Effects of unilateral or bilateral lesions within the anteroventral third ventricular region on c-fos expression induced by dehydration or angiotensin II in the supraoptic and paraventricular nuclei of the hypothalamus

This paper reports the effects of AV3V lesions on the pattern of c-fos induced by 24 h dehydration. As expected, bilateral electrolytic lesions within the AV3V region (the ventral median preoptic nucleus) suppressed water intake following 24 h water deprivation. C-fos expression was also suppressed in the supraoptic (SON) and (less completely) in the paraventricular (PVN) nuclei, but not in the subfornical organ (SFO). Unilateral lesions of the AV3V region suppressed c fos expression in the ipsilateral SON, but this selective ipsilateral effect was less in the PVN. The SFO was again unaffected. Unilateral lesions also suppressed c-fos expression in the ipsilateral SON and PVN (to a lesser degree) following intraventricular infusions of angiotensin 11 (250 pmol). These results suggest that the cellular response of supraoptic neurons to osmotic stimuli require inputs from the AV3V region, but that this is less absolute for the PVN; that the projection from the ventral AV3V area to the SON is ipsilateral, but that to the PVN may be less lateralised. Activation of the SFO by dehydration is not dependent upon the integrity of the ventral AV3V region. These results are closely comparable to the effects of similar lesions on c-fos expression following intraventricular infusions of angiotensin 11, and suggest that the effect of dehydration on forebrain c-fos expression may be related to the central actions of angiotensin II.     

Water deprivation induces regional expression of c-fos protein in the brain of inbred polydipsic mice

We studied the effects of water deprivation on the expression of c-fos protein (Fos) in the brain of inbred polydipsic mice, STR/N strain, that show extreme polydipsia without a lack of vasopressin in the body. Non-polydipsic mice, ICR strain, were used as controls. All male animals were deprived of water for 24 and 48 h. Fos-like immunoreactivity (Fos-LI) in the brain was studied by immunohistochemical techniques. In both groups of mice water deprivation induced a remarkable increase in Fos-LI in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, the median preoptic nucleus (MnPO), the organum vasculosum laminae terminalis (OVLT) and the subfornical organ (SFO). A far more increase, however, was seen in the MnPO, the SFO and the area postrema (AP) of the polydipsic mice compared to those of the non-polydipsic control mice. In the nucleus of the tractus solitarius (NTS) and in the anteroventral part of the PVN (avPVN), water deprivation caused a clear increase in Fos-LI in the polydipsic mice, while in the non-polydipsic mice the same treatment induced no Fos-LI in the NTS and no change in the avPVN. These results indicate that neurons in the circumventricular organs and the NTS are strongly activated by water deprivation in the polydipsic mice, suggesting that these brain structures play an important role in the polydipsia.     

Effects of intracerebroventricular dizocilpine (MK801) on dehydration-induced dipsogenic responses, plasma vasopressin and c-fos expression in the rat forebrain

This study determined the interaction between glutamate receptors and dehydration-induced drinking, vasopressin (AVP) release, plasma osmolality and c-fos expression in the brain of conscious rats. The NMDA receptor antagonist dizocilpine (100 nmol infused into the cerebral ventricles) suppressed drinking following either 22 h water deprivation or intragastric injection of hypertonic saline (1.5 M), attenuated the increased plasma vasopressin induced by dehydration, but had no effects on peripheral hyperosmolality caused by either water deprivation or injections of hypertonic saline. Dizocilpine had no inhibitory effects on feeding after 24 h food deprivation. Dizocilpine also suppressed c-fos expression induced by dehydration in the median preoptic nucleus (MPN), the supraoptic and paraventricular nuclei (SON and PVN), but did not influence c-fos expression in the subfornical organ (SFO). The non-NMDA receptor antagonists CNQX (400 nmol) or DNQX (60 nmol) affected neither the animals' drinking nor c-fos expression induced by dehydration. Double staining showed that suppression of c-fos expression following dizocilpine occurred in the NMDA R1 receptor containing neurons in the hypothalamus. These results suggest that the NMDA-type glutamate receptors may be involved in dehydration induced dipsogenic and neuroendocrinological responses. They complement our earlier findings that dizocilpine also attenuated drinking and c-fos expression following intraventricular infusions of angiotensin II.     

Water-deprived white-footed mice express c-fos on a day/night cycle graded according to the duration of deprivation

Mammals respond to electrolyte and water imbalance by a variety of neural and endocrine mechanisms that regulate water and salt intake and loss. We used the expression of c-fos and Fos-related antigens to indicate neuronal activation in hypothalamic neurons of members of an outbred laboratory population of white-footed mice (Peromyscus leucopus) deprived of water for biologically reasonable periods of time (6–18 h). We examined Fos-like immunoreactivity (Fos-LIR) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). During the dark period, when these animals are normally active, 6 h of water deprivation produced near-maximal increases in the number of cells positive for Fos-LIR in the SON and PVN. In contrast, during the light period, when these mice are normally inactive and do not have access to water, 6 h of water deprivation only slightly affected Fos-LIR. During the day, it required as much as 12 h of water deprivation to produce increases in Fos-LIR cells approaching those achieved at night. Plasma osmolarity was directly related to the number of Fos-LIR cells. In addition, mice lost weight more rapidly at night than during the day when water-deprived, and also recovered that lost weight more rapidly when access to water was returned. Our results show (1) that biologically reasonable levels of water restriction (and resulting changes in blood osmolarity) induce changes in Fos-LIR in this wild mouse species, and (2) that these mice have a daily cycle of sensitivity to water deprivation that is demonstrated by both behavioral, psychological and immunohistological assessment of reactions to water deprivation.     

Effect of lesions of forebrain circumventricular organs on c-fos expression in the central nervous system to plasma hypernatremia

Experiments were carried out on conscious adult male Wistar rats to investigate the effect of selective ablation of the subfornical organ (SFO), and/or the anteroventral third ventricular (AV3V) region on the induction of Fos in central structures in response to plasma hypernatremia. Fos induction, detected immunohistochemically, was used as a marker for neuronal activation. Intravenous infusions of hypertonic saline resulted in dense Fos-like immunoreactivity in several forebrain (paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus (SON), median preoptic nucleus (MnPO), medial preoptic nucleus, organum vasculosum of the laminae terminalis and SFO) and brainstem (nucleus of the solitary tract, ventrolateral medulla, and parabrachial nucleus) structures. Intravenous infusions of the hypertonic saline solution into animals with lesions of either the SFO, the AV3V or both resulted in a decreased number of Fos-like immunoreactive neurons in the MnPO, PVH and SON. In addition, the number of Fos-labeled neurons in the SON after lesions of both the SFO and the AV3V was significantly greater than that observed in isotonic saline infused controls. Finally, lesions of the forebrain circumventricular structures did not alter the Fos labeling in brainstem structures as a result of the infusion of the hypertonic solution. These data suggest that changes in plasma osmolality and/or concentration of sodium alter the activity of SON and brainstem neurons in the absence of afferent inputs from the SFO and AV3V.     

Central renin injections: effects on drinking and expression of immediate early genes

This study investigated the drinking response and the expression of Fos- and Egr-1-immunoreactivity (Fos-ir; Egr-1-ir) in the brain induced by endogenous angiotensin generated by intracerebroventricular (i.c.v.) injection of renin. Renin induced Fos-ir in the subfornical organ (SFO), median preoptic (MnPO), supraoptic and paraventricular nuclei (SON and PVN), area postrema (AP), nuclei of the solitary tract (NTS) and lateral parabrachial nuclei (LPBN). Renin-induced Egr-1-ir exhibited a similar pattern of distribution as that observed for Fos-ir. The dose of i.c.v. renin that induced expression of immediate early gene (IEG) product immunoreactivity also produced vigorous drinking. When renin-injected rats were pretreated with captopril, an angiotensin converting enzyme inhibitor, drinking was blocked. With the same captopril pretreatment, both Fos- and Egr-1-ir in the SFO, MnPO, SON, PVN, AP and LPBN were also significantly reduced.     

Altered c-fos expression in autonomic regulatory centers of genetically obese (ob/ob) mouse brain

This study examined c-fos-like immunoreactivity (FLI) in the brain of obese (ob/ob) and lean (+/+) mice during states of satiety and hunger. The results demonstrated that there is an altered neural activity primarily in the hypothalamus of the obese mouse brain. Most interestingly, only obese mice had an increased FLI in the dorsomedial and supramammillary nuclei. Conversely, only lean mice showed increase in FLI in the medial part of perifornical nucleus following food deprivation for 24 h.     

Regional differences in the expression of Fos-like immunoreactivity after central salt loading in conscious rats: modulation by endogenous vasopressin and role of the area postrema

In this study, we examined the quantitative relationship between centrally administered hypertonic saline (HS) concentrations and the expression of Fos-like immunoreactivity (FLI) in brain regions involved in the homeostasis of body fluids. The regions examined were the organum vasculosum laminae terminalis (OVLT), the median preoptic nucleus (MnPO), the subfornical organ (SFO), the paraventricular nucleus (PVN), the supraoptic nucleus of the hypothalamus, the nucleus of the solitary tract (NTS), and the area postrema (AP). The experiments were performed in conscious rats with attention to the actual changes in central [Na(+)]. Hypertonic saline (0.3, 0.67, or 1.0 M) was delivered at 1 microl/min for 20 min. The changes in cerebrospinal fluid [Na(+)] during i.c.v. administration of 0.3 M hypertonic saline were compatible with those expected for thermal dehydration. FLI increased in a dose-dependent manner in the dorsomedial cap of the PVN and NTS. Although the pressor responses during central salt loading were not significantly affected by pretreatment with the peripheral vasopressin V(1) receptor antagonist OPC-21268, FLI expression in the PVN was significantly augmented. In addition, in AP-lesioned rats, FLI expression in the lateral magnocellular part of the PVN and NTS was significantly enhanced after central salt loading. These results suggest that the peripheral vasopressin system participates in negative feedback to modulate neuronal activities in the PVN, probably through the AP or direct action at the PVN in response to central osmotic and/or Na(+) stimulation.     

Area postrema removal abolishes stimulatory effects of intravenous interleukin-1β on hypothalamic-pituitary-adrenal axis activity and c-fos mRNA in the hypothalamic paraventricular nucleus

This study examined the role of the area postrema (AP) in transducing peripheral immune signals, represented by intravenous (i.v.) interleukin-1β (IL-1), into neuroendocrine responses. The AP, a circumventricular organ with a leaky blood–brain barrier, lies adjacent to the nucleus of the solitary tract (NTS) in the medulla. The AP was removed by aspiration, and 2 weeks later, AP-lesioned or sham-lesioned rats were injected i.v. with 0.5 μg/kg IL-1 or sterile saline. After 30 min, brains were removed and analyzed for c-fos mRNA levels in various structures implicated in the hypothalamic-pituitary-adrenal axis response to peripheral cytokine challenge. The sham-lesioned animals responded to IL-1 with large elevations in adrenocorticotropic hormone (ACTH) and corticosterone levels in the plasma and c-fos mRNA levels in cells of the AP, NTS, central nucleus of the amygdala, bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus (PVN), and meninges. Prior AP removal abolished the IL-1-induced increases in ACTH and corticosterone in the plasma and c-fos mRNA levels in the NTS and PVN. However, AP removal had no effect on IL-1-induced increases in c-fos mRNA levels in the other areas examined. The selective AP lesion effects suggest that the AP and adjacent NTS play a pivotal role in transducing a circulating IL-1 signal into hypothalamic-pituitary-adrenal axis activation by a pathway that may be comprised of known anatomical links between the AP, NTS, and corticotropin-releasing hormone neurons of the PVN.     

Neuronal expression of Fos protein in the hypothalamus of rats with heart failure

We sought to identify the areas that have altered neuronal activity within the hypothalamus of rats with heart failure (HF) by mapping neuronal staining of c-Fos protein (Fos) 6-8 weeks following coronary artery ligation (HF group; n=17) or sham surgery (sham-operated control group, n=15). Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic nucleus (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) using a standard ABC immunocytochemical protocol. The rats in the HF group displayed infarcts averaging 34+/-2% of the outer circumference and 41+/-1% of the inner circumference of the left ventricular wall. Sham-operated control rats had no observable damage to the myocardium. Rats with chronic heart failure (n=5) but no manipulation (no surgery) had a similar number of Fos-staining cells in PVN SON, MnPO, AH and PH compared to sham-operated rats. Acute surgery for isolation of vagus nerves and anesthesia for 90 min increased the number of Fos positive cells in PVN, SON and MnPO of both sham-operated rats and rats with HF. Furthermore, rats with heart failure (n=5) had significantly higher number of Fos-staining cells in PVN (four times), SON (4.5 times) and MnPO (1.5 times) compared to sham-operated rats after acute surgery for isolation of the vagus. The number of Fos-staining cells remained unaltered in AH and PH in both groups of rats. However, in a third series of experiments vagotomy reduced the number of Fos-staining cells in the PVN, SON or MnPO of rats with HF (n=5) to those observed in sham-operated vagotomized rats. This study shows that: (1) there is augmented neuronal activity as indicated by increased number of Fos staining neurons in the PVN, SON and MnPO due to acute surgical stress in rats with HF, and (2) vagal afferents are responsible for the increased neuronal activity in PVN, SON and MnPO of rats with HF during acute surgical stress. These data support the conclusion that vasopressin producing neurons and autonomic areas within the hypothalamus influenced by vagal afferents are activated during HF and are sensitive to 'acute surgical stress' and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in heart failure.     

Fos-like immunoreactivity and thirst following hyperosmotic loading in rats with subdiaphragmatic vagotomy

If receptors in the gut relay information about increases in local osmolality to the brain via the vagus nerve, then vagotomy should diminish this signaling and reduce both thirst and brain Fos-like immunoreactivity (Fos-ir). Water intake in response to hypertonic saline (i.p. or i.g., 1 M NaCl, 1% BW; i.g., 0.6 M NaCl, 0.5% BW) was reduced during 120 min in rats with subdiaphragmatic vagotomy (VGX) compared to sham-VGX rats. Brain Fos-ir was examined in response to both i.g. loads. After the smaller load, VGX greatly reduced Fos-ir in the supraoptic nucleus (SON) and the magnocellular and parvocellular areas of the paraventricular nucleus (PVN). Fos-ir in the subfornical organ (SFO) and nucleus of the solitary tract (NTS) was not affected. After the larger load, VGX significantly reduced Fos-ir in the parvocellular PVN and in the NTS, but not in the other regions. Thus, decreased water intake by VGX rats was accompanied by decreased Fos-ir in the parvocellular PVN after the same treatments, indicating a role for the abdominal vagus in thirst in response to signaling from gut osmoreceptors. The decreased water intake in the VGX group was not reflected as a decrease in Fos-ir in the SFO. Absorption of the larger i.g. load may have activated Fos-ir through more rapidly increasing systemic osmolality, thereby obscuring a role for the vagus at this dose in the SON and magnocellular PVN.     

Expression of Fos in rat brain in relation to sodium appetite: furosemide and cerebroventricular renin

Two experiments were performed to investigate the relationship between the expression of sodium appetite and the appearance of Fos-like immunoreactivity (Fos-IR) in the brain of rats. In the first experiment, rats were depleted of sodium by treatment with furosemide 24 h prior to sacrifice and without access to either food or sodium solution. Some rats had access to distilled water, and others had no fluids available during the 24 h. All of the furosemide-treated rats showed Fos-IR in both the subfornical organ (SFO) and around the organum vasculosum laminae terminalis (OVLT). Rats with access to distilled water during the depletion period showed no Fos-IR in the supraoptic (SON) or paraventricular hypothalamic nuclei (PVN) and, in parallel behavioral studies, comparably-treated rats consumed only 0.3 M NaCl solution at the end of the 24 h. In rats that had no fluids during the deprivation period, only about one half showed Fos-IR in SON and PVN and, in parallel behavioral studies, comparably treated rats consumed both water and 0.3 M NaCI solution at the end of 24 h. In a second experiment, cerebroventricular administration of renin stimulated short latency intake of 0.3 M NaCI and water. The relative intakes of water and NaCl were comparable at a low dose of renin, but intake of water exceeded that of NaCl after higher doses. Renin induced Fos-IR in SFO, MnPO, peri-OVLT region, SON and PVN. Both Fos-IR and fluid intake were antagonized by administration of losartan, an angiotensin 11 type 1 receptor antagonist. Thus, only the circumventricular organs of the lamina terminalis showed Fos-IR during each natriorexigenic regimen in these studies. These data support the view that Ang 11 of both central and peripheral origin activates the SFO and/or peri-OVLT region and contributes to sodium appetite.     

Indomethacin Attenuates Oxytocin and Hypothalamic-Pituitary-Adrenal Axis Responses to Systemic Interleukin-1β

Systemic administration of the cytokine IL-1β produces a significant release of ACTH into the plasma and activation of hypothalamic oxytocin (OT) and corticotropin releasing factor (CRF) cells. However, the mechanism(s) by which systemic IL-1β induces these responses is not clear. In the present study, we have investigated the proposal that catecholamine cells of the ventrolateral medulla (VLM) and nucleus of the solitary tract (NTS) can relay circulating IL-1 signals via a prostaglandin-dependent mechanism to effect the HPA axis responses in the rat. Intra-arterial administration of IL-1β (1 pg/kg) to otherwise untreated animals produced a prominent release of ACTH into the plasma, substantial c-fos expression in paraventricular medial parvocellular (mPVN) corticotropin releasing factor (CRF) cells, supraoptic (SON) and paraventricular nucleus (PVN) OT cells, area postrema cells, NTS and VLM catecholamine cells and cells of the central amygdala. Pretreatment with the prostaglandin synthesis inhibitor, indomethacin (10 mg/kg body weight ia) 15 min before IL-1β administration (1 pg/kg ia) significantly reduced plasma ACTH release and c-fos expression in PVN and SON OT cells and MPVN CRF cells. In addition, the area postrema, A1 and C1 catecholamine cell groups of the VLM and A2 and C2 catecholamine cell groups of the NTS, all exhibited concomitant reductions in c-fos expression. Conversely indomethacin administration did not alter the IL1β-induced expression of c-fos in the central amygdala. These data suggest that central pathways involved in the IL-1β-induced activation of the HPA axis and OT cells are, at least in part, dependent upon prostaglandin synthesis. It is proposed that neurons in the area postrema, NTS and VLM might mediate this IL-1β-induced activation of hypothalamic CRF and OT cells and release of ACTH into the plasma.     

Regional suppression by water intake of c-fos expression induced by intraventricular infusions of angiotensin II

Intracerebroventricular (i.c.v.) infusions of angiotensin II (AII) reliably induced c-fos expression in the supraoptic (SON) and paraventricular (PVN) nuclei, as well as other areas of the basal forebrain including the OVLT, subfornical organ (SFO), and bed nucleus (BNST). Double-labelling showed that AII-induced c-fos was observed in both vasopressin (AVP-) and oxytocin (OXY)-containing neurons of the SON and PVN in male rats. Allowing rats to drink water after AII infusions suppressed c-fos expression both AVP- and OXY-stained magnocellular neurons. Intragastric infusions of water were also effective, showing that oro-pharyngeal stimuli were not critical. Maximal suppression occurred in rats in whom water had been infused intragastrically about 5 min before i.c.v. AII infusions, suggesting that changes in osmolarity were responsible. i.c.v. AII also induced c-fos expression in a number of brainstem structures, including the solitary nucleus (NTS), lateral parabrachial nucleus (LPBN), locus coeruleus (LC), and the area postrema (AP). These results indicate that AVP and OXY-containing neurons in the magnocellular parts of the SON and PVN alter their immediate-early gene response to AII after water intake, and that this does not depend upon oro-pharyngeal factors. Furthermore, AII can induce c-fos expression in a number of brainstem nuclei associated with autonomic function, and these do not respond to water intake.     

Oxytocin neurons in the rat hypothalamus exhibit c-fos immunoreactivity upon osmotic stress

In order to evaluate the responses to osmotic stress of oxytocinergic neurons in vivo, we have studied oxytocin (OXY) and c-fos protein expression in the brain by means of double-immunostaining. C-fos immunoreactivity was detected in a subset of OXY neurons, as well as in other neurons non-immunoreactive for OXY, as early as 90 min after intraperitoneal injection of a hypertonic saline solution. C-fos expression was found in approx. 70% of OXY-immunoreactive neurons in the supraoptic (SON), lateral subcommisural (LSN) and paraventricular (PVN) nuclei, and not in OXY neurons in other hypothalamic areas. The expression of c-fos may be used as a means to map the circuitry by which osmotic stimulation activates OXY-containing neurons, and thus provide further insights into the functions with which OXY may be associated.     

Neuronal expression of fos protein in the forebrain of diabetic rats

We sought to identify the areas that have altered neuronal activity within the hypothalamus of diabetic rats by mapping neuronal expression of c-fos protein (Fos) and Fos-related antigens. After a standard PAP immunocytochemical protocol, Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) of control (vehicle; n=6) and diabetic rats (Sprague-Dawley rats injected with STZ 65 mg/kg/ip 4 weeks prior to the experiment; n=6). Blood glucose levels were significantly elevated in the diabetic group (370+/-8 mg/dl) compared to control group (104+/-3 mg/dl). Diabetic rats had a significantly higher number of Fos-positive cells in PVN (2.5x), SON (7x) and MnPO (2x) compared to the control rats. However, diabetic rats had significantly fewer Fos-positive cells in the AH (0.3x) and no difference was observed in the PH between the diabetic and control rats. Despite the elevated number of Fos-positive cells in the diabetic rats, dehydration (water withdrawal for 24 h) or hypertonic challenge (1.5 ml of 0.1 M NaCl i.p. injection) produced a further increase in the number of Fos-positive cells in the PVN, SON and MnPO. Dehydration did not alter the number of Fos-positive cells in the AH or PH, but hypertonic challenge produced a significant increase in the Fos-positive cells in both the AH and PH of diabetic rats. This study demonstrates that: (1) there is increased basal neuronal activity in the PVN, SON and MnPO, a decrease in neuronal activity in the AH and no change in neuronal activity in the PH as indicated by Fos staining in diabetic rats; and (2) dehydration or hypertonic challenge produces a further increase in the number of Fos-positive cells in the PVN, SON, and MnPO which is comparable to control rats. These data support the conclusion that vasopressin producing neurons in the PVN and SON and autonomic areas within the lamina terminalis and hypothalamus are activated during diabetes and may contribute to the elevated levels of vasopressin and autonomic dysfunction during diabetes.