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.

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.

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.

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.

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.

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.

Glutamate stimulation of arcuate nucleus inhibits responses of subfornical organ neurons to plasma hypernatremia and angiotensin II.

Experiments were done in urethane anesthetized rats to investigate the effect of glutamate (Glu) stimulation of arcuate nucleus of the hypothalamus (Arc) on the discharge rate of subfornical organ (SFO) neurons during changes in plasma sodium concentration and angiotensin II (ANG II) levels. Extracellular recordings were made from 67 histologically verified single neurons within SFO. Of these, 17 (25.4%) were excited by intracarotid infusion of hypertonic NaCl and 21 (31.3%) by intracarotid ANG II infusion. Five (29.4%) of the units excited by hypertonic NaCl were also excited by Glu stimulation of Arc. Similarly, seven (33.3%) of the units excited by ANG II were also excited by Arc stimulation. Additionally, four (19.0%) of the units excited by ANG II were inhibited by Glu stimulation of Arc. The remaining SFO units did not alter their discharge rate to activation of Arc neurons. The response of units to hypertonic NaCl or to ANG II was attenuated during simultaneous activation of Arc. These data suggest that Arc may be involved in modulating the activity of SFO neurons that function in the detection of blood-borne signals from the depletion of intra- and extracellular fluid volumes.

Neuronal regeneration and estrous cycle restoration after locus coeruleus-periventricular gray substance section.

The locus coeruleus (LC) was anatomically separated from the periventricular gray substance (PVG) by means of knife cuts in the adult female rat presenting regular estrous cycling. This resulted in a transient suppression of the estrous cycling that lasted 10-13 days after surgery. After this period, irregular or regular cycling activity was observed. The regular cycling was restored 30-45 days after the knife cuts. Golgi impregnation of some of the brains of these rats revealed regenerative elements in the knife-cut-insulted area. Thus, blood vessels, macrophagic-like elements, and glial-like elements were observed in close relation with the knife-cut pathway. Additionally, well-defined stained neurons typical of the LC and PVG were observed in close proximity to the knife-cut pathway. Dendritic and axon projections towards the insulted area were observed. Well defined axons were seen across the knife-cut pathway. These data support, first, that the LC-PVG communication is part of a circuitry for the modulation of gonadotropic activity, and second, that in the restoration of the estrous cyclicity after the knife cut, regenerative processes leading to a LC-PVG functional reconnection occurred after the knife cut.

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.

Fos induction in brainstem neurons by intravenous hypertonic saline in the conscious rat.

Experiments were done in conscious rats to investigate the effect of intravenous infusion of hypertonic saline on the induction of the protein Fos, in brainstem neurons. Neurons containing Fos-like immunoreactivity were observed in the caudal nucleus of the solitary tract (NTS), the caudal and rostral ventrolateral medulla, and parabrachial nucleus after an infusion of solutions containing 1.4 M NaCl. Little or no expression of Fos was detected in brainstem neurons after intravenous infusions of either physiological (143 mM) or hypotonic (106 mM) NaCl solutions. These data provide evidence for the involvement of brainstem structures in osmoregulatory functions and suggest that brainstem neuronal circuits that function in cardiovascular regulation may also be shared by those involved in body fluid homeostasis.

Effect of arcuate nucleus activation on neuronal activity in subfornical organ.

Experiments were done in urethane anesthetized rats to investigate the effect of activation of the arcuate nucleus (Arc) on the discharge rate of single units in the subfornical organ (SFO). Extracellular recordings were made from 51 spontaneously active neurons histologically verified in the SFO. Of these units, 19 (37%) were inhibited (mean latency 7.3 +/- 1.1 ms) and 14 (28%) were excited (mean latency 12.8 +/- 3.2 ms) during electrical stimulation of the Arc. The remaining 18 units did not alter their firing frequency during stimulation of the Arc. These data suggest that the Arc may be involved in the modulation of neural and humoral events related to body fluid homeostasis and cardiovascular regulation by altering the activity of SFO neurons.

Olfactory bulb neurons respond to cervicovaginal distension.

Mitral cell layer neuronal activity in the olfactory bulb (OB) of the anesthetized rat is modulated by cervicovaginal distension. Data are reported on 22 cells that decreased and 6 that increased in response to the distension. These results provide support for the existence of a functional interaction between the reproductive tract and the olfactory system.

Recovery of vestibular function in young guinea pigs after streptomycin treatment. Glutamate decarboxylase activity and nystagmus response assessment.

Fifty-day streptomycin (STP) treatment in guinea pigs causes specific vestibular hair cell (VHC) types I and II (HCI; HCII) degeneration, depletion of glutamate decarboxylase (GAD) and a gradual disappearance of postrotatory nystagmus response (PRNR), which is a sign of vestibular function alteration. In order to look for a possible spontaneous reversibility and its time course guinea pigs receiving 300 mg/kg STP daily were monitored for PRNR and vestibular GAD loss. Once PRNR was lost, STP was interrupted and the animal was allowed to recover; at the time that PRNR was completely reestablished, vestibular GAD was measured. PRNR was lost within 22-25 days of STP treatment. Vestibular GAD showed a loss that, with time of treatment, gave two slopes: a fast decrement (45% in 20 days) and a slow one (40% in the remaining 30). Stopping of the STP treatment after 22-25 days and animal recovery resulted in the return of both PRNR and GAD activity 22 days after STP stoppage. These results suggest two STP-susceptible GAD-containing VHC populations, one more sensitive than the other, possibly HCI followed by hair cell II (HCII). As hypothetic HCI loss and PRNR disappearance is simultaneous, the important role of the former for vestibular function could be inferred. Interruption of STP treatment after PRNR loss results in a long range restoration of both GAD activity and vestibular function, and thus recovery of HCI, the first evidence of its occurrence in a mammalian vestibule, could be suggested. The intimate mechanism of this recovery remains to be seen.

Natriuresis after a water load in humans under different sodium body content.

The present study was aimed at observing the diuretic and natriuretic responses after a water load (2% body weight) in four groups of young consenting volunteers submitted previously, during three days, to a hypersodic (500 mEq Na/day), hyposodic (35 mEq Na/day), and normosodic (200 mEq Na/day) diet, or treated with furosemide (Lasix, 40 mg/day). During the treatment urine was collected each day. On the fourth day, in the morning, the bladder was emptied, the water load was ingested, and the urine collected during 10 periods of 20 min each. The urinary, sodium, and chloride flows were determined. The four groups displayed diuretic curves following a similar pattern. In contrast, the natriuretic curves of the four groups were completely different; totally flat with low values for the furosemide group and a large initial natriuretic curve for the hypersodic group with a gradual decrease but maintaining high values. The results indicate that the way the organism compensates for the excess of water by means of urinary water loss is independent of the body sodium content, whereas the way in which sodium loss is accomplished is determined by its body content and is independent of the way in which the water is lost.

Role of the paraventricular nucleus in the projection from the nucleus of the solitary tract to the olfactory bulb.

Electrophysiological experiments were performed on anesthetized rats to determine the effects of lesions of the paraventricular nucleus on the amplitude of evoked potentials recorded in the periglomerular layer of the olfactory bulb after nucleus of the solitary tract electrical stimulation. Lesions of the paraventricular nucleus enhance the amplitude of both the positive and negative components of the evoked potential in the olfactory bulb. The pathway from the paraventricular nucleus to the olfactory bulb seems to exert a suppressive influence over the projection from the nucleus of the solitary tract to the olfactory bulb under these conditions.

Vagus nerve afferent and efferent innervation of the rat uterus: an electrophysiological and HRP study.

To determine a possible brainstem connection with the uterus, a study with electrophysiological techniques and horseradish peroxidase (HRP) tracing was performed in the rat. Neurons of the nucleus of the tractus solitarius decreased in discharge frequency during cervicovaginal distension. HRP injections into the uterine walls resulted in the appearance of labelled cells in the nodose ganglion and in the dorsal motor nucleus of the vagus nerve. The results demonstrate a direct bidirectional vagal complex-uterus connection via the vagus nerve. Results are discussed in terms of a complex uterus control system in which the paraventricular nucleus might play an integrative role.

Electrophysiological evidences of a bidirectional communication between the locus coeruleus and the suprachiasmatic nucleus.

To assess a possible relation between the suprachiasmatic nucleus (SCN) and the locus coeruleus (LC), a study with evoked potentials was performed in the rat. An evoked potential was recorded in the SCN area after electrical stimulation of the LC. Also, an evoked potential was recorded in the LC after electrical stimulation of the SCN area. The results indicate specificity of the projecting regions and of the activated regions, suggesting the existence of a possible bidirectional communication between the LC and the SCN. The pathways possibly involved are discussed.

Nucleus of the tractus solitarius projections to the olfactory tubercle: an HRP study.

Labelled cells were found in the nucleus of the tractus solitarius (NTS) after horseradish peroxidase injections in the olfactory tubercle (OT) of the rat. These results suggest a direct pathway from the NTS to the OT. The importance of this pathway in a neural circuit related to autonomic functions is discussed.

Effect of locus coeruleus and periventricular gray substance lesions on suprachiasmatic damage-induced persistent estrous.

To assess the relation between the suprachiasmatic nucleus and the locus coeruleus, persistent estrous was induced in female rats by damage of the suprachiasmatic nucleus followed with electrolytic damage of the locus coeruleus. Locus coeruleus lesions resulted in a transient loss of persistent estrous indicated by sustained diestrous aspect of the vaginal smears. There was a direct correlation between duration of estrous loss and the amount of locus coeruleus damage. Simultaneous damage to the periventricular gray substance, adjacent to the locus coeruleus, prevented the suppression effects of locus coeruleus damage upon persistent estrous. These results suggest a physiological role of the locus coeruleus and periventricular gray substance in the modulation of the neuroendocrine control of ovarian function due to suprachiasmatic nucleus activity.

Effect of locus coeruleus and periventricular gray substance lesions on bright light-induced persistent estrous.

To assess the relation between the suprachiasmatic nucleus and the locus coeruleus, persistent estrous was induced in female rats by exposure to constant bright light followed with electrolytic damage of the locus coeruleus. Locus coeruleus lesions resulted in a transient loss of persistent estrous indicated by sustained diestrous aspect of the vaginal smears. There was a direct correlation between duration of estrous loss and the amount of locus coeruleus damage. Simultaneous damage to the periventricular gray substance adjacent to the locus coeruleus prevented the suppression effects of locus coeruleus damage upon persistent estrous. These results suggest a physiological role of the locus coeruleus and periventricular gray substance in the modulation of the neuroendocrine control of ovarian function due to suprachiasmatic nucleus activity.