L.Pastor Solano-Flores

Fos induction in central structures after afferent renal nerve stimulation.

Experiments were done in the conscious and unrestrained rat to identify central structures activated by electrical stimulation of afferent renal nerves (ARN) using the immunohistochemical detection of Fos-like proteins. Fos-labelled neurons were found in a number of forebrain and brainstem structures bilaterally, but with a contralateral predominance. Additionally, Fos-labelled neurons were found in the lower thoracolumbar spinal cord predominantly ipsilateral to the side of ARN stimulation. Within the forebrain, neurons containing Fos-like immunoreactivity after ARN stimulation were primarily found along the outer edge of the rostral organum vasculosum of the laminae terminalis, in the medial regions of the subfornical organ, in the median preoptic nucleus, in the ventral subdivision of the bed nucleus of the stria terminalis, along the lateral part of the central nucleus of the amygdala, throughout the deeper layers of the dysgranular insular cortex, in the parvocellular component of the paraventricular nucleus of the hypothalamus (PVH), and in the paraventricular nucleus of the thalamus. Additionally, a smaller number of Fos-labelled neurons was observed in the supraoptic nucleus, in the magnocellular component of the PVH and along the lateral border of the arcuate nucleus. Within the brainstem, Fos-labelled neurons were found predominantly in the commissural and medial subnuclei of the nucleus of the solitary tract and in the external subnucleus of the lateral parabrachial nucleus. A smaller number were observed near the caudal pole of the locus coeruleus, and scattered throughout the ventrolateral medullary and pontine reticular formation in the regions known to contain the A1, C1 and A5 catecholamine cell groups. The final area observed to contain Fos-labelled neurons in the central nervous system was the thoracolumbar spinal cord (T9-L1) which contained cells in laminae I-V of the dorsal horn ipsilateral to side of stimulation and in the intermediolateral cell column at the same levels bilaterally, but with an ipsilateral predominance. Few, if any Fos-labelled neurons were observed in the same structures of control animals in which the ARN were stimulated, but the renal nerves proximal to the site of stimulation were transected, or in the sham operated animals. These data indicate that ARN information originating in renal receptors is conveyed to a number of central areas known to be involved in the regulation of body fluid balance and arterial pressure, and suggest that this afferent information is an important component of central mechanisms regulating these homeostatic functions.

Co-localization of estrogen and angiotensin receptors within subfornical organ neurons.

A double-staining immunocytochemical study was done in ovariectomized (OVX) female rats that were either treated with 17beta-estradiol (E(2)) (OVX+E(2)) to produce an approximate circulating level of 30 pg/ml plasma, or not-treated with E(2) (OVX), to investigate the distribution of subfornical organ (SFO) neurons that contained estrogen receptors (ER), and to determine whether these neurons also contained the angiotensin II AT(1)-receptor (AT(1)R). Neurons that contained either ER-like immunoreactivity only, AT(1)R-like immunoreactivity only, or both ER and AT(1)R immunoreactivity were found throughout the extent of the SFO in both the OVX+E(2) and OVX rats. However, some regional differences were apparent in both groups of female rats. Neurons containing the ER were predominantly found in the peripheral regions of the SFO, near large blood vessels and the ependymal layer of the third ventricle. A number of lightly stained ER containing neurons was also observed scattered throughout the central core region of the SFO. OVX only animals were found to have a larger number of ER containing neurons in the SFO compared to the E(2) treated animals. Neurons containing AT(1)R were also found throughout the SFO, but without a distinct distribution pattern in either group of rats, although there were more neurons that exhibited AT(1)R immunoreactivity in the OVX animals. Finally, a distinct group of SFO neurons was found that exhibited both ER and AT(1)R immunoreactivity in both groups of animals, although a larger number of these double labelled neurons was found in the OVX animal. Most of these neurons were also found along the peripheral border of the SFO in close proximity to blood vessels and the ventricular lining. These data have demonstrated the co-existence of ER and AT(1)R in SFO neurons of the female rat, and suggest that circulating level of E(2) alter the expression of both the ER and AT(1)R in these neurons. In addition, these data suggest that E(2) may alter the physiological responses of SFO neurons to angiotensin II by down regulating the number of AT(1)R.

Direct projections to subfornical organ from catecholaminergic neurons in the caudal nucleus of the solitary tract

Experiments were done to investigate the distribution of neurons within the nucleus of the solitary tract (NTS) that projected directly to the subfornical organ (SFO) and to determine whether these neurons were components of the catecholaminergic cell groups in the dorsal medial medulla. Microinjection of the fluorescent tract-tracers fluorogold or rhodamine latex micro-beads were made into the SFO of the rat. Brainstem sections were then processed immunocytochemically for the identification of neuronal cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT). Retrogradely labelled cell bodies that projected to the SFO were found in the NTS, bilaterally, extending from approximately the level of the rostral aspect of the area postrema (obex) caudally to the calamus scriptorius. These neurons were located predominantly in the medial, lateral and dorsolateral subdivisions of NTS. Approximately one-third of these retrogradely labelled neurons were immunoreactive to TH and DBH. On the other hand, less than 7% of the retrogradely labelled neurons were found to contain TH and PNMT immunoreactivity. These data have demonstrated that noradrenergic neurons of the A2 cell group in the caudal NTS innervate the SFO and suggest that these neurons may be involved in relaying cardiovascular afferent information directly to a forebrain circumventricular organ involved in arterial pressure and body fluid homeostasis.

Identification of neurons containing orexin-B (hypocretin-2) immunoreactivity in limbic structures.

Orexins (hypocretins) are neuropeptides which have recently been identified exclusively within lateral hypothalamic and perifornical neurons, and these orexin (ox) containing neurons appear to have extensive projections to all levels of the neuraxis. In this study, we report the identification of two distinct clusters of neurons containing ox-B-like immunoreactivity within the amygdaloid complex of the rat. A cluster of small to medium size ovoid shaped neurons containing ox-B-like immunoreactivity was found predominantly within the lateral division of the central nucleus of the amygdala (ACe). A second distinct, but smaller group of ox-B labelled neurons with similar shapes and sizes to those in ACe was also identified in the anterior lateral subnucleus of the bed nucleus of the stria terminalis (BST) immediately adjacent the internal capsule, and in an area just ventral to the lateral ventricle. Neurons containing ox-A-like immunoreactivity were not observed in either structure. However, both structures contained ox-A- and ox-B labelled varicose fibers. Unilateral electrolytic lesions of the lateral hypothalamic area that contained ox-A and ox-B neurons did not alter the labelling of either ACe or BST ox-B pericarya. As both the ACe and BST are known to be involved in integrating complex homeostatic mechanisms associated with behaviours, these data suggest that a specific subset of ox-B neurons within the amygdaloid complex may serve as a component of neuronal circuits coordinating these responses.

Estrogen alters the bradycardia response to hypocretin-1 in the nucleus tractus solitarius of the ovariectomized female

Experiments were performed to investigate the effect of 17beta-estradiol (E; 30 pg/ml plasma) treatment (15-25 days) in the ovariectomized (OVX) female Wistar rat on the cardiovascular responses to hypocretin-1 (hcrt-1) in the nucleus tractus solitarius (NTS). In an initial series of experiments, the distribution of hcrt-1-like immunoreactivity within the region of the NTS was mapped in both OVX only and OVX+E animals. Hcrt-1 immunoreactivity was found throughout the NTS region in both groups of females, predominantly within the caudal interstitial, commissural, medial and lateral subnuclei of the NTS. The relative density of hcrt-1 immunoreactivity in all NTS subnuclei was similar in both female groups. Microinjections of hcrt-1 (0.5-10 pmol) into the caudal lateral and medial subnuclei of the NTS complex of the alpha-chloralose of the urethane-anaesthetized E-treated OVX rat elicited a dose-related decrease in heart rate (HR). On the other hand, although a dose-response effect on arterial pressure was evident, significant arterial pressure responses were observed only at the higher dose of hcrt-1 (>2.5 pmol). In the OVX only female rat, microinjection of hcrt-1 into similar NTS sites elicited a bradycardia and depressor response only at the highest dose of hcrt-1, and these responses were significantly smaller in magnitude than those elicited in the OVX+E animal. In addition, in the OVX only animals, a few sites within the caudal commissural subnucleus of the NTS complex were found at which hcrt-1 elicited tachycardia and pressor responses. Finally, it was found that the reflex bradycardia to the activation of arterial baroreceptors as a result of increasing systemic arterial pressure with phenylephrine (2-4 microg/kg) was significantly potentiated in the OVX+E animals only. These data suggest that hcrt-1 in the NTS of the female activates a neuronal circuit that controls the circulation and that the circulating level of E alters the sensitivity of these cardiovascular circuits to hcrt-1.

c-Fos induction in spinal cord neurons after renal arterial or venous occlusion

Experiments were done in the anesthetized rat to identify the dorsal root ganglia (DRG) and the spinal cord segments that contain neurons activated by either renal venous occlusion (RVO) or by renal arterial occlusion (RAO). Fos induction, detected immunohistochemically in DRG and the spinal cord neurons, was used as a marker for neuronal activation. RVO induced Fos immunoreactivity in neurons in the DRG of spinal segments T8-L2 on the side ipsilateral to that of occlusion. The largest number of Fos-labeled neurons was found in the T11 DRG. In the spinal cord the largest number of Fos-labeled neurons was found in the ipsilateral dorsal horn of spinal segments T11-T12, predominantly in a cluster near the dorsomedial edge of laminae I-II. A few additional Fos-labeled neurons were observed in laminae IV and V. After RAO Fos-labeled neurons were found in the ipsilateral DRG of spinal segments similar to those observed to contain neurons after RVO. However, most of the Fos-labeled neurons were observed within the T12-L1 DRG. In the spinal cord Fos-labeled neurons were scattered throughout lamina I-II of the ipsilateral dorsal horn of spinal segments T8-L2, although the largest number was observed at the T13 level. Additionally, a distinct cluster of Fos-labeled neurons was observed predominantly in the region of the ipsilateral intermediolateral cell column, although a few neurons were found scattered throughout the nucleus intercalatus, central autonomic areas, and laminae IV and V of the cord bilaterally. No Fos labeling was observed in the complementary contralateral DRG or dorsal horns after either RVO or RAO. In addition, renal nerve transection prevented Fos labeling in the ipsilateral DRG and dorsal horns after RVO or RAO. Taken together, these data suggest that functionally different renal afferent fibers activate DRG neurons that may have distinct projections in the spinal cord.Experiments were done in the anesthetized rat to identify the dorsal root ganglia (DRG) and the spinal cord segments that contain neurons activated by either renal venous occlusion (RVO) or by renal arterial occlusion (RAO). Fos induction, detected immunohistochemically in DRG and the spinal cord neurons, was used as a marker for neuronal activation. RVO induced Fos immunoreactivity in neurons in the DRG of spinal segments T8-L2on the side ipsilateral to that of occlusion. The largest number of Fos-labeled neurons was found in the T11 DRG. In the spinal cord the largest number of Fos-labeled neurons was found in the ipsilateral dorsal horn of spinal segments T11-T12, predominantly in a cluster near the dorsomedial edge of laminae I-II. A few additional Fos-labeled neurons were observed in laminae IV and V. After RAO Fos-labeled neurons were found in the ipsilateral DRG of spinal segments similar to those observed to contain neurons after RVO. However, most of the Fos-labeled neurons were observed within the T12-L1DRG. In the spinal cord Fos-labeled neurons were scattered throughout lamina I-II of the ipsilateral dorsal horn of spinal segments T8-L2, although the largest number was observed at the T13 level. Additionally, a distinct cluster of Fos-labeled neurons was observed predominantly in the region of the ipsilateral intermediolateral cell column, although a few neurons were found scattered throughout the nucleus intercalatus, central autonomic areas, and laminae IV and V of the cord bilaterally. No Fos labeling was observed in the complementary contralateral DRG or dorsal horns after either RVO or RAO. In addition, renal nerve transection prevented Fos labeling in the ipsilateral DRG and dorsal horns after RVO or RAO. Taken together, these data suggest that functionally different renal afferent fibers activate DRG neurons that may have distinct projections in the spinal cord.

Arcuate nucleus inputs onto subfornical organ neurons that respond to plasma hypernatremia and angiotensin II

Experiments were done in urethane anesthetized rats to investigate the effect of electrical and glutamate stimulation of arcuate nucleus (Arc) on the discharge rate of subfornical organ (SFO) neurons that responded to either plasma hypernatremia or angiotensin II (ANG II). Extracellular recordings were made from 253 histologically verified single neurons in SFO. Of these, 40.3% (102/253) responded with excitation and 10% (25/253) with inhibition to Arc stimulation. Thirty-five (34.3%) of the units excited by Arc were also excited by intracarotid infusion of hypertonic (0.5 M) NaCl. In addition, 37 (36.3%) of the units excited by Arc were also excited by intracarotid infusion of ANG II. Furthermore, 10 (40.0%) of the units inhibited by Arc were found to be excited by ANG II. None of the units inhibited by Arc stimulation were responsive to plasma hypernatremia. These data indicate that inputs from Arc neurons converge onto SFO neurons that alter their discharge rate during changes in plasma concentration of Na+ or ANG II. These results suggest that Arc may be involved in body fluid balance and circulatory regulation by modulating the activity of SFO neurons that function in the detection of blood-borne signals from the depletion of intra- and extra-cellular fluid volumes.

Medullary pathways mediating the parasubthalamic nucleus depressor response

The parasubthalamic nucleus (PSTN) projects extensively to the nucleus of the solitary tract (NTS); however, the function of PSTN in cardiovascular regulation is unknown. Experiments were done in alpha-chloralose anesthetized, paralyzed, and artificially ventilated rats to investigate the effect of glutamate (10 nl, 0.25 M) activation of PSTN neurons on mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). Glutamate stimulation of PSTN elicited depressor (-20.4 +/- 0.7 mmHg) and bradycardia (-26.0 +/- 1.0 beats/min) responses and decreases in RSNA (67 +/- 17%). Administration (intravenous) of atropine methyl bromide attenuated the bradycardia response (46%), but had no effect on the MAP response. Subsequent intravenous administration of hexamethonium bromide blocked both the remaining bradycardia and depressor responses. Bilateral microinjection of the synaptic blocker CoCl(2) into the caudal NTS region attenuated the PSTN depressor and bradycardia responses by 92% and 94%, respectively. Additionally, prior glutamate activation of neurons in the ipsilateral NTS did not alter the magnitude of the MAP response to stimulation of PSTN, but potentiated HR response by 35%. Finally, PSTN stimulation increased the magnitude of the reflex bradycardia to activation of arterial baroreceptors. These data indicate that activation of neurons in the PSTN elicits a decrease in MAP due to sympathoinhibition and a cardiac slowing that involves both vagal excitation and sympathoinhibition. In addition, these data suggest that the PSTN depressor effects on circulation are mediated in part through activation of NTS neurons involved in baroreflex function.

C-fos expression in arcuate nucleus following intracerebroventricular hypertonic saline injections

Experiments were done in conscious rats to investigate the effect of i.c.v. infusions of hypertonic NaCl solutions on the induction of the protein Fos in the arcuate nucleus (Arc). Neurons containing Fos-like immunoreactivity were observed throughout the rostrocaudal extent of Arc after i.c.v. infusions of hypertonic saline solutions (337-744 mM). However, most of the labelled neurons were confined to the middle third of the nucleus, in the region of the dorsomedial and ventromedial subnuclei. Few, if any Fos-labelled neurons were observed in Arc of animals that received i.c.v. infusions of isotonic (142 mM) or mild hypertonic (173 mM) saline solutions or a hyperosmotic (660 mOsm/kg) saline solution of mannitol. No Fos-labelled neurons were found in the subfornical organ, although a few were observed scattered throughout the organum vasculosum laminae terminalis (OVLT) in all the animals studied. The density nor the distribution pattern of Fos-labelled neurons in OVLT was altered in animals receiving i.c.v. infusions of hypertonic saline or hyperosmotic solutions. These data demonstrate that Arc neurons are activated during a hypertonic saline challenge and suggest that Arc may function as a sodium-sensitive structure that is involved in body-fluid and circulatory homeostasis.

Neurotensin projections to subfornical organ from arcuate nucleus

Two series of experiments were done in the rat to investigate whether neurons in arcuate nucleus of the hypothalamus (Arc) containing neurotensin (NT)-like immunoreactivity projected to subfornical organ (SFO). In the first series, the anterograde tract-tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was microiontrophoresed into the region of Arc that contains NT neurons. After a 9-12 day survival period the animals were sacrificed and forebrain sections that contained SFO were processed for combined PHA-L and NT immunoreactivity. In the second series of experiments, unilateral or bilateral electrolytic lesions of Arc were made and after a 10-17 day survival period SFO was examined to determine the relative contribution of NT Arc neurons to NT immunoreactivity within SFO. PHA-L labelled fibers with terminal-like boutons were found in SFO primarily on the side ipsilateral to the site of injection. A small number of the PHA-L labelled fibers in the lateral aspect of SFO was also immunoreactive to NT. Unilateral lesions reduced, whereas bilateral lesions of Arc eliminated most of the NT-like immunoreactivity within SFO. These data demonstrate the existence of a direct pathway from Arc to SFO that contains the putative neurotransmitter NT. These results suggest that this pathway may function in the modulation of neural and/or humoral events related to cardiovascular regulation and body fluid homeostasis by influencing the activity of SFO neurons.