Organum Vasculosum Lamina Terminalis Region Research Articles

Activation of renal afferent pathways following furosemide treatment: II. Effect of angiotensin blockade

The goal here and in the accompanying paper was to evaluate the two pathways used by the kidney to provide information to the central nervous system (CNS); e.g., the indirect, hormonal route via activation of the renin–angiotensin system and the direct pathway via activation of sympathetic afferents in the caudal thoracic spinal cord. Here, three experiments were designed to evaluate the actions of angiotensin elicited by subcutaneous injection of furosemide on neural activation of the CNS. The number of neurons immunocytochemically staining for the protein product (Fos) of the c- fos gene was used as an index of neuronal activation. In the first experiment, furosemide injection was preceded by treatment with a dose of Captopril, CAP, (an angiotensin-converting enzyme (ACE) inhibitor) that blocks the peripheral but not the central formation of angiotensin II. In the second experiment, furosemide injection was preceded by treatment with a higher dose of CAP; this dosage blocks the peripheral and central formation of angiotensin II. In the third experiment, furosemide injection was preceded by treatment with Losartan, a competitive receptor antagonist of type I angiotensin II receptors at a dose that would block central and peripheral angiotensin receptors. Control animals in each experiment received injections of vehicle (sterile isotonic saline) instead of furosemide. In each experiment, rats were sacrificed 1.75 h following furosemide or saline injection by transcardial perfusion and tissues were immunocytochemically processed for demonstration of Fos antigen. Rats receiving furosemide plus the low CAP dose showed more Fos-positive cells than control rats in the subfornical organ (SFO), organum vasculosum lamina terminalis (OVLT), supraoptic nucleus (SON), magnocellular region of the paraventricular nucleus, nucleus of the solitary tract (NTS), and caudal thoracic/rostral lumbar spinal cord dorsal horn. Rats receiving furosemide plus Losartan or furosemide plus the higher CAP dose did not show increased Fos immunoreactivity in any of the abovementioned structures relative to their respective control animals. We conclude that the receptor-mediated action of angiotensin II is in some way involved in the activation of the pathway that occurs in the SFO, OVLT, SON, and magnocellular region of the paraventricular nucleus (PVN) in response to furosemide treatment. It is possible that the furosemide-induced activation in the SON and PVN is not due to direct actions of angiotensin II on angiotensin receptors in those structures, but instead occurs synaptically as a result of inputs from the SFO and OVLT, which have themselves been activated directly by angiotensin II. In the accompanying paper, furosemide-induced activation in the NTS and caudal thoracic spinal cord is abolished by prior bilateral renal denervation, meaning that these neurons are likely part of a renal afferent pathway. Here, these structures did not elaborate Fos in animals injected with furosemide plus the high CAP dose or furosemide plus Losartan. Thus, the present results also suggest that the central blockade of the formation of angiotensin II or blockade of the actions of angiotensin II prevents in some way the activation of the renal afferent pathway mediated by the renal nerves (the direct pathway) in response to the actions of furosemide. Therefore, these results suggest that central angiotensin II is somehow involved in “priming” or increasing the sensitivity of the direct renal afferent pathway. Taken together with the accompanying paper, our results indicate that interruption of the direct pathway via renal denervation did not interfere with the elaboration of Fos in the lamina terminalis; in contrast, modification of the humoral renal afferent pathway can affect the sensitivity of the direct pathway. These results may have important implications for pathophysiological changes associated with fluid balance disorders including renal hypertension.

Synaptic control of rat supraoptic neurones during osmotic stimulation of the organum vasculosum lamina terminalis in vitro.

1. The effects of osmotic or electrical stimulation of the organum vasculosum lamina terminalis (OVLT) were examined during intracellular recordings (32 degrees C) obtained from ninety-five supraoptic nucleus magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. 2. Brief (10-20 s) applications of hypertonic and hypotonic solutions to the area of the OVLT caused prolonged (> 1 min) increases and decreases, respectively, in electrical activity in seventy of seventy-four trials performed on neurones with membrane potentials near spike threshold (approximately -55 mV). Changes in firing frequency were related to changes in external osmolality in a dose-dependent manner between 275 and 355 mosmol kg-1. 3. When 30 s periods recorded immediately before, and 30 s following, the application of an osmotic stimulus were examined, the frequency of spontaneous EPSPs (sEPSPs) was related in a dose-dependent manner to the osmolality of the solution superfusing the OVLT region. The increased EPSP frequency was maintained and did not adapt if the osmolality of the medium was raised for periods of > 10 min. In contrast, the frequency of spontaneous IPSPs (sIPSPs) was virtually unaffected by changes in external osmotic pressure. 4. Osmotically evoked changes in MNC firing were strongly correlated with accompanying changes in the frequency of sEPSPs (slope, 0.9; correlation coefficient (r) = 0.7), but not sIPSPs (r = 0.2), suggesting that changes in firing rate following osmotic stimulation of the OVLT are selectively mediated by changes in synaptic excitation. 5. In the presence of bicuculline (5-10 microM), electrical stimulation of the OVLT evoked fast EPSPs in forty-seven of forty-eight MNCs tested. These responses were reversibly reduced by application of 20-40 microM kynurenic acid (n = 3) or 20-40 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; n = 11). Similarly, bath application of CNQX (n = 3) or kynurenic acid (n = 4) reversibly abolished the excitatory response of supraoptic neurones following hypertonic stimulation of the OVLT. 6. Brief (10-15 s) applications of gamma-aminobutyric acid (GABA) over the OVLT reversibly abolished increases in sEPSP frequency and action potential firing rate evoked by hyperosmotic stimulation of the OVLT. In the presence of GABA, the rates of sEPSP and sIPSP frequency were reduced to 37 +/- 10 and 44 +/- 13% (means +/- S.E.M.), respectively, of those observed under isotonic conditions (295 mosmol kg-1). 7. These results suggest that inhibitory and excitatory pathways originating from neurones located within the OVLT are tonically active under resting osmotic conditions in rat hypothalamic explants. Osmotically evoked changes in MNC firing, however, are selectively mediated through increases or decreases in the intensity of the excitatory component of OVLT-derived inputs.

Thermal and PGE2 sensitivity of the organum vasculosum lamina terminalis region and preoptic area in rat brain slices.

1. The effects of local applications of prostaglandin E2 (PGE2) on the unit activity of fifty-one neurones in the organum vasculosum lamina terminalis (OVLT) region and fifty-eight neurones in the preoptic area (POA) were investigated in small tissue slices from the rat hypothalamus containing the OVLT and POA isolated from each other. 2. Of these, thirty OVLT and twenty-eight POA neurones were warm sensitive and increased their discharge rate in response to a rise in tissue temperature. One OVLT neurone and one POA neurone were cold sensitive and showed the opposite type of responses to changes in temperature. The thermosensitivity of these neurones was still observed in a Ca2+ free-high Mg2+ solution. 3. Perfusion with PGE2 in doses between 1 and 250 nM changed the discharge rate in forty-two of fifty-one OVLT neurones and in thirty-two of fifty-eight POA neurones in a dose-dependent manner. The responses to PGE2 were not lost during synaptic blockade. The threshold dose of PGE2 to alter the discharge rate of the OVLT neurones (4.8 +/- 1.1 (S.E.M.) nM, n = 16) was significantly lower than that of the POA neurones (40.9 +/- 12.2 nM, n = 16). 4. Fifteen of forty-two OVLT neurones exhibited the responses with a slower onset (latency 5-13 min) and a longer duration (20 min to 3 h), but such responses were observed in only one of thirty-two POA neurones. 5. The responses of OVLT and POA neurones to PGE2 (50-250 nM) were reversibly blocked by a concurrent application of AH6809, a prostanoid EP1 and/or a DP receptor antagonist. 6. While there was no clear correlation between the type of thermosensitivity and the type of response to PGE2 among the POA neurones, a significantly higher incidence of inhibitory response to PGE2 was found among the warm-sensitive neurones in the OVLT region. 7. The lower threshold responses to PGE2 and the higher incidence of PGE2 responsiveness among OVLT neurones are consistent with previous findings which showed that the highest density of PGE2 receptor binding and the highest pyrogenic sensitivity to microinjected PGE2 were observed in the OVLT region. The results provide further evidence for the critical involvement of the OVLT region in mediating the febrile responses to blood-borne endogenous pyrogen through the local release of PGE2.

Enhancement of the febrile responses of rats to endogenous pyrogen occurs within the OVLT region

The febrile responses of male Sprague-Dawley rats to a semi-purified endogenous pyrogen (EP) derived from human monocytes are markedly enhanced 3 days after the animals are intravenously injected with a variety of immunoadjuvants. The present study was designed to investigate the site within the body at which these substances act to produce this febrile-enhancing phenomenon. Stainless steel microinjection cannula guide tubes were implanted within the region of the organum vasculosum lamina terminalis (OVLT) of the rats and control febrile dose-response curves to EP were established. Minute quantities of the immunoadjuvants zymosan, lipopolysaccharide endotoxin, and the synthetic adjuvant peptide, muramyl dipeptide, were microinjected into the OVLT region and 3 days later, the febrile responses of the animals were retested. In each case the febrile response elicited by a standard dose of EP was more than doubled, the slope of the fever dose-response curve was tripled, and the dose threshold was lowered by a factor of four to five. These responses are identical with those produced when much larger amounts of these immunoadjuvants are injected intravenously, and, thus, we conclude that the site of action of these substances in enhancing fever in response to EP resides in or near the OVLT region. It is proposed that EP stimulates a type of reticuloendothelial cell residing within the OVLT to release prostaglandin E, which in turn crosses the blood-brain barrier to effect the changes in the thermoregulatory neurons of the preoptic anterior hypothalamic area that result in fever.

Altered angiotensin II sensitivity of neurons in the organum vasculosum lamina terminalis region of the spontaneously hypertensive rat

Using in vitro hypothalamic brain slices, differences in angiotensin II (AII) sensitivity of neurons in the organum vasculosum lamina terminalis (OVLT) region were compared between spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY). AIIm the AII competitive antagonist saralasin, and l-glutamate were micropressure-applied onto OVLT neurons. AII excitation of SHR neurons was blocked or antagonized by simultaneous application of saralasin, evoked at significantly lower thresholds and displayed exaggerated periods of postactivity compared to OVLT neurons in preparations taken from WKY controls. Neuronal responses to l-glutamate were similar between the two rat strains. Differences in neuronal sensitivity to AII may be causally linked to hypertension in SHR.

Vasoconstrictor and vasodilator sites within anteroventral third ventricle region.

Previous studies have shown that electrical stimulation of the rat anteroventral third ventricle (AV3V) region produces a characteristic pattern of hemodynamic effects, i.e., renal and mesenteric vasoconstriction, and hindquarters vasodilation. In the present study, we localized the vasoconstrictor and vasodilator effects to specific subregions of the AV3V. In urethan-anesthetized rats prepared with arterial catheters and pulsed Doppler flow probes, we assessed the effects of electrical stimulation of four nuclei within AV3V on mean arterial pressure and renal, mesenteric, and hindquarters resistance. These nuclei were the organum vasculosum lamina terminalis (OVLT), ventral nucleus medianus (median preoptic nucleus), anterior (precommissural) nucleus medianus (median preoptic nucleus), and periventricular preoptic nuclei. Stimulation was carried out by use of a tungsten microelectrode. Stimulation of the OVLT consistently provoked stimulus-locked increases in arterial pressure coupled with increases in mesenteric and renal vascular resistance. Ganglionic blockade with chlorisondamine prevented these responses, demonstrating that they were mediated neurogenically. Stimulation of the three remaining nuclei produced decreases in arterial pressure, hindquarters vasodilation, and little change in mesenteric and renal vascular resistance. No changes in heart rate were observed with stimulation of any of the four nuclei. These results suggest that the vasoconstrictor and pressor functions of the AV3V region are localized in or near the OVLT region, whereas the remaining nuclei of the AV3V region mediate vasodilator and depressor responses.

Quantitativein VivoAutoradiographic Localization of [125I_Tyr11] Somatostatin-14-and [Leu8,d-Trp22-125ITyr-125] Somatostatin-28-Binding Sites in Rat Brain*

Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14 section) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28 section) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14 section (with or without unlabeled S-14) or 17 pmol S-28 section (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14 section- and S-28 section-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14 section and S-28 section (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14 section was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina terminalis region. Median eminence (ME) contained virtually no specific S-14 section ARG. As with S-14 section, the highest ARG density of S-28 section-binding sites was also found in the area postrema, which labeled approximately equally with the two radioligands. This was followed by the ME, subfornical organ, and organum vasculosum lamina terminalis. The overall patterns of labeling of the CVOs with S-14 section and S-28 section showed significant differences, especially in the ME. Within the ME, labeled S-28 section was concentrated in a broad band throughout the external zone in a location identical to that of immunoreactive S-14. Analysis of dose-response curves obtained with 0.3-30 nmol unlabeled S-14 or S-28 revealed IC50 values for S-14 3- to 6-fold lower than those for S-28 for all labeled CVOs. With both S-14 section and S-28 section, the labelling density of the adrenal glands was double that of the area postrema. Adrenal binding of the radioligands was confined to the cells of the zona glomerulosa. We conclude: specific high affinity binding sites for S-14 section and S-28 section exist in the CVOs and the adrenal glomerulosa; and the 3- to 6-fold higher affinity of binding of S-14 to CVOs compared to S-28 together with the dissimilar patterns of labelling of the different CVOs by the two radioligands suggest the existence of separate populations of S-14 and S-28 receptors.

Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin secretion in the female rat.

The present study examines the function of several cytologically distinct suprachiasmatic structures in the regulation of ovulation and positive feedback effects of estrogen and progesterone on gonadotropin release in the rat. Small (0.6-0.8 mm dia.) electrolytic lesions were placed at four loci along the rostrocaudal extent of the suprachiasmatic region in regularly cycling female rats. Anovulatory persistent estrus occurred only when lesions were located either in the suprachiasmatic nucleus (SCN) or the medial preoptic nucleus (MPN), a small periventricular cell group lying immediately caudal to the organum vasculosum lamina terminalis (OVLT). Lesions restricted to the OVLT and adjacent ventral prechiasmatic region (VPC-L), or the anterior suprachiasmatic region (ASR) between the MPN and SCN resulted in irregular estrous cycles frequently marked by periods of prolonged diestrus. Following administration of 50 microgram estradiol benzoate (EB) a daily afternoon surge of gonadotropin was observed in control animals. This circadian release of gonadotropins was completely abolished by SCN, ASR and MPN lesions. EB-induced gonadotropin surges were also greatly attenuated by VPC-L lesions. Subsequent administration of 1.5 mg progesterone (P) induced large surges of luteinizing hormone and follicle-stimulating hormone in VPC-L and ASR lesioned animals as well as controls. P administration also elicited gonadotropin surges in SCN lesioned animals, although surges were markedly attenuated in magnitude compared to controls. Only lesions that destroyed the MPN and immediately adjacent periventricular tissue completely and invariably eliminated P-induced gonadotropin release. Thus, anovulatory-persistent estrus appears to be associated specifically with lesions that interfere with the-positive feedback effect of P (MPN and SCN lesions). Animals with lesions that block or attenuate EB effects without interfering with P sensitive neural substrates can maintain long-term spontaneous ovulation (VPC-L and ASR lesions). An hypothesis is advanced to account for the differential effect of MPN and SCN lesions on P-induced gonadotropin release.