Hypothalamic orexin-A (hypocretin-1) neuronal projections to the vestibular complex and cerebellum in the rat.

Immunohistochemistry combined with retrograde tract-tracing techniques were used to investigate the distribution of orexin-A (OX-A)- and OX-A receptor-like (OX1) immunoreactivity within the vestibular complex and cerebellum, and the location of hypothalamic OX-A neurons sending axonal projections to these regions in the Wistar rat. OX-A immunoreactive fibers and presumptive terminals were found throughout the medial (MVe) and lateral (LVe) vestibular nuclei. Light fiber labeling was also observed in the spinal and superior vestibular nuclei. Within the cerebellum, dense fiber and presumptive terminal labeling was observed in the medial cerebellar nucleus (Med; fastigial nucleus), with less dense labeling in the interposed (Int) and lateral cerebellar nuclei (Lat; dentate nucleus). A few scattered OX-A immunoreactive fibers were also observed throughout the cortex of the paraflocculus. OX1-like immunoreactivity was found densely concentrated within LVe, moderate in MVe, and scattered within the spinal and superior vestibular nuclei. Within the cerebellum, OX1-like immunoreactivity was also observed densely within Med and in the dorsolateral aspects of Int. Additionally, OX1 like-labeling was found in Lat, and within the granular layer of the caudal paraflocculus cerebellar cortex. Fluorogold (FG) microinjected into these vestibular and cerebellar regions resulted in retrogradely labeled neurons throughout the ipsilateral hypothalamus. Retrogradely labeled neurons containing OX-A like immunoreactivity were observed dorsal and caudal to the anterior hypothalamic nucleus and extending laterally into the lateral hypothalamic area, with the largest number clustered around the dorsal aspects of the fornix in the perifornical area. A few FG OX-A like-immunoreactive neurons were also observed scattered throughout the dorsomedial, and posterior hypothalamic nuclei. These data indicate that axons from OX-A neurons terminate within the vestibular complex and deep cerebellar nuclei of the cerebellum and although the function of these pathways is unknown, they likely represent pathways by which hypothalamic OX-A containing neurons co-ordinate vestibulo-cerebellar motor and autonomic functions associated with ingestive behaviors.

Distribution of stanniocalcin binding sites in the lamina terminalis of the rat.

Stanniocalcin (STC-1), a 50 kDa glycoprotein hormone that regulates calcium/phosphate homeostasis in bony fish and mammals, has been shown to be expressed in central neurons and choroid plexus, and to exert a protective effect against hypercalcemic and hypoxic damage to neurons. Circumventricular organs are known to function in the regulation of ion and body fluid balance. Therefore, the possibility exists that STC-1 may be involved in the regulation of calcium/phosphate and fluid homeostasis through its actions on these central sites. In the present study, the distribution of STC-1 binding sites in forebrain circumventricular organs of the rat were investigated by in situ ligand binding using a stanniocalcin-alkaline phosphatase (STC-AP) fusion protein. Cells exhibiting STC-1 binding sites were found throughout the lamina terminalis. Dense cytoplasmic staining was observed predominantly within ependymal cells lining the anterior third ventricle region (AV3V), as well as cells of the choroid plexus. Additionally, neurons of the organum vasculosum of the lamina terminalis, the dorsal and ventral components of the median preoptic nucleus and the rostral aspects of the subfornical organ exhibited dense STC-1 cytoplasmic staining. STC-1 binding sites were also found in cells of the supraoptic nucleus, suprachiasmatic nucleus and anteroventral preoptic nucleus. These data suggest that STC-1 binding sites localized on the ependymal cells of the AV3V region and neurons of circumventricular organs may be associated with neuronal pathways involved in calcium/phosphate and fluid homeostasis.

Induction of c-fos in forebrain circumventricular organs after renal artery stenosis.

Experiments were done in the anaesthetized rat to determine the effect of activation of renal receptors following renal arterial occlusion (RAO) on the induction of c-fos in neurons of the lamina terminalis in the forebrain. Following RAO, fos labeled neurons were found in both the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT). Transection of the renal nerves ipsilateral to RAO reduced ( approximately 61%) the number of fos labeled neurons in the SFO and prevented the fos labeling in the OVLT. Similarly, administration of the angiotensin II converting enzyme inhibitor enalapril maleate prior to RAO also reduced ( approximately 27%) the number of fos labeled neurons in the SFO to RAO. However, the number of fos labeled neurons was not altered in the OVLT. The number of fos labeled neurons in the SFO of the intact animals after RAO was found to be greater than the algebraic sum of the number of fos labeled neurons in the renal nerve transected and enalapril treated animals. These results suggest that neurons in the SFO are activated by at least two different mechanisms following renal artery occlusion; those involving the activation of afferent renal nerves and those due to changes in circulating levels of angiotensin II. In addition, afferent renal nerve inputs combined with the effect of increased circulating levels of angiotensin II produce a greater activation of the SFO than either input alone. On the other hand, the OVLT appears to be selectively activated by afferent renal nerve inputs following RAO. Taken together, these data suggest that neural inputs from the kidney may play an important role in controlling body fluid balance and arterial pressure (AP) by influencing the activity of forebrain circumventricular organs neurons that function in the detection of blood borne signals associated with changes in extracellular fluid volume.

Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius.

Hypocretin-1 (hcrt-1)-containing axons have been shown to have an extensive distribution within the central nervous system, although the total number of hypothalamic hcrt-1 neurons has been shown to be small. This suggests that hcrt-1 neurons may innervate central structures with similar function through collateral axonal projections. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether hypothalamic hcrt-1-containing neurons send collateral axonal projections to cardiovascular sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb) in the rat. Fluorogold- (FG) and/or rhodamine (Rd)-labeled latex microspheres were microinjected into either the NTS or Amb at sites that elicited bardycardia responses (L-glutamate; 0.25 M; 10 nl). After a survival period of 10-15 days, the rats were sacrificed and tissue sections of the hypothalamus were processed immunohistochemically for the identification of hcrt-1-containing cell bodies. After injection of the tract-tracers into the NTS or Amb, retrogradely labeled neurons were observed within several hypothalamic regions; the paraventricular hypothalamic nucleus, lateral hypothalamic area, perifornical hypothalamic area, and posterior hypothalamus, bilaterally, but with an ipsilateral predominance. In addition, after NTS injections, retrogradely labeled neurons were found within the ipsilateral caudal arcuate nucleus. Of the total number (1107+/-97) of hcrt-1-immunoreactive neurons found bilaterally within the lateral and perifornical hypothalamic nuclei, 7.9+/-1.4% were found to be retrogradely labeled from the NTS, 16.4+/-1.8% from the Amb, and 3.1+/-0.5% from both medullary sites. Hcrt-1 neurons projecting to the NTS were found mainly in and around the perifornical hypothalamic region, with a smaller number in the caudal lateral hypothalamic area. On the other hand, those innervating the Amb were primarily observed within the caudal lateral hypothalamic area, with a smaller number in the perifornical hypothalamic area. Neurons with collateral axonal projections to NTS and Amb were observed within two specific hypothalamic areas: one group of neurons was found in the perifornical hypothalamic area, and the other was observed in the lateral hypothalamic region just dorsal to the retrochiasmatic component of the supraoptic nucleus. These data indicate that axons from hcrt-1 neurons bifurcate to innervate functionally similar cardiovascular-responsive sites in the NTS and Amb. Although the function of these hcrt-1-containing hypothalamic-medullary pathways is not known, they likely represent the anatomical substrate by which the lateral hypothalamic hcrt-1 neurons simultaneously coordinate autonomic-cardiovascular responses to different behaviors.