Increased renal Na-K-ATPase, NCC, and beta-ENaC abundance in obese Zucker rats.

Renal sodium retention, as a result of increased abundance of sodium transporters, may play a role in the development and/or maintenance of the increased blood pressure in obesity. To address this hypothesis, we evaluated the relative abundances of renal sodium transporters in lean and obese Zucker rats at 2 and 4 mo of age by semiquantitative immunoblotting. Mean systolic blood pressure was higher in obese rats relative to lean at 3 mo, P < 0.02. Furthermore, circulating insulin levels were 6- or 13-fold higher in obese rats compared with lean at 2 or 4 mo of age, respectively. The abundances of the alpha(1)-subunit of Na-K-ATPase, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC), and the beta-subunit of the epithelial sodium channel (ENaC) were all significantly increased in the obese rats' kidneys. There were no differences for the sodium hydrogen exchanger (NHE3), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC1), the type II sodium-phosphate cotransporter (NaPi-2), or the alpha-subunit of ENaC. These selective increases could possibly increase sodium retention by the kidney and therefore could play a role in obesity-related hypertension.

Mineralocorticoid treatment attenuates activation of oxytocinergic and vasopressinergic neurons by icv ANG II.

Central oxytocin (OT) neurons limit intracerebroventricular (icv) ANG II-induced NaCl intake. Because mineralocorticoids synergistically increase ANG II-induced NaCl intake, we hypothesized that mineralocorticoids may attenuate ANG II-induced activation of inhibitory OT neurons. To test this hypothesis, we determined the effect of deoxycorticosterone (DOCA; 2 mg/day) on icv ANG II-induced c-Fos immunoreactivity in OT and vasopressin (VP) neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and also on pituitary OT and VP secretion in male rats. DOCA significantly decreased the percentage of c-Fos-positive (%c-Fos+) OT neurons in the SON and PVN, both in the magnocellular and parvocellular subdivisions, and the %c-Fos+ VP neurons in the SON after a 5-ng icv injection of ANG II. DOCA also significantly reduced the %c-Fos+ OT neurons in the SON after 10 ng ANG II and tended to attenuate 10 ng ANG II-induced OT secretion. However, the %c-Fos+ OT neurons in DOCA-treated rats was greater after 10 ng ANG II, and DOCA did not affect the %c-Fos+ OT neurons in the PVN nor VP secretion or c-Fos immunoreactivity in either the SON or PVN after 10 ng ANG II. DOCA also did not significantly alter the effect of intraperitoneal (ip) cholecystokinin (62 microg) on %c-Fos+ OT neurons or of ip NaCl (2 ml of 2 M NaCl) on the %c-Fos+ OT and VP neurons. These findings indicate that DOCA attenuates the responsiveness of OT and VP neurons to ANG II without completely suppressing the activity of these neurons and, therefore, support the hypothesis that attenuation of OT neuronal activity is one mechanism by which mineralocorticoids enhance NaCl intake.

Estradiol attenuates angiotensin-induced aldosterone secretion in ovariectomized rats.

Estrogen replacement therapy significantly reduces the risk of cardiovascular disease in postmenopausal women. Previous studies indicate that estradiol (E2) decreases angiotensin II (AT) receptor density in the adrenal and pituitary in NaCl-loaded rats. We used an in vivo model that eliminates the potentially confounding influence of ACTH to determine whether the E2-induced decrease in adrenal AT receptor expression affects aldosterone responses to angiotensin II (Ang II). Female rats were ovariectomized, treated with oil (OVX) or E2 (OVX+E2; 10 microg, s.c.) for 14 days, and fed a NaCl-deficient diet for the last 7 days to maximize adrenal AT receptor expression and responsiveness. On days 12-14 rats were treated with dexamethasone (DEX; 25 microg, i.p., every 12 h) to suppress plasma ACTH. On day 14 aldosterone secretion was measured after a 30-min infusion of Ang II (330 ng/min). Ang II infusion increased the peak plasma aldosterone levels to a lesser degree in the OVX+E2 than in the OVX rats (OVX, 1870 +/- 290 pg/ml; OVX+E2, 1010 +/- 86 pg/ml; P < 0.05). Ang II-induced ACTH and aldosterone secretion was also studied in rats that were not treated with DEX. In the absence of DEX, the peak plasma aldosterone response was also significantly decreased (OVX, 5360 +/- 1200 pg/ml; OVX+E2, 2960 +/- 570 pg/ml; P < 0.05). However, E2 also reduced the plasma ACTH response to Ang II (P < 0.05; OVX, 220 +/- 29 pg/ml; OVX+E2, 160 +/- 20 pg/ml), suggesting that reduced pituitary ACTH responsiveness to Ang II contributes to the effect of E2 on Ang II-induced aldosterone secretion. Adrenal AT1 binding studies confirmed that E2 significantly reduces adrenal AT1 receptor expression in both the presence and absence of DEX in NaCl-deprived rats. These results indicate that E2-induced decreases in pituitary and adrenal AT1 receptor expression are associated with attenuated pituitary ACTH and adrenal aldosterone responses to Ang II and suggest that estrogen replacement therapy may modulate Ang II-stimulated aldosterone secretion as part of its well known cardioprotective actions.

Estrogen regulates angiotensin AT1 receptor expression via cytosolic proteins that bind to the 5' leader sequence of the receptor mRNA.

Two of the most highly recognized factors implicated in the pathogenesis of hypertension, atherosclerosis, congestive heart failure and associated cardiovascular disease are the renin angiotensin system (RAS) and estrogen. A major effect of estrogen results from its influence on the RAS. Beta-estradiol (E2) replacement in ovariectomized (OVX) rats significantly decreased type 1 angiotensin (AT1) receptor expression in the pituitary and adrenal, whereas it significantly increased receptor expression in the uterus when compared to OVX controls. Additional evidence demonstrated an important influence of estrogen on a recently discovered post-transcriptional mechanism for regulating expression of the AT1 receptor. This mechanism consists of cytosolic RNA binding proteins (BPs) that recognize the 5' leader sequence (5'LS) of the receptor mRNA. The activities of these 5'LS BPs were modulated by estrogen in an inverse manner to AT1 receptor regulation. Moreover, in vitro translation assays in wheat germ lysates suggested that the 5'LS BPs inhibited AT1 receptor translation. Our data therefore indicate that hormonal regulation of AT1 receptors involves modulation of 5'LS BPs by estrogen. These findings may in part account for the observed protective effects of estrogen on cardiovascular disease.

Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct.

Previously, we demonstrated that escape from vasopressin-induced antidiuresis ("vasopressin escape") in rats is associated with a large, selective decrease in whole kidney expression of aquaporin-2, the vasopressin-regulated water channel. Here, we show that isolated perfused inner medullary collecting ducts (IMCDs) from vasopressin-escape rats desamino-[D-arginine]vasopressin (DDAVP)/water-loaded have dramatically reduced vasopressin-dependent osmotic water permeabilities [46% of control rats (DDAVP alone)], which coincides with a fall in inner medullary aquaporin-2 protein abundance as measured by immunoblotting in the opposite kidney. Furthermore, we demonstrate in IMCD suspensions that cAMP accumulation in response to DDAVP is substantially reduced in the vasopressin-escape rats both in the presence and absence of the phosphodiesterase inhibitor IBMX. By immunoblotting, we show that the abundance of two proteins important in cAMP generation: the stimulatory heterotrimeric G protein subunit Gs and adenylyl cyclase type VI, do not change. We conclude that vasopressin escape is associated with relative vasopressin resistance of the collecting duct cells manifested by decreased intracellular cAMP levels. The decreased cAMP levels can contribute to the demonstrated decrease in collecting duct water permeability in two ways: 1) by causing a decrease in aquaporin-2 expression and 2) by limiting the acute action of vasopressin to increase collecting duct water permeability.

Medullary c-Fos activation in rats after ingestion of a satiating meal.

The distribution and chemical phenotypes of hindbrain neurons that are activated in rats after food ingestion were examined. Rats were anesthetized and perfused with fixative 30 min after the end of 1-h meals of an unrestricted or rationed amount of food, or after no meal. Brain sections were processed for localization of the immediate-early gene product c-Fos, a marker of stimulus-induced neural activation. Hindbrain c-Fos expression was low in rats that ate a rationed meal or no meal. Conversely, c-Fos was prominent in the medial nucleus of the solitary tract (NST) and area postrema in rats that ate to satiety. There was a significant positive correlation between postmortem weight of gastric contents and the proportion of NST catecholaminergic neurons expressing c-Fos. Cells in the ventrolateral medulla (VLM) were not activated in rats after food ingestion, in contrast with previous findings that stimulation of gastric vagal afferents with anorexigenic doses of cholecystokinin activates c-Fos expression in both NST and VLM catecholaminergic cells. These findings indicate that anatomically distinct subsets of hindbrain catecholaminergic neurons are activated in rats after food ingestion and that activation of these cells is quantitatively related to the magnitude of feeding-induced gastric distension.

Central c-Fos expression in neonatal and adult rats after subcutaneous injection of hypertonic saline.

Centrally-mediated responses to plasma hyperosmolality include compensatory drinking and pituitary secretion of vasopressin and oxytocin in both adult and neonatal rats. However, the anorexia that is produced by plasma hyperosmolality in adult rats is not evident in neonates, perhaps due to functional immaturity of osmoresponsive hindbrain circuits. To examine this possibility, the present study compared treatment-induced brain expression of the immediate-early gene product c-Fos as a marker of neural activation in adult and two-day-old rats after subcutaneous injection of 2 M NaCl (0.1 ml/10 g body weight). This treatment produced marked hypernatremia in adult and two-day-old rats without altering plasma volume. Several brain regions (including components of the lamina terminalis, the paraventricular and supraoptic nuclei of the hypothalamus, and the area postrema) were activated to express c-Fos similarly in adult and two-day-old rats after 2 M NaCl injection, consistent with previous reports implicating a subset of these regions in osmotically-stimulated drinking and neurohypophyseal secretion. In contrast, other areas of the brain that were activated to express c-Fos in adult rats after 2 M NaCl injection were not activated in neonates: these areas included the central nucleus of the amygdala, the parabrachial nucleus and catecholamine cell groups within the caudal medulla. This study demonstrates that certain brain regions that are osmoresponsive in adult rats (as defined by induced c-Fos expression) are not osmoresponsive in two-day-old rats. When considered in the context of known differences between the osmoregulatory capacities of adult and neonatal rats, our results are consistent with the idea that osmoresponsive forebrain centres are primarily involved in osmotically-stimulated compensatory drinking and neurohypophyseal secretion, whereas osmoresponsive regions of the hindbrain are important for concomitant inhibition of feeding and gastric emptying.

Cholecystokinin induces Fos expression in catecholaminergic neurons of the macaque monkey caudal medulla.

Systemic administration of cholecystokinin octapeptide (CCK) slows gastric emptying, inhibits feeding, and stimulates pituitary hormone release in rats and primates. To characterize the central neural circuits that mediate these effects in primates, the present study analyzes the distribution and chemical phenotypes of caudal medullary neurons that are activated in rhesus and cynomolgus macaque monkeys after CCK treatment. Monkeys were injected intravenously with CCK (3 or 15 micrograms/kg b.wt) or vehicle solution (0.15 M NaCl), then were anesthetized and perfused with fixative 75 min later. Coronal tissue sections through the caudal medulla were processed for immunocytochemical localization of the immediate-early gene product Fos as a marker of stimulus-induced neuronal activation, and were double-labeled for tyrosine hydroxylase to identify catecholaminergic cells. Many neurons in the area postrema, nucleus of the solitary tract, and ventrolateral medulla were activated to express Fos in monkeys after CCK treatment, similar to previous reports in rats. Treatment-activated neurons included substantial proportions of the A1/C1 and A2/C2 catecholaminergic cell groups, whereas neurons in the locus coeruleus (A6 cell group) were not activated. These results indicate that the autonomic, behavioral, and neuroendocrine effects produced by systemic administration of CCK involve hindbrain neural systems whose anatomical and chemical features are comparable in rats and primates.

Osmotic inhibition of prolactin secretion in rats.

Chronic hyponatremia is known to cause inhibition of pituitary vasopressin (AVP) and oxytocin (OT) secretion in response to most physiological stimuli, as well as a marked inhibition of synthesis of these peptides. Because many studies have implicated neurohypophyseal peptides in the regulation of pituitary prolactin (PRL) secretion, we investigated the effects of chronic hyponatremia on basal and stimulus-induced PRL secretion in rats. Hyponatremia was induced by subcutaneous infusion of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) (5 ng/h) to rats fed a nutritionally balanced liquid diet, and plasma [Na+] was maintained < or = 115 mmol/l for 10-12 days. After this period, hyponatremic rats and normonatremic controls fed the same diet without dDAVP were subjected to one of the following stimuli known to stimulate PRL release in rats: 3 min exposure to ether, hemorrhage (20 ml/kg), intravenous injection of 5-hydroxytryptophane (5-HTP, 10 mg/kg), or intravenous injection of estradiol (5 micrograms/kg). A baseline blood sample was collected before each stimulus, and 3-6 additional blood samples were collected at selected intervals after the stimulus. Baseline levels of plasma PRL were not different between normonatremic and hyponatremic rats. However, PRL responses induced by either or estradiol, but not those induced by hemorrhage or 5-HTP, were very significantly blunted in the chronically hyponatremic rats. Plasma AVP and OT responses were measured as an index of magnocellular secretion, but did not correlate with the PRL responses for any of the stimuli tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of estrogen on stimulated salt appetite in rats.

Adult male rats consumed 50-150% more 0.5 M NaCl solution than females did during a 7-hr drinking test when robust salt appetite was elicited by dietary sodium deprivation for 8 days, daily injections of deoxycorticosterone, or adrenalectomy followed by 2 days of sodium deprivation. In contrast, male rats drank much less saline after systemic treatment with the natriuretic agent furosemide, adrenalectomy followed by 1 day of sodium deprivation, or sc treatment with colloid solution after 2 days of sodium deprivation, and female rats drank comparably small volumes. Conversely, 30-day-old prepubescent male and female rats showed equally robust salt appetites after 8 days of sodium deprivation. These and other findings support an inhibitory role of estrogen on salt appetite in rats, which appears to occur only when the appetite is especially pronounced.

Exogenous cholecystokinin activates cFos expression in medullary but not hypothalamic neurons in neonatal rats.

The central distribution of neurons activated to express cFos immunoreactivity in response to peripheral administration of cholecystokinin octapeptide (CCK) was examined in 2-day-old rats. Similar to previous results in adult rats, neurons in specific subregions of the area postrema and nucleus of the solitary tract (NST) expressed cFos after CCK treatment. However, in marked contrast to results in adult rats, CCK treatment in neonates did not stimulate cFos expression in hypothalamic neurons or in other forebrain areas, and did not increase plasma oxytocin levels. These results suggest that vagal sensory activation of intrinsic brainstem circuits may be sufficient for the known inhibitory effects of exogenous CCK on gastric motility and feeding in neonatal rats. The prominent forebrain activation produced by CCK administration in adult rats likely reflects later maturation of direct and relayed ascending neural projections from the NST.

Distribution and neurochemical phenotypes of caudal medullary neurons activated to express cFos following peripheral administration of cholecystokinin.

Immunocytochemical localization of the protein product of the proto-oncogene c-fos allows anatomical identification of physiologically activated neurons. The present study examined the subnuclear distribution of cFos protein in the rat caudal medulla following peripheral administration of cholecystokinin octapeptide, which reduces feeding and gastric motility by a vagally mediated mechanism. To begin phenotypic characterization of neurons activated to express cFos following cholecystokinin treatment, double-labeling techniques were used to identify vagal motor neurons and neurons immunoreactive for tyrosine hydroxylase, neuropeptide Y, and neurotensin. Activated cells were most prevalent in the subnucleus medialis of the nucleus of the solitary tract, less prevalent in the subnucleus commissuralis, and virtually absent in the subnuclei centralis and gelatinosus. Many activated cells occupied the caudal area postrema; some of these were catecholaminergic. In contrast, activated cells were sparse within the medial rostral area postrema. Other activated cells occupied the dorso- and ventrolateral medulla and the midline raphe nuclei. Retrograde labeling of vagal motor neurons confirmed that very few were activated. Those that were activated occupied the caudal dorsal motor nucleus. In the dorsomedial medulla, 51% of catecholaminergic neurons and 39% of neurons positive for neuropeptide Y were activated, but no neurotensin-positive neurons were activated. In the ventrolateral medulla, 25% of catecholaminergic neurons and 27% of neuropeptide Y-positive neurons were activated. By characterizing the subnuclear distribution and chemical phenotypes of neurons activated by exogenous cholecystokinin, these data contribute to elucidation of the neural circuits mediating the behavioral, physiological, and neuroendocrine effects produced by this peptide.

Central oxytocin inhibition of salt appetite in rats: evidence for differential sensing of plasma sodium and osmolality.

Sodium chloride ingestion is stimulated during conditions of sodium deficiency to maintain body fluid and electrolyte balance. Recent studies have indicated that salt appetite in rats is often inversely related to peripheral and central secretion of the hormone oxytocin (OT). We studied the potential role of central OT on salt and water ingestion by treating rats intracerebroventricularly with OT conjugated to the A chain of the plant cytotoxin ricin (rAOT) to produce a chronic selective inactivation of brain cells containing OT-receptive elements. The rats treated with rAOT and control rats treated with the ricin A chain alone were given 5-hr two-bottle (water and 0.5 M NaCl) drinking tests 30 min after they were made hyperosmolar by injections of hypertonic (2M) mannitol solution, which elevated plasma osmolality but reduced plasma Na+ concentration. In the control rats only water intake was stimulated in response to the induced hyperosmolality, but in the rAOT-treated rats hypertonic mannitol caused a robust salt appetite as well as thirst. Analogous results were obtained in rats treated with two different OT-receptor antagonists prior to induction of hyperosmolality with mannitol. In contrast to these results, when hyperosmolality was induced by administration of equivalently hypertonic (1M) NaCl, which elevated both plasma osmolality and plasma Na+ concentration, only water intake but not salt intake was stimulated in both control and OT-receptor antagonist-treated rats. When salt appetite was stimulated by the physiological stimulus of polyethylene glycol-induced hypovolemia, hypertonic mannitol similarly inhibited salt ingestion in control animals but not in rAOT-treated rats, whereas hypertonic NaCl inhibited subsequent salt ingestion in both groups. These results suggest that salt appetite is regulated by both Na(+)- and osmolality-sensing mechanisms in rats. In addition, they indicate that central OT likely mediates a significant component of osmolality-related inhibition of salt appetite but does not appear to be essential for Na(+)-related inhibition of this important homeostatic behavior.

c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems.

Expression of c-Fos, or other immediate early gene products, by individual neurons can be used as a marker of cell activation, making staining of these proteins an extremely useful technique for functional anatomical mapping of neuroendocrine systems. Because these proteins are located in the nucleus, identification of the phenotype of the activated neuron using substances located within the cytoplasm can be accomplished with standard double-labeling immunocytochemical techniques. Although it is clear that neurons have the capacity to express a number of immediate early gene products, what remains to be established is whether there is a different pattern of expression following various stimuli. In our studies, we focus primarily on expression of one immediate early gene product, the c-Fos protein. We also include some experiments using expression of other members of the Fos family and Jun proteins as markers for neuronal activation. Our studies describe uses of c-Fos expression in both parvocellular and magnocellular hypothalamic systems to address the following issues: (a) identification of neuroendocrine cells activated by specific treatments and conditions, (b) ascertainment of functional differences in subpopulations activated by specific stimuli, (c) evaluation of neuronal activity in complex areas containing multiple neuroendocrine systems, (d) identification of other brain areas activated in conjunction with neuroendocrine systems following specific stimuli, (e) analysis of connectivity of activated neuroendocrine systems with other parts of the brain, and (f) identification of stimuli that decrease neuronal activity. The neuroendocrine systems studied include those that secrete arginine vasopressin (AVP), oxytocin (OT), corticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), and dopamine (DA). The use of c-Fos expression has permitted functional neuroanatomical mapping of these systems in response to specific stimuli such as cholecystokinin (CCK), hyperosmolality, and volume depletion, or during various physiological states such as the proestrous ovulatory luteinizing hormone (LH) surge and lactation. Although the use of c-Fos as a marker of neuronal activation will continue to be an extremely powerful technique, future studies will also be directed at relating immediate early gene expression to changes in neuroendocrine gene expression. To this end, we have shown that both c-Fos and c-Jun are expressed in neuroendocrine neurons in response to a number of stimuli, setting the stage for potential regulatory drive to genes containing AP-1 binding sites.

Cholecystokinin stimulates gonadotropin-releasing hormone release in the monkey (Macaca mulatta).

There is evidence to suggest that the arginine vasopressin release observed in association with emesis after i.v. injection of cholecystokinin (CCK) and N-methyl-D-aspartate (NMDA) is mediated by stimulation of emetic centers in the brainstem. That the GnRH-releasing action of NMDA is also sometimes accompanied by emesis led to the suggestion that stimulation of this parvocellular system by the glutamate agonist may also be mediated in part via activation of brainstem pathways. If this is the case, then other nauseogenic agents, such as CCK, should similarly elicit GnRH release. The foregoing prediction was tested in the castrated juvenile male monkey, an experimental model characterized by the absence of spontaneous GnRH release. GnRH discharges were monitored indirectly by measuring changes in circulating LH concentrations after the responsivity of the gonadotroph had been heightened by a chronic intermittent i.v. infusion of the decapeptide. An i.v. bolus of CCK at 10 and 30 micrograms/kg BW led to a distinct discharge of GnRH accompanied by emesis or other behaviors suggestive of nausea. Lower doses of CCK (1 and 3 micrograms/kg BW) failed to significantly perturb GnRH release or cause emesis, although NMDA (5 mg/kg BW; racemic form) injected i.v. 3 h after the CCK challenge led to a robust rise in GnRH. In a parallel study, three repetitive i.v. CCK injections at 2h intervals maintained intermittent GnRH release. Pretreatment with a long-acting GnRH receptor antagonist ([AcD2Nal1,4ClPhe2,DTrp3,DArg6,DAla10]GnRH -HOAc) abolished the LH response to CCK, confirming that the action of this peptide was mediated by GnRH release. Although a direct hypothalamic site of action for these agents remains the most likely possibility, since both NMDA and CCK receptors are present in the infundibular region, the present data are consistent with the notion that CCK and, by inference, NMDA may activate GnRH release in part via the stimulation of brainstem emetic centers. Plasma GH, PRL, and cortisol concentrations were also monitored during the course of some of these experiments, and the release of these hormones was observed after the administration of either the 10 or 30 micrograms/kg BW dose of CCK.

c-Fos Expression in Rat Brain and Brainstem Nuclei in Response to Treatments That Alter Food Intake and Gastric Motility.

Expression of the proto-oncogene protein c-Fos was evaluated immunocytochemically in individual brain cells as a marker of treatment-related neuronal activation following pharmacological and physiological treatments that are known to alter food intake and gastric motility in rats. c-Fos expression in response to each treatment was analyzed in the brainstem dorsal vagal complex, the limbic system, and the hypothalamus, representing the areas thought to be involved in coordinating the autonomic, behavioral, and neuroendocrine responses that occur during conditions of stimulated or inhibited food intake. Our results indicate that the patterns of brain c-Fos expression associated with treatments that inhibit food intake differ significantly from the patterns produced by treatments that potentiate food intake. Treatments that inhibited food intake (administration of the anorexigenic agents cholecystokinin, LiCl, and hypertonic saline, and food ingestion following fasting or insulin treatment) were associated with widespread stimulation of c-Fos expression in cells in the nucleus tractus solitarius (NTS), and to a more variable degree the area postrema (AP), but without significant activation of neurons in the dorsal motor nucleus of the vagus nerve (DMN). In contrast, treatments that potentiated food intake (food deprivation and insulin-induced hypoglycemia) were associated with marked stimulation of c-Fos expression in DMN neurons, but little or no activation in cells in the NTS or the AP. Pharmacological treatments that inhibited food intake and gastric motility also were associated with pronounced c-Fos expression in several forebrain areas, including the parvocellular and magnocellular subdivisions of the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala (CeA), and the bed nucleus of the stria terminalis (BNST). In contrast, more physiological inhibition of food intake resulting from spontaneous food ingestion did not cause significant activation of c-Fos expression in these forebrain regions, nor did treatments that stimulated food intake. Central administration of oxytocin, which also is known to inhibit food intake, was associated with a pattern of c-Fos activation analogous to that produced by spontaneous food ingestion after fasting (c-Fos expression in the NTS and AP, but without significant activation in the DMN or forebrain areas). Finally, acute gastric distension produced complex results, in that it was associated with activation of c-Fos expression in all areas of the brainstem (NTS, AP, and DMN), as well as in multiple forebrain areas (PVN, CeA, and BNST). Our results therefore demonstrate that specific patterns of brain c-Fos expression are associated with treatments that alter food intake in rats, and indicate that assessment of c-Fos immunoreactivity in different brain areas can identify common functional neuroanatomical networks that are activated by diverse treatments which nonetheless produce similar behavioral, autonomic, and neuroendocrine effects in animals.

Central oxytocin inhibition of angiotensin-induced salt appetite in rats.

In several models of salt appetite in the rat, stimulated NaCl intake can be severely blunted by treatments associated with pituitary release of oxytocin (OT). Central administration of the potent dipsogen angiotensin II (ANG II) is known to elicit a limited salt appetite as well as thirst, but it has also been reported to stimulate pituitary OT secretion. These results suggest the possibility that the expression of ANG II-induced salt appetite in rats may be inhibited by a simultaneous central release of OT in response to this stimulus. To investigate this possibility, rats were given intracerebroventricular injections of OT-receptor antagonists before administration of 5 ng ANG II intracerebroventricularly in a 1-h two-bottle (water and 0.3 M NaCl) drinking test. This pretreatment resulted in a three- to fourfold potentiation of ANG II-induced saline ingestion, which was most prominent during the first 15 min of the test. OT-receptor antagonism did not, however, interfere with the dipsogenic properties of ANG II, nor did it stimulate saline ingestion alone in the absence of ANG II. Immunocytochemical studies demonstrated that central administration of ANG II at this dose caused pronounced c-fos expression in hypothalamic magnocellular OT and vasopressin neurons and also in OT neurons in parvocellular subdivisions of the paraventricular nucleus. These results therefore demonstrate that central administration of small doses of ANG II activates both magnocellular and parvocellular OT neurons in rats and indicate that some of the activated central OT pathway(s) may mediate an inhibitory effect that limits the salt ingestion induced by this treatment.

Functional neurolobectomy induced by controlled compression of the pituitary stalk.

Degeneration of magnocellular nerve terminals in the neurohypophysis was induced by compressing the pituitary stalk of anesthetized rats for 30 s using a triangle-shaped wire. Immediately after stalk compression (SC), rats exhibited markedly increased water intake characteristic of diabetes insipidus, followed by a triphasic pattern of fluid intake. In SC rats, arginine vasopressin (AVP) and oxytocin (OT) contents of the neurointermediate lobe (NIL) of the pituitary gland were significantly reduced to approximately 2.5% and approximately 10% of sham-operated controls, respectively. In contrast, OT, but not AVP, content of the stalk-median eminence (SME) of SC rats was significantly increased. Histological examination of the pituitaries showed substantial degeneration of the neural lobe with very scarce AVP-neurophysin and OT-neurophysin immunoreactivity, while both the anterior and the intermediate lobes appeared to be intact. Plasma AVP and OT responses to infusion of hypertonic NaCl were significantly blunted in SC rats compared to sham-operated controls. However, two days after surgery the secretory patterns of LH in SC rats were similar to those in the controls. These results indicate that controlled compression of the pituitary stalk results in selective degeneration of the neural lobe without causing permanent ischemic damage to the anterior pituitary, and produces marked sustained functional deficits in pituitary AVP and OT secretion. Consequently, SC provides an alternative means to achieve selective neurolobectomy in rats.

Cholecystokinin induces c-fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex.

Systemic administration of cholecystokinin (CCK) decreases gastric motility and stimulates pituitary secretion of oxytocin (OT). Although peripheral OT does not affect gastric function, increasing evidence suggests that central OT secretion acting within the dorsal vagal complex (DVC) can alter gastric motility. To evaluate whether systemically administered CCK is capable of activating oxytocinergic neurons projecting to the DVC, we utilized fluorogold retrograde labeling from the DVC in combination with c-fos and OT immunocytochemical staining to quantitatively analyze paraventricular nucleus (PVN) neurons of rats following injection of CCK at a dose known to cause maximal pituitary OT secretion (100 micrograms/kg i.p.). Our results showed that 2320 +/- 63 PVN neurons were retrogradely labeled from the DVC; 146 +/- 21 (6.3%) of these contained OT, and these cells were predominantly located in the medial parvocellular subdivision of the PVN. Of all retrogradely labeled cells, 671 +/- 112 (28.9%) expressed c-fos after CCK stimulation, and 68 +/- 14 of these (10.1%) contained OT. Approximately 50% of the OT-containing neurons retrogradely labeled from the DVC stained positively for c-fos. Many magnocellular OT neurons in the PVN that were not retrogradely labeled from the DVC also expressed c-fos after CCK stimulation. These results demonstrate that parvocellular OT neurons projecting to the DVC are co-activated along with magnocellular OT neurons projecting to the pituitary following administration of a large dose of CCK, and lend support to a possible functional role for OT as a central neurotransmitter that modulates vagal efferent traffic to the gastrointestinal tract.