DiI tracing of the hypothalamic projection systems during perinatal development.

The hypothalamus is the higher neuroendocrine center of the brain and therefore possesses numerous intrinsic axonal connections and is connected by afferent and efferent fiber systems with other brain structures. These projection systems have been described in detail in the adult but data on their early development is sparse. Here I review studies of the time schedule and features of the development of the major hypothalamic axonal systems. In general, anterograde tracing experiments have been used to analyze short distance projections from the arcuate and anteroventral periventricular nuclei (Pe), while hypothalamic projections to the posterior and intermediate pituitary lobes (IL) and median eminence, mammillary body tracts and reciprocal septohypothalamic connections have been described with retrograde tracing. The available data demonstrate that hypothalamic connections develop with a high degree of spatial and temporal specificity, innervating each target with a unique developmental schedule which in many cases can be correlated with the functional maturity of the projection system.

Prenatal carbocyanine dye tracing of septo-hypothalamic connections.

This is the first study of the prenatal development of septal projections to the hypothalamus in rats, using carbocyanine dyes (DiI and DiA) as retrograde tracers. First septal neurons send axons to the preoptic area and anterior hypothalamus on embryonic day 14,5 (E14,5) and on E15 numerous labeled neurons are visualized in the septum after DiI insertion into the preoptic region. On E18 and E20 these neurons develop numerous spiny dendrites that occupy all rostrocaudal extension of the septum with concentration in the ventral part of the septum. Only a few septal neurons send their axons to the mediobasal hypothalamus at E15 confirmed by double-labeling (DiI+DiA) experiments on E20-E21. All septo-hypothalamic connections are unilateral and the number of the neurons revealed in the septum correlates with the place and size of the DiI insertion in the hypothalamus: more lateral and anterior hypothalamic marker insertions always resulted in significant neuronal labeling in the septum. No septal connections with the posterior hypothalamus specifically, the mammillary bodies are formed prenatally. We have demonstrated that the development of septal projections to various rostrocaudal regions of the hypothalamus take place during different stages of development. Prominent parts of the septal projections are to the preoptic area and anterior hypothalamus while few connections with the mediobasal hypothalamus are formed prenatally. These data provide basic knowledge of early steps of the development of the septo-hypothalamic connections.

Normalization of hypothalamic serotonin (5-HT 1B) receptor and NPY in cancer anorexia after tumor resection: an immunocytochemical study.

Tumor growth leads to anorexia and decreased food intake, the regulation of which is via the integrated hypothalamic peptidergic and monoaminergic system. Serotonin (5-HT), an anorectic monoamine acts primarily via 5-HT 1B-receptors in hypothalamic nuclei while neuropeptide Y (NPY) acts an orexigenic peptide. We previously reported that 5-HT 1B-receptors are up regulated while NPY is down regulated in tumor-bearing (TB)-related anorexia, contributing to food intake reduction. In anorectic TB rats we hypothesize that after tumor resection when food intake has reverted to normal, normalization of 5-HT 1B-receptor and NPY will occur. The aim of this study was to demonstrate normalization of these hypothalamic changes compared to Controls. In anorectic tumor-bearing rats after tumor resection (TB-R) and in sham-operated (Control) rats, distribution of 5-HT 1B-receptors and NPY in hypothalamic nuclei was analyzed using peroxidase antiperoxidase immunocytochemical methods. Image analysis of immunostaining was performed and the data were statistically analyzed. Immunostaining specificity was controlled by omission of primary or secondary antibodies and pre-absorption test. Our results show that after TB-R versus Controls a normalization of food intake, 5-H-1B-receptor and NPY expression in the hypothalamus occurs. These data, discussed in context with our previous studies, support the hypothesis that tumor resection results not only in normalization of food intake but also in reversible changes of anorectic and orexigenic hypothalamic modulators.

Axonal projections from the hypothalamus to the pituitary intermediate lobe in rats during ontogenesis: DiI tracing study.

This study has determined ontogenetic schedule of axonal arrival from the hypothalamus in the pituitary intermediate lobe (IL) in rats using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. The brains with attached pituitaries were dissected in rats from the 20th embryonic day (E20) to the 20th postnatal day (P20). Anterior lobe was mechanically detached from the IL, material fixed in paraformaldehyde, and DiI crystals were applied on the IL laying on the posterior lobe (PL). The labeling of IL + PL resulted in staining of hypothalamic magnocellular neurons, which send their axons to the PL, and hypothalamic parvocellular neurons contributing to the innervation of the IL. Therefore, the magnocellular neurons were not taken into account when identifying the neurons projecting axons to the IL. Rare fluorescent neurons projecting their axons to the IL were detected as early as on E20 in the ventral part of the periventricular nucleus (Pe) and in the rostral part of the arcuate nucleus. Few DiI-labeled neurons were seen in Pe from P1 to P3. At P5, the fluorescent neurons were accumulated giving rise to the prominent cluster in the Pe, which was enlarged on later stages and occupied all the Pe. In addition to the Pe, fluorescent neurons first appeared in the retrochiasmatic region and around the ventromedial nucleus in young rats. Thus, the axons of hypothalamic neurons of the Pe and mediobasal hypothalamus first arrive in the IL in rats at the end of intrauterine development, although the principal innervation of the IL is the postnatal event.

Hypothalamic 5-HT1B-receptor changes in anorectic tumor bearing rats.

Serotonin (5-HT) is an anorectic monoamine and its regulatory effects on feeding are mediated primarily via 5-HT1B-receptors localized in the hypothalamic nuclei, which, apart from the brain stem, are among the most crucial areas of food intake regulation. The distribution of 5-HT1B-receptors in the hypothalamic nuclei was studied in tumor-bearing (TB) rats at the onset of anorexia and in sham-operated control rats, using the peroxidase-anti-peroxidase immunocytochemical method and specific polyclonal antiserum. Semiquantitative image analysis of 5-HT1B-receptor immunostaining was performed on high-resolution digital photomicrographs using the NIH Scion Image analysis program and the data were compared using Student's t-test. Immunostaining detected 5-HT1B-receptor proteins in the same hypothalamic structures in the Controls as in the TB rats. Qualitative and semiquantitative analysis revealed a significant increase in 5-HT1B-receptor expression in the magnocellular neurons of paraventricular and supraoptic hypothalamic nuclei in TB rats versus Controls. In contrast, changes were not significant in the parvocellular portion of paraventricular nucleus or in the lateral hypothalamus including perifornical region. These findings emphasize serotonin's influence on the magnocellular hypothalamic nuclei during developing of cancer anorexia, which is associated with a decrease in food intake.

Physiological and morphological characteristics of the rhythmic contractions of the amnion in veiled chameleon (Chamaeleo calyptratus) embryogenesis.

A morpho-functional study of the veiled chameleon (Chamaeleo calyptratus) amnion was performed as part of a general comparative study of amnion rhythmic contractions (ARC) in reptile and bird embryogenesis. Eggs incubated at 27.5 degrees C were used at different developmental stages from day 80 (D80) to D184 for the recording of ARC using a force transducer. Slow ARC, about 1 min in duration, were revealed from D88 (stage 31) to the near-hatching stages (incubation time was 183-198 days). The frequency and amplitude of slow ARC increased significantly towards the end of incubation. This ARC enhancement correlated with the differentiation of smooth muscle elements in the amniotic membrane from single spindle-shaped cells to complex "star-like" structures and with the development of the actin fibers, revealed by phalloidin, in the amniotic muscle layer. Short-term changes in temperature influenced ARC and heart rate (HR). Cooling to 25 degrees C from the control (27.5 degrees C) resulted in a significant decrease in both ARC frequency and HR. Heating to 30 degrees C significantly increased the embryonic HR, but not ARC frequency. Within the temperature range from 25 to 30 degrees C the temperature effect on ARC frequency and HR was reversible.

Decreased NPY innervation of the hypothalamic nuclei in rats with cancer anorexia.

Whether the decrease in food intake that occurs at the onset of anorexia in tumor bearing (TB) rats is related to a change in the hypothalamic neuropeptide Y (NPY) system was tested by comparing NPY expression in sham operated Fischer Control and anorectic TB rats. Coronal cryocut sections of their fixed brain were processed by the peroxidase-antiperoxidase method with NPY polyclonal antibodies. NPY-immunoreactive fibers were widely distributed throughout the forebrain, but were most prominent in the hypothalamic paraventricular, suprachiasmatic, arcuate and periventricular nuclei. NPY-immunoreactive neurons were visualized in Control and anorectic TB rats in the preoptic region, the arcuate nucleus, and occasionally in the lateral hypothalamus. Semiquantitative image analysis showed a significant decrease in the NPY immunostaining in some hypothalamic nuclei of the anorectic TB rats, most prominently in the supraoptic nucleus, the parvocellular portion of the paraventricular nucleus, and, to a lesser extent, the suprachiasmatic and arcuate nuclei. No changes in NPY innervation were seen in the ventromedial nucleus and the lateral hypothalamus. The data support the hypothesis of an altered hypothalamic NPY system at the onset of anorexia in TB rats and also reveal the hypothalamic nuclei through which NPY influences food intake.

Neurons possessing enzymes of dopamine synthesis in the mediobasal hypothalamus of rats. Topographic relations and axonal projections to the median eminence in ontogenesis.

We evaluated the topographic relations between tyrosine hydroxylase (TH)- and/or aromatic L-amino acid decarboxylase (AADC)-immunoreactive neurons in the arcuate nucleus (AN), as well as between TH- and/or AADC-immunoreactive axons in the median eminence (ME) in rats at the 21st embryonic day, 9th postnatal day, and in adulthood. The double-immunofluorescent technique in combination with confocal microscopy was used. Occasional bienzymatic neurons but numerous monoenzymatic TH- or AADC-immunoreactive neurons were observed in fetuses. There was almost no overlap in the distribution of monoenzymatic neurons, and therefore few appositions were observed in between. In postnatal animals, numerous bienzymatic neurons appeared in addition to monoenzymatic neurons. They were distributed throughout the AN resulting in the increased frequency of appositions. Furthermore, specialized-like contacts between monoenzymatic TH- and AADC-immunoreactive neurons appeared. The quantification of the fibers in the ME showed that there were large specific areas of the monoenzymatic TH-immunoreactive fibers and bienzymatic fibers in fetuses, followed by the gradual reduction of the former and the increase of the latter to adulthood. The specific area of the monoenzymatic AADC-immunoreactive fibers in fetuses was rather low, and thereafter increased progressively to adulthood. The fibers of all the types were in apposition in the ME at each studied age. Close topographic relations between the neurons containing individual complementary enzymes of dopamine synthesis at the level of cell bodies and axons suggest functional interaction in between.

Central mechanisms involved with catabolism.

PURPOSE OF REVIEW: Catabolism conjures up an end-metabolic process in which muscle and fat tissue are broken down into their constituent parts to provide nutrients for the body, secondary to a noxious stimulus that prevents the organism from adequately nourishing itself. However, catabolism is a primary event, initiated in the brain in response to perceived or real stresses or noxious stimuli, which has a secondary effect of inhibiting food intake and consequently the break down of skeletal muscle and adipose tissues to provide nutrients for the body to survive. RECENT FINDINGS: This is achieved via a cascade of neurohormonal monoaminergic and peptidergic mediators in the central nervous system, invoking the cortex, the limbic system and the hypothalamus. Among the most detailed mediators studied are corticotropin-releasing factor and serotonin which, via the hypothalamic-pituitary-adrenal axis and the sympathetic and parasympathetic nervous system, stimulate catecholamines and cortisol and inhibit anabolic hormones, insulin, leptin, ghrelin, including neuropeptide Y and other neuropeptides, among them the paracrine-acting cytokines. Simultaneously, there occurs stimulation of the counter-regulatory hormones cortisol, glucagon and the melanocortin family of neuropeptides. SUMMARY: The net effect is anorexia, with the inhibition of food intake, body weight loss, delayed gastric emptying and functions, the stimulation of gluconeogenesis, glycogenolysis and ketogenesis as sources of metabolic fuel, which if unabated leads ultimately to cachexia. The use of antagonists and the removal of stress or noxious stimuli experimentally test different pathways of this dynamic metabolic picture. Several studies have demonstrated important progress towards our understanding of the central mechanisms involved in anorexia and weight loss, which we summarize in this review.