Embryonic Ethanol Exposure Affects the Early Development, Migration, and Location of Hypocretin/Orexin Neurons in Zebrafish.

BACKGROUND: Embryonic ethanol (EtOH) exposure is known to increase alcohol drinking later in life and have long-term effects on neurochemical systems in the brain. With zebrafish having marked advantages for elucidating neural mechanisms underlying brain disorders, we recently tested and showed in these fish, similar to rodents, that low-dose embryonic EtOH stimulates voluntary consumption of EtOH while increasing expression of hypocretin/orexin (hcrt) neurons, a neuropeptide that promotes consummatory and reward-related behaviors. The goal of the present study was to characterize how embryonic EtOH affects early development of the hcrt system and produces persistent changes at older ages that may contribute to this increase in EtOH consumption. METHODS: We utilized live imaging and Imaris software to investigate how low-dose embryonic EtOH (0.5%), administered from 22 to 24 hours postfertilization, affects specific properties of hcrt neurons in hcrt:EGFP transgenic zebrafish at different ages. RESULTS: Time-lapse imaging from 24 to 28 hpf showed that embryonic EtOH increased the number of hcrt neurons, reduced the speed, straightness, and displacement of their migratory paths, and altered their direction early in development. At older ages up to 6 dpf, the embryonic EtOH-induced increase in hcrt neurons was persistent, and the neurons became more widely dispersed. These effects of embryonic EtOH were found to be asymmetric, occurring predominantly on the left side of the brain, and at 6 dpf, they resulted in marked changes in the anatomical location of the hcrt neurons, with some detected outside their normal position in the anterior hypothalamus again primarily on the left side. CONCLUSIONS: Our findings demonstrate that low-dose embryonic EtOH has diverse, persistent, and asymmetric effects on the early development of hypothalamic hcrt neurons, which lead to abnormalities in their ultimate location that may contribute to behavioral disturbances, including an increase in EtOH consumption.

Variations of nNOS and its mRNA in the neurons of the hypothalamic supraoptic nucleus in response to stress by immobilization

Using rats subjected to immobilization stress, the aim of the present study was to investigate the expression of neuronal nitric oxide synthase and of its mRNA in the magnocellular neurons of the supraoptic nucleus of the hypothalamus, which does not have parvocellular neurons or direct neurohaemal connections with the anterior lobe of the hypophysis. nNOS expression was studied with immunohistochemistry using sheep antinNOS serum, and the intraneural detection of nNOS mRNA was accomplished using a nonisotopic in situ hybridization technique, employing a specific biotinylated probe. The acute stress elicited by restraint induced an increase in the overall size of the supraoptic nucleus, together with an increase in the number of magnocellular neurons expressing nNOS immunoreaction. The optical densitometry values of the nNOS immunoreaction, the nuclear areas of the immunoreactive neurons, and the density of neurons showing nNOS mRNA hybrids were higher in the supraoptic nucleus of the stress-treated animals than in the controls, suggesting that nitric oxide of nNOS origin plays an active role in the magnocellular neurons of the hypophyseal-hypothalamic system and the involvement of this system in the stress axis (AU)

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Brief daily suckling shifts locomotor behavior and induces PER1 protein in paraventricular and supraoptic nuclei, but not in the suprachiasmatic nucleus, of rabbit does.

Nursing in the rabbit is a circadian event during which mother and pups interact for a period of < 5 min every day. Here we explored behavioral and neuronal changes in the mother by analyzing the suprachiasmatic nucleus (SCN), and oxytocinergic (OT) neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). We maintained lactating does in a light-dark cycle (lights on at 07 : 00 hours; ZT0); they were scheduled to nurse during either the day (ZT03) or the night (ZT19). Groups of intact and nursing females was perfused, one at each 4-h point through a 24-h cycle. We explored, by immunohistochemistry, the PER1 expression and double-labeling, with OT antibody, of neurons in the PVN and SON at lactation on day 7. In the SCN, intact and lactating groups had peak PER1 expression at ZT11; however, there was a reduction in PER1 at peak time in the nursing groups. There was a locomotor activity rhythm with increased activity around the time of lights-on in intact subjects and around the time of suckling in lactating does. There was an induction of PER1 in OT cells in the PVN and SON that shifted in phase with timing of nursing. We further explored the maintenance of the PER1 expression in OT cells in nursing-deprived does and found a significant decrease at 24 and 48 h after the last nursing. We conclude that suckling induced PER1 in the PVN and SON, but not in the SCN, in nursing does, and also shifted their locomotor behavior.

The central vasopressinergic system: examining the opportunities for psychiatric drug development.

Arginine vasopressin (AVP) is a cyclic nonapeptide synthesized exclusively by neurosecretory cells of the central nervous system (CNS). Two functionally distinct vasopressinergic systems can be defined based on differences in the sites of action and release of AVP. The peripheral vasopressinergic system encompasses the sites of action for AVP released into peripheral circulation (e.g. vascular smooth muscle, liver, kidney) from nerve terminals in the posterior pituitary. Peripherally circulating AVP is responsible for the classic endocrine functions ascribed to this neurohormone (e.g. vasoconstriction, glycogen metabolism, antidiuresis). The central vasopressinergic system, on the other hand, includes the sites of AVP synthesis and release within the CNS, where AVP acts as a neuromodulator/neurotransmitter regulating an array of CNS-mediated functions (e.g. learning and memory, neuroendocrine reactivity, social behaviors, circadian rhythmicity, thermoregulation, and autonomic function). Historically, pharmaceutical interest has focused on drug development efforts that sought to exploit the peripheral effects of AVP. Evidence, however, from clinical studies and animal models of CNS disorders has directly implicated disturbances in vasopressinergic activity in the pathophysiology of a number of human psychiatric disorders (mood, anxiety, and cognitive disorders). This review will examine the available evidence of central vasopressinergic system involvement in psychiatric disorders, and the potential opportunities for development of novel psychopharmaceuticals around this system will be discussed. Specific lines of evidence will be presented which rationalize each AVP receptor subtype (V(1)R or V(1a), V(2)R, V(3)R or V(1b)) as a molecular target for particular psychiatric indications.

The volume of a sexually dimorphic nucleus in the ovine medial preoptic area/anterior hypothalamus varies with sexual partner preference.

Sheep are one of the few animal models in which natural variations in male sexual preferences have been studied experimentally. Approximately 8% of rams exhibit sexual preferences for male partners (male-oriented rams) in contrast to most rams, which prefer female partners (female-oriented rams). We identified a cell group within the medial preoptic area/anterior hypothalamus of age-matched adult sheep that was significantly larger in adult rams than in ewes. This cell group was labeled the ovine sexually dimorphic nucleus (oSDN). In addition to a sex difference, we found that the volume of the oSDN was two times greater in female-oriented rams than in male-oriented rams. The dense cluster of neurons that comprise the oSDN express cytochrome P450 aromatase. Aromatase mRNA levels in the oSDN were significantly greater in female-oriented rams than in ewes, whereas male-oriented rams exhibited intermediate levels of expression. Because the medial preoptic area/anterior hypothalamus is known to control the expression of male sexual behaviors, these results suggest that naturally occurring variations in sexual partner preferences may be related to differences in brain anatomy and capacity for estrogen synthesis.

Cyto- and chemoarchitecture of the hypothalamus of a wallaby ( Macropus eugenii) with special emphasis on oxytocin and vasopressinergic neurons.

We have studied the organization of the hypothalamus in an Australian diprotodontid metatherian mammal, the wallaby ( Macropus eugenii), using cytoarchitectural, histochemical and immunohistochemical techniques. Coronal sections of adult brains were processed for Nissl staining, histochemical reactivity (cytochrome oxidase, nicotinamide adenine dinucleotide phosphate diaphorase and acetylcholinesterase) and immunohistochemistry (antibodies to tyrosine hydroxylase, calbindin, calretinin, non-phosphorylated neurofilament protein, oxytocin and vasopressin). The distribution of immunoreactive neurons for these substances was mapped with the aid of a computer-linked microscope. In general, the wallaby hypothalamus showed a similar nuclear organization to that seen in rodents. The paraventricular nucleus could be divided into several subdivisions based on the different cellular parcellation, similar to that described in rodents. The ventromedial hypothalamic nucleus had cell-sparse dorsomedial and cell-dense ventrolateral subdivisions as seen in eutheria, suggesting a similar functional compartmentalization in all theria. The positions of tyrosine hydroxylase-positive neurons in the wallaby hypothalamus were also similar to those in eutheria. Oxytocin and vasopressinergic neurons were found in all the same major nuclear groups as seen in eutheria, although a nucleus circularis could not be identified. The general similarities between wallaby and eutherian hypothalamus indicate that the basic chemo- and cytoarchitectural features of the hypothalamus are common to eutheria and metatheria and validate the use of the wallaby as a mammalian model of wide applicability in investigations of hypothalamic functional development.

Facilitative role of prolactin-releasing peptide neurons in oxytocin cell activation after conditioned-fear stimuli.

Emotional stress activates oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei and stimulates oxytocin release from the posterior pituitary. Oxytocin neurons in the hypothalamus have synaptic contact with prolactin-releasing peptide (PrRP) neurons. Intracerebroventricular administration of PrRP stimulates oxytocin release from the pituitary. These observations raise the possibility that PrRP neurons play a role in oxytocin response to emotional stress. To test this hypothesis, we first examined expression of Fos protein, an immediate early gene product, in the PrRP neurons in the medulla oblongata after conditioned-fear stimuli. Conditioned-fear stimuli increased the number of PrRP cells expressing Fos protein especially in the dorsomedial medulla. In order to determine whether PrRP cells projecting to the supraoptic nucleus are activated after conditioned-fear stimuli, we injected retrograde tracers into the supraoptic nucleus. Conditioned-fear stimuli induced expression of Fos protein in retrogradely labeled PrRP cells in the dorsomedial medulla. Finally we investigated whether immunoneutralization of endogenous PrRP impairs oxytocin release after emotional stimuli. An i.c.v. injection of a mouse monoclonal anti-PrRP antibody impaired release of oxytocin but not of adrenocorticotrophic hormone or prolactin and did not significantly change freezing behavior in response to conditioned-fear stimuli. From these data, we conclude that PrRP neurons in the dorsomedial medulla that project to the hypothalamus play a facilitative role in oxytocin release after emotional stimuli in rats.

RANTES: a new prostaglandin dependent endogenous pyrogen in the rat.

Fever, a hallmark of disease, is a highly complex process initiated by the action of a number of endogenous pyrogens on the thermosensitive cells of the brain. We describe the activity of RANTES, a chemotactic cytokine, as intrinsically pyrogenic in the rat, when it is delivered directly to the thermosensitive region of the rat's anterior hypothalamic, pre-optic area (AH/POA). RANTES, microinjected into the AH/POA in a dose of 1, 5, 10, 15, 25 or 50 pg, produces an immediate and intense dose-related fever following injection. Increasing the dose to 100 pg did not result in a further increase in the febrile response. No significant change in body temperature was produced by heat-inactivated RANTES. The intrahypothalamic injection of antibodies against RANTES (2.0 microg, 15 min prior to RANTES) significantly blocked the fever induced by this chemokine. Pretreatment with ibuprofen blocked the fever induced by RANTES. In order of potency, the magnitude of the febrile response induced by RANTES was greater than that produced with equipotent doses of either macrophage inflammatory protein-1beta or interleukin-6. The results thus demonstrate that RANTES is the most potent endopyrogen discovered thus far and exerts its action directly on pyrogen-sensitive cells of the AH/POA through a prostaglandin-dependent pathway.

Visceral inputs to neurons in the anterior hypothalamus including those that project to the periaqueductal gray: a functional anatomical and electrophysiological study.

The present study was designed to examine peripheral, in particular noxious visceral, inputs to neurons in the hypothalamus that project to the midbrain periaqueductal gray. The induction of Fos protein was used to localize hypothalamic neurons that were activated by noxious visceral stimulation. This was combined with retrograde transport of fluorescent latex microspheres from identified "pressor" and "depressor" sites in the dorsolateral/lateral or ventrolateral columns of the periaqueductal gray. A second series of electrophysiological experiments examined the receptive field characteristics, including the incidence of viscerosomatic convergence, of neurons in the ventral part of the anterior hypothalamus. Noxious visceral stimulation (intraperitoneal acetic acid) induced Fos-like immunoreactivity in significantly more neurons in the hypothalamus than control stimuli (intraperitoneal saline and intravenous phenylephrine). Particularly high numbers of Fos-positive neurons were found in the paraventricular nucleus, the supraoptic nucleus and ventral regions of the anterior hypothalamus. When combined with retrograde tracing from "depressor" sites in the ventrolateral periaqueductal gray, the highest numbers of double-labelled neurons were localized in the paraventricular nucleus and the lateral area of the anterior hypothalamus. However, the regions that contained the greatest proportions of Fos-positive neurons that projected to "depressor" sites in the ventrolateral periaqueductal gray were the lateral area of the anterior hypothalamus and its rostral extension, the lateral preoptic area. Fewer double-labelled neurons were localized in the hypothalamus after retrograde transport from sites in the dorsolateral/lateral periaqueductal gray compared to the results obtained from injections of tracer in the ventrolateral periaqueductal gray. Furthermore, the numbers of Fos-positive hypothalamic neurons that projected to the dorsolateral/lateral periaqueductal gray were very similar in experimental and control animals. The electrophysiological study confirmed that a large proportion of neurons in and around the lateral area of the anterior hypothalamus can be driven by noxious visceral stimulation and demonstrated a high incidence of viscerosomatic convergence in these cells (66% of cells driven from somatic structures were also driven by electrical stimulation of the splanchnic nerve). Somatic receptive fields of these neurons were generally large, often including all four limbs and the face. The results of the functional anatomical and electrophysiological studies have identified neurons in an area of the ventral anterior hypothalamus that are a focus of nociceptive visceral input and which project to the midbrain periaqueductal gray, in particular to its ventrolateral column. These results are discussed in relation to the roles of the anterior hypothalamus and the different longitudinal columns of the periaqueductal gray in co-ordinating autonomic and sensory functions in response to visceral pain.

The interstitial nuclei of the human anterior hypothalamus: an investigation of sexual variation in volume and cell size, number and density.

The four interstitial nuclei of the anterior hypothalamus (INAH) have been considered as candidate human nuclei for homology with the much studied sexually dimorphic nucleus of the preoptic area of the rat. Assessment of the INAH for sexual dimorphism has produced discrepant results. This study reports the first systematic examination of all four INAH in the human for sexual variation in volume, neuronal number and neuronal size. Serial Nissl-stained coronal sections through the medial preoptic area and anterior hypothalamus were examined from 18 males and 20 females who died between the ages of 17 and 65 without evidence of hypothalamic pathology or infection with the human immunodeficiency virus. A computer-assisted image-analysis system and commercial stereology software package were employed to assess total volume, neuronal number and mean neuronal size for each INAH. INAH3 occupied a significantly greater volume and contained significantly more neurons in males than in females. No sex differences in volume were detected for any of the other INAH. No sexual variation in neuronal size or packing density was observed in any nucleus. The present data corroborate two previous reports of sexual dimorphism of INAH3 but provide no support for previous reports of sexual variation in other INAH.

Serotonin regulation of aggressive behavior in male golden hamsters (Mesocricetus auratus).

These studies examined the neurochemistry and neuroanatomy of the serotonin (5-HT) system innervating the anterior hypothalamus (AH) and the interaction of 5-HT receptor agonists with arginine vasopressin (AVP) in the regulation of offensive aggression in golden hamsters. Because specific 5-HT1A, 5-HT1B, and AVP V1A binding sites were observed within the AH by in vitro autoradiography, the hamsters were tested for offensive aggression after microinjections of AVP in combination with either the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetraline (DPAT) or the 5-HT1B agonist CGS-12066A (CGS) directly within the AH. Though treatment with DPAT resulted in a dose-dependent inhibition of AVP-facilitated offensive aggression, CGS was ineffective. In addition, a retrograde tracer was injected within the AH to localize the distribution of 5-HT neurons projecting to the area. Retrogradely labeled 5-HT neurons were found within the dorsal, median, and caudal linear raphe nuclei and are suspected to inhibit AVP-facilitated offensive aggression by an activation of 5-HT1A receptors in the AH.

The medial preoptic and anterior hypothalamic regions of the rhesus monkey: cytoarchitectonic comparison with the human and evidence for sexual dimorphism.

Examination of thionin-stained sections through the hypothalamus of the rhesus monkey revealed nuclei that resemble the first, second and third interstitial nuclei of the anterior hypothalamus (INAH1-3) of the human. Volumetric analysis of these nuclei in a small sample of monkeys suggests that the nucleus that resembles INAH3 is larger in males than in females. INAH1-3 have each been reported to be larger in men than in women and each has been considered as a potential candidate for homology with the much-studied sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat. Positional and cytoarchitectonic criteria suggest that of these nuclei, INAH3 and its potential counterpart in the rhesus monkey are the best candidates for homology with the SDN-POA. While the criteria employed in the present study may be used to suggest homologies, they are not adequate to confirm them. Confirmation of the homologies suggested here must rely on other considerations such as connectivity, neurotransmitter and peptide content, and function. It is hoped that the present report will stimulate interest in further examinations of the rhesus hypothalamus that will test both the suggested homologies and the evidence for sexual dimorphism.

Cell death in the sexually dimorphic dorsal preoptic area/anterior hypothalamus of perinatal male and female ferrets.

A sexually dimorphic male nucleus (MN) is present in Nissl-stained sections through the dorsal (d) preoptic area/anterior hypothalamus (POA/AH) of male ferrets. The MN-POA/AH is composed of a cluster of large cells which is organized in males by the action of estradiol, formed via the neural aromatization of circulating testosterone (T), during the last quarter of a 41-day gestation. Several recent studies using rodent species have raised the possibility that the hormone-induced masculinization of POA/AH morphology is mediated at least in part by a perinatal modulation of cell death. We asked whether a perinatal reduction in cell death contributes to the differentiation of the MN-POA/AH in the male ferret, which is a carnivore species. The appearance of internucleosomal DNA fragmentation, detected by in situ end labeling (ISEL) using the ApopTag kit (Oncor Corp.) and of pyknotic cell nuclei in Nissl-stained sections were used to estimate the occurrence of cell death. Male and female ferret kits were killed at four different ages spanning the perinatal period during which the MN-POA/AH is organized and assumes an adult phenotype. A peak density of dying cells was present in both sexes at postnatal day (P) 2, which is nearly 1 week after the age, embryonic day (E) 37, when the MN-POA/AH is first visible in male ferrets using Nissl stains. The density of cells in the sexually dimorphic dPOA/AH which were either ISEL-positive or pyknotic was similar in males and females on E34, as well as on P2, 10, and 20. In the nondimorphic ventral POA/AH, the highest density of dying cells was present in both sexes at E34, and there were significantly more ISEL-positive cells present in males than females at this particular age. In contrast to previous studies using rodents, our results suggest that in fetal male ferrets a modulation of the incidence of cell death contributes little to estradiol's organizational action in the dPOA/AH.

The effects of anterior hypothalamic lesions on short-day responses in Siberian hamsters given timed melatonin infusions.

The suprachiasmatic nucleus (SCN) of the hypothalamus is an area of dense 2-[125I]Iodomelatonin binding in Siberian hamsters (Phodopus sungorus sungorus) that is suggestive of a possible role in the reception and/or relaying of melatonin (MEL) signals. Indeed, in pinealectomized male Siberian hamsters given short day (SD) MEL signals (long-duration MEL infusions), lesions of the SCN (SCNx) block testicular regression and decreases in body and fat pad masses seen in identically treated hamsters with sham lesions (SCNs). In similar studies using Syrian hamsters (Mesocricetus auratus), anterior hypothalamic lesions (AHx), but not SCNx, blocked SD MEL signal-induced gonadal regression despite the similarity in the 2-[125I]Iodomelatonin binding pattern between the two species. The discrepancy between the ability of SCNx to block the reception of SD MEL signals between the two species is puzzling, given the similarity in the reproductive status of the Syrian and Siberian hamsters to systemically administered and timed MEL infusions. One possible way of reconciling the differences between these studies was that ancillary damage to areas neighboring the SCN, including the AH, may have occurred in our attempt to achieve complete SCNx in Siberian hamsters. Therefore, the purpose of the present study was to challenge AHx Siberian hamsters with SD MEL signals. Adult male hamsters were pinealectomized, fitted with subcutaneous catheters, and given daily timed infusions of MEL for 5 or 10 h (long day-like and short day-like, respectively) or the saline vehicle for 6 wk following bilateral electrolytic, or sham (AHs) lesions of the AH. Hamsters receiving 10 h MEL infusions that lacked evidence of anatomical or functional damage to the SCN showed SD-like gonadal regression, decreases in body and fat pad mass, and food intake similar to that observed in AHs animals. In contrast, 10 h MEL-infused SCNx hamsters did not exhibit SD-like responses, a finding confirming our previous report. These data suggest that interspecies differences exist between Syrian and Siberian hamsters in central nervous system sites and pathways involved in the reception/transmission of SD MEL signals.

Attenuation of medical forebrain bundle reward by anterior lateral hypothalamic lesions.

Psychophysical data consistent with rostro-caudal conduction along reward-relevant neurons linking the lateral hypothalamus (LH) and ventral tegmental area (VTA) have lead to the hypothesis that some of the directly activated neurons responsible for medial forebrain bundle (MFB) self-stimulation arise anterior to the level of the LH. This hypothesis has been challenged on the grounds that lesions to the anterior LH (ALH) often fail to degrade the rewarding value of stimulating more posterior MFB sites. The present study was aimed at investigating the effect of lesion location and stimulation current on the efficacy of ALH lesions in an effort to account for the inconsistencies in the earlier data. Self-stimulation thresholds were obtained for LH and VTA sites by estimating the number of pulses per stimulation train required for half-maximal responding at each of 3 currents. Electrolytic lesions (anodal, 1.0 mA for 10 s) were then made to the ALH at varying medial-lateral coordinates. In 7 of the 14 rats with MFB stimulation sites, lesions to the ALH produced increases in threshold which often declined over the next several days to weeks; in 5 cases thresholds remained elevated by 0.1 to 0.25 log10 units above baseline up to end of testing. In all but one case, the effective lesions were centered in the lateral ALH. Increases in threshold were more likely to be detected when stimulating at low currents; at low currents fewer neurons are recruited and the lesion can have a greater proportional effect on threshold. These data support the hypothesis that cell bodies, terminals, or fibers of passage in the ALH contribute to the rewarding effect of stimulating more posterior MFB sites.

Retinal projections to the subcortical nuclei in the Japanese field vole (Microtus montebelli).

Retinal projections in the Japanese field vole (Microtus montebelli) were determined by anterograde transport of horseradish peroxidase (HRP). Injection of HRP into the unilateral vitreous body demonstrated that the terminal labeling of the optic projections was seen bilaterally in the suprachiasmatic nucleus (SCH), the ventral (GLv) and dorsal (GLd) lateral geniculate nuclei, the intergeniculate leaflet (IGL), the medial pretectal nucleus (NTOM) of the pretectum (PT) and the superficial layer of the superior colliculus (CS), with contralateral predominance, and only contralaterally labeled terminals were found in the lateroposterior thalamic nucleus (LP), the lateral pretectal nucleus (NTOL) of the PT, the dorsal (DTN) and medial (MTN) terminal nuclei of the accessory optic system (AOS). The distribution area of the retinofugal terminals was divided into a three laminar arrangement in the GLd, i.e., layers 1 and 3 and layer 2, received the retinal input from contralateral and ipsilateral eye, respectively, as in arboreal squirrels. The contralateral CS received retinal fibers in the superficial layer, while ipsilateral optic fibers projected sparsely to the stratum opticum of the colliculi. Retinal connections to the DTN and MTN of the AOS were clearly discerned but no lateral terminal nucleus with retinal afferents was found. In addition, the AOS had no inferior fasciculus. These findings indicate that the vole has a contradictory features of a well- and a less-developed sense of vision. Namely, the image forming visual system such as the retino-GLd was as well-developed as in a squirrel, on the other hand, the non-image forming visual system such as the retino-AOS was less-developed as in an insectivore's brain.

High-frequency ultrasonic vocalization induced by intracerebral glutamate in rats.

Direct injection of glutamate, a neuroexcitatory agent, into the anterior hypothalamic-preoptic area of the rat brain induced ultrasonic vocalization. This vocalization was characterized by short-duration calls (below 60 ms) of high sound frequency (pitch), mostly above 40 kHz, and was similar to the known 50-kHz vocalization observed in natural situations. The glutamate-induced vocalization was dose dependent within the dose range of 16.9-67.6 micrograms and was antagonized by local pretreatment with MK-801, an NMDA antagonist. The increasing dosage of glutamate induced more calls and had a significant influence on frequency and intensity of emitted ultrasound. The average sound frequency increased whereas the mean sound intensity decreased with the dosage of glutamate. On the other hand, the mean duration of a single call and the bandwidth did not significantly change with doses of glutamate. Injection of carbachol, a muscarinic cholinomimetic agent, into the same brain sites as glutamate, induced a different type of ultrasonic vocalization with low sound frequency and long call duration, known as 22-kHz calls. The results suggest that high sound frequency, short-duration calls (50 kHz) and low sound frequency, long-duration calls (22 kHz) have different neurophysiological and neurochemical mechanisms.

A direct pretectosuprachiasmatic projection in the rat.

The major afferent projections of the suprachiasmatic nuclei originate in the retina and the intergeniculate leaflet of the lateral geniculate nucleus and are important in the entrainment of endogenous circadian rhythms. A characteristic feature of the suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus is that they are bilaterally innervated from the retina. However, parts of the olivary and posterior pretectal nuclei have been shown to be bilaterally innervated from the retina as well. We therefore aimed to explore whether these two nuclei, in the rat, were anatomically related to the suprachiasmatic nucleus. The anterograde neuronal tract-tracer, Phaseolus vulgaris-leucoagglutinin, was injected iontophoretically into different pretectal nuclei. Pretectal injections centered only in the medial part of the pretectum, i.e. involving the olivary and posterior pretectal nuclei, gave rise to a substantial bilateral innervation of the suprachiasmatic nucleus. From the site of injection, Phaseolus vulgaris-leucoagglutinin-immunoreactive nerve fibers coursed laterally and rostrally into the optic tract, and within the optic tract and chiasm, under the diencephalon to penetrate dorsally into the suprachiasmatic nucleus. Varicose Phaseolus vulgaris-leucoagglutinin-labeled nerve fibers were found exclusively in the ventrolateral part of the suprachiasmatic nucleus, mostly on the ipsilateral side. To determine the precise location of the projecting neurons, the retrograde tracer Cholera toxin, subunit B, was iontophoretically injected into the suprachiasmatic nucleus. The presence of of labeled neurons scattered in both the posterior and olivary pretectal nuclei was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and developmental pattern of vasoactive intestinal polypeptide binding sites in the human hypothalamus.

Using a quantitative in vitro autoradiographic approach, vasoactive intestinal polypeptide (VIP) binding site densities were compared in the post-mortem hypothalamus of human neonate/infant and adult. The densities were similar during development in most of the hypothalamic nuclei and areas examined underlying the stability of 125I-VIP binding sites in the post-mortem hypothalamus of young and adult individuals. However, the ventral part of the medial preoptic area, the medial, lateral, and supramammillary nuclei were characterized by an increase of 125I-VIP binding with age. In young and adult individuals, the highest densities of hypothalamic 125I-VIP binding sites were detected in the supraoptic and infundibular nuclei; the ependyma; the organum vasculosum of the lamina terminalis; the horizontal limb of the diagonal band of Broca; the ventral part of the medial preoptic area (in adult); the suprachiasmatic, paraventricular, and periventricular nuclei; and the medial and lateral mammillary nuclei in adult. Moderate densities were found in the vertical limb of the diagonal band of Broca, the bed nucleus of the stria terminalis, the ventral part of the medial preoptic area in neonate/infant, the medial and lateral mammillary nuclei in neonate/infant, the supramammillary nucleus in adult, the dorsal hypothalamic area, and the ventromedial nucleus. Low to moderate binding site densities were observed in the other hypothalamic regions of young or adult individuals. The nonspecific binding ranged from 15% of the total binding in the anterior hypothalamus to 20% in the mediobasal and posterior hypothalamic levels. Taken together, these results provide evidence for a large distribution of VIP binding sites in neonate/infant and adult human hypothalamus suggesting the implication of VIP in the development of this brain structure and the maintenance of its various functions.