six-day dehydrated groups were compared. The invagination factor of the nuclear membrane, a nuclear shape indicator, decreased significantly in dehydrated rats, indicating a tendency towards spherical nuclei. It is noteworthy that the nuclear profile perimeter was constant, about 32 microns, in control and osmotically simulated rats. The higher nuclear accumulation of Fos-related antigens after six days of dehydration suggests that in chronically stimulated supraoptic nucleus neurons there is a sustained induction of cell-specific genes. Moreover, the transcription rate of the target genes containing the consensus DNA sequence TGAC/GTCA or c-AMP responsive elements recognition sites may depend upon the nuclear concentration of Fos-related antigens in supraoptic nucleus neurons. Our results also suggest that the initial Fos-related antigen expression and nuclear size increase are triggered concomitantly in supraoptic nucleus neurons after a short period of osmotic stimulation. On the other hand, we propose that nuclear envelope invaginations represent a reservoir of nuclear membrane which allows dynamic changes in nuclear size and shape depending on the metabolic status of the supraoptic nucleus neurons.

[Age-related change of synaptic number in the suprachiasmatic nucleus of the rat hypothalamus].

Numerical changes of the synapses in the hypothalamic suprachiasmatic nuclei (SCN) of rats at different ages were investigated with electron microscope, The population of the major SCN synapses formed with dendrite and total number of synapses were found to be reduced with advancing age. Compared with the young animal group, the synaptic number was statistically different from that in the adult or senescent group. As a result of these changes, the function of SCN might decline with aging.

Ultrastructural changes in the hypothalamic secretory nuclei of rats depending on the duration of clinical death.

The studies were carried out on neurosecretory nuclei of rat hypothalamus following complete circulatory arrest for 5 min (group I) and 10 min (group II). The surviving time of the animals after the experiment was 6 weeks. In group I, the ultrastructural appearance of the perikaryonic areas of the neurons indicates increased metabolic activity of these cells. Crinophagocytic bodies were noted near the Golgi apparatus. In the processes of neurons changes in the structure of the cytoskeletal elements were observed. In group II significant differences were noted, as compared with group I. They consisted in the desolation of the rough endoplasmic reticulum membranes from ribosomes, dilatation of the Golgi area cisternae, and swelling of mitochondria. In the perivascular region cells with the surface of the cytoplasmic processes membranes covered by the product of the Alcian blue reaction were noticed. In our opinion these cells may represent "cerebral macrophages". The ultrastructural changes were more pronounced in animals of group II, as compared with animals of group I.

Efferent projections from the lateral septal nucleus to the anterior hypothalamus in the rat: a study combining Phaseolus vulgaris-leucoagglutinin tracing with vasopressin immunocytochemistry.

The tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the lateral septum of the rat at different rostrocaudal locations to study the efferent septal projections to the anterior hypothalamus. For spatial correlation of these septofugal elements with the vasopressinergic system a dual immunocytochemical technique was used (i) to demonstrate nerve fibers and their corresponding bouton-like structures labeled with the tracer, and (ii) to identify vasopressin in the same section. The hypothalamic paraventricular and supraoptic nuclei, the accessory hypothalamic magnocellular system, and the suprachiasmatic nucleus are recipients of PHA-L-labeled fibers from all parts of the lateral septum. Close appositions between (i) these axons and their varicosities, and (ii) vasopressin-immunoreactive perikarya and their processes, putatively indicating functional interrelationships, were observed in all these nuclear areas, especially in their neuropil formations.

Immunohistochemical evidence for synaptic connections between neuropeptide Y-containing axons and periventricular somatostatin neurons in the anterior hypothalamus in rats.

By employing a pre-embedding double immunolabeling technique, we examined light and electron microscopically synaptic associations between neuropeptide Y (NPY)-containing axons and somatostatin (SRIH)-containing neurons in the anterior periventricular area (APV) of the rat hypothalamus. For light microscopy, the immunoreactions for NPY and SRIH were visualized with silver-gold and diaminobenzidine (DAB), respectively, and the reverse labeling was used for electron microscopy. Light microscopy disclosed many brown SRIH perikarya surrounded by several black beads of NPY fibers in the APV. In electron microscopy, immunoreactive SRIH neurons revealed silver-gold particles scattered throughout the cytoplasm and accumulated in the Golgi area and the secretory granules. SRIH perikarya and dendritic processes indicated synaptic associations with DAB-labeled NPY fiber terminals and immunonegative fibers. NPY presynaptic terminals possessed numerous small clear vesicles and a few dense core vesicles; vesicular membranes and cores were labeled with DAB chromogen. Both the pre- and postsynaptic membranes were thickened equally to be a symmetric synapse. These findings suggest that NPY neurons are involved in the regulation of growth hormone secretion from the pituitary by affecting periventricular SRIH neurons.

Innervation of somatostatin synthesizing neurons by adrenergic, phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive axons in the anterior periventricular nucleus of the rat hypothalamus.

The adrenergic innervation of somatostatin synthesizing neurons located in the anterior region of the rat hypothalamic periventricular nucleus was studied by means of a light and electron microscopic immunocytochemical double labelling technique. This region which is the source of hypophysiotrophic somatostatin immunoreactive (IR) neurons also receives a dense plexus of adrenergic axons as determined by immunocytochemistry of phenylethanolamine-N-methyltransferase (PNMT), the marker enzyme for the central adrenergic system. The simultaneous detection of PNMT and somatostatin antigens in hypothalamic sections of colchicine pretreated animals revealed a congruency in the distribution of the labelled elements and also close juxtaposition of PNMT-IR axons to somatostatin producing neurons. At the ultrastructural level, axo-somatic and axo-dendritic synaptic connections were found between PNMT-containing axons and somatostatin expressing neurons. These morphological findings support the view that the central adrenergic system might influence the production and secretion of growth hormone in the pituitary gland by a direct monosynaptic interaction with somatostatin synthesizing neurons.

Ultrastructural evidence for gamma aminobutyric acid-immunoreactive synapses on somatostatin-immunoreactive perikarya in the periventricular anterior hypothalamus.

Somatostatin-immunoreactive perikarya in the periventricular anterior hypothalamus were demonstrated to be surrounded by gamma aminobutyric acid GABA-immunoreactive nerve terminals, by combining pre-embedding immunocytochemistry for somatostatin and gold labelling post-embedding immunocytochemistry for GABA. Ultrastructural studies revealed that in each 100-nm section, cells immunoreactive for somatostatin (n = 62) were contacted by a mean of 7.6 +/- 0.4 terminal profiles of which 3.0 +/- 0.3 (40%) were GABA-immunoreactive. Most GABA-immunoreactive terminals contained clear rounded vesicles and, where synaptic specializations were well demonstrated, appeared to be symmetric. The finding provides evidence that there is a significant GABA input to somatostatin neurons, an observation strengthening the hypothesis that GABA may inhibit somatostatin neurons, thereby causing increased secretion of growth hormone.

The hypothalamic magnocellular system in the domestic fowl. Study on semithin sections.

Neuronal characteristics and location of the neurosecretory, magnocellular, fuchsin-paraldehyde-positive (FA+) system of the fowl are described at the light-microscopic level on serial semithin sections. Three nuclei make up this system, the nucleus supraopticus, n. magnocellularis interstitialis and n. paraventricularis. These nuclei display magnocellular neurons, not showing a parvocellular component. The neurons of the three nuclei showed a scattered pattern of distribution and a dense surrounding neuropil. Groups formed by magnocellular neurons were found in the three nuclei and groups formed by one magnocellular and a parvocellular neurons were only found in the n. magnocellularis interstitialis and in the n. paraventricularis. The presence of neurons in apposition to blood vessels was frequent in the magnocellular FA+ system of the domestic fowl.

The anterior periventricular hypothalamus is the site of somatostatin inhibition of its own release: an in vitro and immunocytochemical study.

The site of action of the inhibitory effect of somatostatin (SRIF) on its own release was studied by: (1) measuring SRIF release in vitro from tissue preparations containing either the proximal (periventricular hypothalamus) or the distal (median eminence) portions of the hypothalamic SRIF neurons, and (2) immunocytochemical investigation of the interconnections occurring between SRIF neuronal elements in these hypothalamic regions. In vitro, a biologically active, but noncross-reacting SRIF analog (D-Trp8 SRIF) in the RIA, inhibited 25 mM K+ induced SRIF release from anterior periventricular hypothalamic tissues. The inhibitory effect of D-Trp8 SRIF was dose-dependent, maximal at 10(-7) M, and restricted to this anterior region, since median-eminence SRIF release was not modified by the presence of D-Trp8 SRIF. Additionally, LHRH release from anterior periventricular hypothalamus was unchanged in the presence of D-Trp8 SRIF. In the periventricular nucleus, perikarya and dendrites of labeled SRIF neurons showed frequent apposition of their limiting membranes. Classical synapses were also observed between SRIF-containing axonal processes and labeled perikarya or dendrites. Although membrane appositions between neighboring SRIF axons frequently occurred in the median eminence, no synaptic-like SRIF-SRIF connections could be detected at this level. The data demonstrate a direct inhibitory action of a SRIF agonist on the anterior periventricular hypothalamic release of the peptide. This effect correlates well with the occurrence of SRIF-SRIF synapses in this region; suggesting that SRIF exerts a negative feedback in the control of its own release through autoreceptors located on the perikarya or dendrites of SRIF-containing neurons.

Peptidergic neurosecretory axons regenerate into sciatic nerve autografts in the rat hypothalamus.

Pieces of intact or degenerated sciatic nerves autografted into contact with transected neurosecretory axons within the hypothalamus were invaded by neurophysin-positive axons. With increasing time after grafting, increasing numbers of axons were present in both types of grafts, but grafts of degenerated sciatic nerves always contained more axons. At the fine-structural level typical neurosecretory as well as nonneurosecretory axons were usually associated with basal lamina-enclosed neurolemmocyte processes; occasional axons occurred among collagen fibrils or within basal lamina scaffolds. Profiles with the fine structural characteristics of axon terminals were present by 20 days after transplantation.

Fetal hypothalamic transplants in diabetic rats analysed by scanning electron microscopy.

Adult male Brattleboro rats with chronic diabetes insipidus underwent stereotaxic surgery wherein minced fragments of anterior hypothalamus from fetal rats, 17 days post-coitus, were stereotaxically positioned into the lumen of the host third cerebral ventricle. Host rats with fetal donor tissue were killed at various times following surgery and were prepared for correlative scanning-transmission electron microscopy. Examination with this technique revealed the presence of large neurografts which grew to occupy the entire lumen of the host third ventricle. Grafts were well vascularized and in addition exhibited remarkable numbers of supraependymal, cerebrospinal fluid-contacting neurons. The physical emergence of this cell line in proximity to viable grafts is discussed with respect to the biochemical influences that a neuropeptide producing fetal transplant has upon a peptide-deficient host.

[Ultrastructure of the neurosecretory cells of the anterior commissural nucleus of the hypothalamus in rats]. / Ul'trastruktura neirosekretornykh kletok perednekomissural'nogo iadra gipotalamusa krys.

The ultrastructure of neurosecretory cells of the anterior commissural nucleus of rat hypothalamus is similar to that of the supraoptic nucleus and of the "magnocellular" part of the paraventricular nucleus. The only difference is a less expressed granular endoplasmatic reticulum and a smaller diameter of elementary neurosecretory granules (80-150 nm in diameter). Such elementary granules are characteristic of neurosecretory terminals located in the external zone of the median eminence. It is suggested that neurosecretory cells of the anterior commissural nucleus project to this neurohemal region.

[Neurocyte reaction of the anterior and mediobasal hypothalamic nuclei in birds to testosterone propionate]. / Reaktsiia neirotsitov iader perednego i mediobazal'nogo gipotalamusa ptits na testosteron-propionat.

Some magnocellular and parvocellular hypothalamic nuclei of cockerel were studied for their reactivity to testosterone-propionate (TP). It has been ascertained that injections of 1 or 5 micrograms/100 g doses of TP activate the neurocytes of preoptic periventricular and tuberal nuclei. This effect is manifested between 45th and 60th day of life and may be regarded as an early event of sexual maturation.