Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy - A focus on energy expenditure.

OBJECTIVE: Bariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study was to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model. METHODS: Diet-induced obese male Sprague-Dawley rats were either sham-operated, underwent VSG surgery or were pair-fed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of chow-fed rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 14C-2-deoxy-d-glucose (14C-2DG). Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals. RESULTS: Following VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in chow-fed animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlighted a patent neural link between the gut and BAT that included groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus. CONCLUSIONS: Collectively, these data support a role for BAT in mediating the metabolic sequelae following VSG surgery, particularly the improvement in glucose regulation, and highlight the need to better understand the contribution from this tissue in human patients.

Neuroendocrine mechanisms underlying bariatric surgery: Insights from human studies and animal models.

Obesity has reached epidemic proportions and, to date, bariatric surgery remains the only effective treatment for morbid obesity in terms of its capacity to achieve durable weight loss. Bariatric surgery procedures, including Roux-en-Y gastric bypass (RYGB), adjustable gastric banding (AGB) and sleeve gastrectomy (SG), have been the primary procedures conducted over the past decade, with SG increasing in popularity over the past 5 years at the expense of both RYGB and AGB. Although these procedures were initially proposed to function via restrictive or malabsorptive mechanisms, it is now clear that profound physiological changes underlie the metabolic improvements in patients who undergo bariatric surgery. Data generated in human patients and animal models highlight the rapid and sustained changes in gut hormones that coincide with these procedures. Furthermore, recent studies highlight the involvement of the nervous system, specifically the vagus nerve, in mediating the reduction in appetite and food intake following bariatric surgery. What is unclear is where these pathways converge and interact within the gut-brain axis and whether vagally-mediated circuits are sufficient to drive the metabolic sequalae following bariatric surgery.

Neural and humoral changes associated with the adjustable gastric band: insights from a rodent model.

BACKGROUND: Bariatric surgical procedures, including the laparoscopic adjustable gastric band (LAGB), are currently the only effective treatments for morbid obesity, however, there is no clear understanding of the mechanisms underpinning the efficacy of LAGB. The aim of this study is to examine changes in activation of the sensory neuronal pathways and levels of circulating gut hormones associated with inflation of an AGB. DESIGN AND RESULTS: The trajectory within the central nervous system of polysynaptic projections of sensory neurons innervating the stomach was determined using the transsynaptically transported herpes simplex virus (HSV). Populations of HSV-infected neurons were present in the brainstem, hypothalamus and cortical regions associated with energy balance. An elevation of Fos protein was present within the nucleus of the solitary tract, a region of the brainstem involved in the control of food intake, following acute and chronic band inflation. Two approaches were used to test (1) the impact of inflation of the band alone (on a standard caloric background) or (2) the impact of a standard caloric meal (on the background of the inflated band) on circulating gut hormones. Importantly, there was a significant elevation of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) following oral gavage of a liquid meal in animals with pre-inflated bands. There was no impact of inflation of the band alone on circulating GLP-1, PYY or ghrelin in animals on a standard caloric background. CONCLUSION: These data are consistent with the notion that the LAGB exerts its effects on satiety, reduced food intake and reduced body weight by the modulation of both neural and hormonal responses with the latter involving an elevation of meal-related levels of GLP-1 and PYY. These data are contrary to the view that the surgery is purely 'restrictive'.

Projections of RFamide-related peptide-3 neurones in the ovine hypothalamus, with special reference to regions regulating energy balance and reproduction.

RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain.

A rodent model of adjustable gastric band surgery-implications for the understanding of underlying mechanisms.

BACKGROUND: Bariatric surgery is currently the only anti-obesity therapy that can deliver weight loss of up to 20-30% of body weight. Laparoscopic adjustable gastric banding (LAGB) and Roux-en-y gastric bypass are the most commonly performed of these surgeries. The mechanisms by which LAGB initiates an increase in satiety remain completely unknown. The aim of this study is to establish a rodent model of adjustable gastric banding (AGB) that will enable investigation of these mechanisms. METHODS: Sprague-Dawley rats were implanted with adjustable gastric bands immediately below the gastro-esophageal junction around the glandular stomach. This band, as in humans, can be inflated via an exteriorized port resulting in an incremental impact on the stomach. RESULTS: Rats with an incremental inflation of the AGB showed a clear stepwise reduction in food intake and body weight. Normal food intake and body weight gain were restored with band deflation. Barium-assisted X-ray of the stomach showed the formation of a small gastric pouch proximal to the inflated band in a manner analogous to the human LAGB. CONCLUSIONS: This is the first animal model of the AGB that allows incremental inflation for optimal tightening of the band in the conscious animal with corresponding effects on food intake and body weight. This model will allow measurement of acute and chronic neural and hormonal changes following activation of the band in the conscious animal and will provide the potential to inform and improve surgical approaches that are at the forefront of obesity treatments.

Effect of central administration of QRFP(26) peptide on energy balance and characterization of a second QRFP receptor in rat.

The recently identified neuropeptide QRFP(26) is predominantly expressed in the hypothalamus and was suggested to play a role in the regulation of food intake following the observation of an acute orexigenic effect after central administration in mice. QRFP(26) exerts its effect via GPR103 and a newly identified receptor in mouse. The aim of our study was (a) to investigate the distribution of QRFP(26) and a newly discovered QRFP receptor mRNA in rat and (b) to further characterize the effects of central administration of QRFP(26) on energy balance in rats. QRFP(26) mRNA was detected in the retrochiasmatic nucleus, periventricular nucleus, arcuate nucleus and restricted areas of the lateral nucleus of the hypothalamus. We found an additional receptor with high homology for GPR103 in rat. This receptor increases inositol triphosphate production in transfected cells in presence of QRFP(26) and its mRNA was particularly enriched in ventral and posterior thalamic groups, anterior hypothalamus and medulla. When QRFP(26) (10 microg and 50 microg) was administered centrally before the start of the light phase both doses increased food intake for 2 h after injection without reaching statistical significance. QRFP(26) caused no changes in locomotor activity or energy expenditure. In summary, central QRFP(26) injection causes slight and transient hyperphagia in rats without changing any other energy balance parameters after 24 h. We conclude that QRFP(26) has limited impact on the central regulation of energy balance in rats and that its essential function remains to be clarified.

The effect of urocortin on ingestive behaviours and brain Fos immunoreactivity in mice.

The influence of urocortin (UCN) on ingestive behaviours and brain neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in mice. Rat UCN was administered by continuous intracerebroventricular (ICV) or subcutaneous (SC) infusion. ICV infusion of UCN (100 ng/h, 14 days) transiently reduced daily food and water intakes (days 1-4) but body weight was reduced from day 2 into the post-infusion period. Sodium intake was reduced from day 3 to the end of infusion. SC infusion of UCN caused similar but smaller reductions in food and water intakes and body weight, without change in sodium intake. In separate experiments, Fos immunoreactivity was increased in several brain nuclei known to be involved in the control of body fluid and energy homeostasis, e.g. central nucleus of the amygdala, median preoptic nucleus, bed nucleus of the stria terminalis and arcuate nucleus. Increased Fos expression was similar for ICV and SC infusions when measured on days 2-3 or 6-7 of infusion. In conclusion, increases of brain activity by UCN may be associated with stimulation of adrenocorticotrophic hormone release and sympathetic nervous activity, but increases may also indicate suppression of ingestive behaviours by stimulating central inhibitory mechanisms located in areas known to control body fluid and energy homeostasis.

Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat.

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 microg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10(-7) M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10(-7) M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood-brain barrier may have regulatory influences on fluid balance during pregnancy in rats.

A method for the identification of pseudorabies virus protein and angiotensin AT(1A) receptor mRNA expression in the same CNS neurons.

Neurotropic viruses have been used over the last 10 years to map the distribution of chains of synaptically connected neurons in the CNS. The peptide content of infected neurons has been determined in a number of cases immunohistochemically. However, it has been unclear whether specific mRNA can be assessed in virus-infected neurons. We have established a technique which enables the identification of viral protein and mRNA in the same neuron. In the present study pseudorabies virus retrogradely transported from the kidney was localised using immunohistochemistry and mRNA for the angiotensin II AT(1A) receptor was detected by hybridisation histochemistry. Virus protein was visualised using an immunohistochemical procedure with diaminobenzidine as the chromogen and the same sections were exposed to radioactively labelled ((35)S) riboprobes, hybridising the angiotensin II AT(1A) receptor. The combination of these two approaches resulted in the identification of neurons shown to project polysynaptically to the kidney and express AT(1A) mRNA. These data provide neuroanatomical support for previous physiological observations that ablation of the lamina terminalis and administration of losartan, the AT(1) receptor antagonist, blocks the inhibition of renal sympathetic nerve activity following centrally injected Ang II in rats and sheep [5].

Activation of kidney-directed neurons in the lamina terminalis by alterations in body fluid balance.

This study was undertaken to determine if neurons in the lamina terminalis, previously identified as projecting to the kidney (35), were responsive to alterations in stimuli associated with fluid balance homeostasis. Neurons in the lamina terminalis projecting to the kidney were identified by the retrograde transynaptic transport of Bartha's strain of pseudorabies virus in anesthetized rats. Rats were also exposed to 24-h water deprivation, intravenous hypertonic saline, or intracerebroventricular ANG II. To determine if "kidney-directed" neurons were activated following each stimulus, brain sections that included the lamina terminalis were examined immunohistochemically for viral antigen and Fos protein. With the exception of ANG II in the subfornical organ, all regions of the lamina terminalis contained neurons that were significantly activated by water deprivation, hypertonic saline, and ANG II. These results provide evidence for a neural substrate, which may underpin some of the effects of hypertonic saline and ANG II on renal function thought to be mediated through the lamina terminalis.

Fos immunoreactivity in the lamina terminalis of adrenalectomized rats and effects of angiotension II type 1 receptor blockade or deoxycorticosterone.

Neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in the lamina terminalis, a thin strip of tissue forming the anterior wall of the third brain ventricle, after adrenalectomy. Several weeks after surgery, the adrenalectomized rats were maintained with access to water and a low sodium diet for five days. In addition, hypertonic (0.5M) NaCl solution was available for the entire five-day period (sodium available) or only during the first four days (sodium unavailable). The number of neurons expressing Fos, determined at the end of the fifth day, was increased in the adrenalectomized rats with or without NaCl solution to drink. Fos activity in the median preoptic nucleus was increased only in adrenalectomized rats without access to NaCl solution. Treatment of adrenalectomized rats with the sodium-retaining mineralocorticoid hormone, deoxycorticosterone, at the end of the fourth day, decreased Fos expression in the subfornical organ and the organum vasculosum of the lamina terminalis when NaCl solution was available but not when the NaCl solution was unavailable. In the adrenalectomized rats with NaCl solution available, mineralocorticoid treatment decreased both urinary sodium excretion and daily sodium intake. Brain nuclei in the lamina terminalis also became activated in intact rats made sodium deplete by treatment with the diuretic, furosemide. Relative to sodium-deplete intact rats, however, sodium-deplete adrenalectomized rats had a greater number of neurons expressing Fos in the organum vasculosum. Treatment of sodium-deplete rats, adrenalectomized or intact, with the angiotensin II-type 1 receptor antagonist, ZD7155, decreased sodium intake and Fos expression in the subfornical organ but not in the organum vasculosum of the lamina terminalis or median preoptic nucleus. In conclusion, the results demonstrated that activation of the brain nuclei located in the lamina terminalis of adrenalectomized rats was primarily related to sodium deficit and not to the absence of the mineralocorticoid hormones, although the adrenal hormones may have a role in limiting the activation of organum vasculosum of the lamina terminalis during sodium depletion. Furthermore, the results obtained with the administration of the angiotensin receptor antagonist are consistent with the proposal that sodium appetite of the sodium-deplete rat, adrenalectomized or intact, is mediated by circulating angiotensin II acting in the subfornical organ.

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)

Distribution of Fos immunoreactivity in the lamina terminalis and hypothalamus induced by centrally administered relaxin in conscious rats.

The effect of intracerebroventricular (ICV) injections of synthetic human or rat relaxin (25 or 250 ng) on the distribution of Fos detected immunohistochemically in the rat forebrain was investigated. Following ICV relaxin, many Fos-positive neurons were observed in the periphery of the subfornical organ, dorsal part of the organum vasculosum of the lamina terminalis, throughout the median preoptic nucleus, supraoptic nucleus and hypothalamic paraventricular nucleus. Such effects did not occur following ICV injection of artificial cerebrospinal fluid or the separated A and B chains of relaxin, nor following the intravenous injection of 250 ng of relaxin. Both vasopressin and oxytocin containing neurons identified immunohistochemically in the supraoptic and paraventricular nuclei exhibited Fos following ICV relaxin, and many neurons in the medial parvocellular part of the paraventricular nucleus contained Fos. The results indicate that centrally administered relaxin may increase neuronal activity in regions of the hypothalamus and lamina terminalis which are associated with cardiovascular and body fluid regulation and oxytocin secretion.

Distribution of Fos-immunoreactivity in rat brain following a dipsogenic dose of captopril and effects of angiotensin receptor blockade.

Immunohistochemical techniques were used to detect Fos in the brain following subcutaneous administration of the angiotensin converting enzyme inhibitors captopril or enalapril at 0.5 mg/kg to conscious rats. Increased Fos-like immunoreactivity was observed in many neurons in the lamina terminalis, and in regions of the hypothalamus. Captopril at this dose also caused water drinking in other rats. Pre-treatment with the angiotensin AT1 receptor antagonist ZD7155 (10 mg/kg) given subcutaneously prevented the captopril-induced increase in Fos in the lamina terminalis. This dose of ZD7155 also prevented captopril-induced drinking in other rats. With a higher dose (50 mg/kg) of captopril or enalapril, there was no increase in Fos in the lamina terminalis. This dose of captopril was not dipsogenic. The results are consistent with the proposal that the lower dose (0.5 mg/kg) of captopril or enalapril increases circulating angiotensin I levels which are then converted to angiotensin II in the organum vasculosum of the lamina terminalis and subfornical organ. Stimulation of neurons at these sites may subserve water drinking and sodium appetite.

Identification of osmoresponsive neurons in the forebrain of the rat: a Fos study at the ultrastructural level.

The aims of this study are twofold. The first is to describe the ultrastructural morphology of putative osmoreceptors concentrated in the ventral aspect of the lamina terminalis in the rat forebrain. The second is to determine whether or not these neurons lie within an area which lacks a blood-brain barrier, i.e. the organum vasculosum lamina terminalis. The results describe a compact population of neurons in the ventral part of the lamina terminalis which both respond to an osmotic challenge and project directly to the supraoptic nucleus. Injection of horseradish peroxidase into the circulation, as a marker to define areas of the brain without a blood-brain barrier, indicates that these neurons are in the dorsal aspect of the organum vasculosum of the lamina terminalis. An ultrastructural analysis of the neurons in this area, which respond to an osmotic challenge with an elevation of Fos protein, show them to have no specific morphological characteristics which differentiate them from other, non-responsive neurons in the organum vasculosum of the lamina terminalis. However, one possible exception is that osmotically sensitive neurons have a less indented nucleus, suggesting that they are in a more active state than their non-osmotically sensitive neighbours. It is concluded that neurons in this region of the brain are candidate structures for the "receptors" which mediate vasopressin release in response to an osmotic challenge. The response of only a subset of neurons in the organum vasculosum of the lamina terminalis to an osmotic stimulus, despite an apparent morphological homogeneity and the ability of blood borne agents to reach all parts of the structure suggests that osmoresponsiveness is conferred by unique membrane properties or intracellular processing events. The presence of synaptic input to osmoresponsive cells indicates a potential for integration of other inputs at this level.

Comparison of c-fos expression in the lamina terminalis of conscious rats after intravenous or intracerebroventricular angiotensin.

Fos immunoreactivity in the rat brain after intracerebroventricular (ICV) angiotensin II (ANG II) was compared with that induced by intravenous ANG II. ANG II was infused into the lateral ventricle (at 1 ng/min) or femoral vein (at 5 micrograms/h) of conscious rats. After 90 min, rats were killed and Fos was detected by immunohistochemistry. Both infusions caused Fos immunoreactivity to be present in the lamina terminalis, hypothalamic supraoptic, and paraventricular nuclei, bed nucleus of the stria terminalis, and central amygdaloid nucleus. However, distributions of Fos immunoreactivity within the lamina terminalis differed with the different routes of infusion. Intravenous ANG II caused intense Fos immunoreactivity mainly in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). By contrast, ICV ANG II caused intense Fos immunoreactivity predominantly in the median preoptic nucleus and juxtaventricular neurons of the SFO and OVLT. These results suggest that IV ANG II induces behavioural and endocrine responses by direct actions on the SFO and OVLT, whereas ICV ANG II directly stimulates neurons in the median preoptic nucleus as well neurons in the SFO and OVLT.

Distribution of Fos in rat brain resulting from endogenously-generated angiotensin II.

The beta adrenergic agonist isoproterenol has been used in these studies to elevate circulating levels of angiotensin II. Neurons in the brain responsive to the subcutaneous infusion of isoproterenol were identified using an antibody to Fos, the protein product of c-fos which is now used extensively as a marker of activated neurons. Fos-positive neurons were present in a range of specific forebrain and hind brain regions. Infusion of losartan (an angiotensin II type receptor antagonist) showed that neurons in the lamina terminalis were activated directly or indirectly by angiotensin II, whereas other neurons in the hypothalamus and brain stem were responsive as a consequence of the peripheral vasodilation caused by isoproterenol. The distribution of activated neurons in the lamina terminalis was consistent with that of neurons thought to be involved in water drinking.

Localization of barosensitive neurons in the caudal ventrolateral medulla which project to the rostral ventrolateral medulla.

A population of depressor neurons in the caudal ventrolateral medulla that project to the rostral ventrolateral medulla may mediate the baroreceptor reflex. The aim of the present study was to determine the anatomical distribution of the population of neurons in the caudal ventrolateral medulla that mediate the baroreceptor reflex. Injection of the retrogradely transported tracer, rhodamine-labelled latex beads, into the pressor area of the rostral ventrolateral medulla of rats was used to identify neurons in the caudal ventrolateral medulla with projections to that area. Barosensitive neurons were identified by immunohistochemical detection of the protein Fos, a marker of neuronal activation, following infusion of the pressor agent phenylephrine (10 micrograms/kg/min, i.v. for 2 h n = 5). Isotonic saline was infused into control animals (n = 4). Neurons in the caudal ventrolateral medulla with projections to the rostral ventrolateral medulla were located at all rostrocaudal levels examined between 1 mm caudal and 0.4 mm rostral of the obex. Compared to saline infused rats, phenylephrine infusion induced a significant increase in the proportion of those neurons that expressed Fos (14% vs. 1% P < 0.000.1). These barosensitive neurons were found mainly at the level of the obex, between the lateral reticular nucleus and the nucleus ambiguus. In conclusion, this study is the first to show the distribution of the population of barosensitive neurons in the caudal ventrolateral medulla that project to the pressor region of the rostroventrolateral medulla. The results suggest there is a subpopulation of depressor neurons, confined to a small region of the rostral part of the caudal ventrolateral medulla, that are likely to be the interneurons that mediate the baroreceptor-reflex response.

Identification of neural pathways activated in dehydrated rats by means of Fos-immunohistochemistry and neural tracing.

The distribution of Fos-immunoreactivity (Fos-IR) was studied in the brains of rats deprived of water for 24 or 48 h and compared with that in brains of water-replete rats. Intense Fos-IR was observed in many neurons of the median preoptic nucleus (MnPO), organum vasculosum of the lamina terminalis (OVLT), supraoptic nucleus and hypothalamic paraventricular nucleus. There was less intense and sparse Fos-IR in the subfornical organ. In water-replete rats, Fos-IR was absent or very low in these regions. In other rats, cholera toxin B-gold conjugate was microinjected bilaterally into the supraoptic nucleus to identify retrogradely labelled neurons in the lamina terminalis projecting to the supraoptic nucleus. Approximately 30% of these retrogradely labelled neurons in the OVLT and MnPO also exhibited Fos-IR after 48 h of water deprivation. These data show that neurons in the MnPO, OVLT and, to a lesser extent, the subfornical organ probably play an important role in homeostatic responses to dehydration, such as vasopressin secretion.

Fos production in retrogradely labelled neurons of the lamina terminalis following intravenous infusion of either hypertonic saline or angiotensin II.

The lamina terminalis consists of neurons which are activated by both osmotic and angiotensinergic stimuli and which project axons to many sites including regions of the hypothalamus responsible for vasopressin production. Combination of retrograde neuronal tracing procedures with the identification of Fos protein following discrete stimuli shows populations of neurons, projecting to the supraoptic nuclei, which are preferentially activated by intravenous infusion of either hypertonic saline or angiotensin II. Following infusion of hypertonic saline, the greatest percentage of neurons both labelled with cholera toxin-gold and having elevated levels of Fos protein occurred in that part of the lamina terminalis called the organum vasculosum lamina terminalis. Conversely, angiotensin infusion resulted in greatest numbers of Fos and cholera toxin-gold-labelled neurons in the subfornical organ with fewer double-labelled cells represented in the other components of the lamina terminalis, the median preoptic nucleus and the organum vasculosum lamina terminalis. While these data do not support more than a general separation of the functions examined among neurons of the lamina terminalis, they do highlight a discrete group of osmoresponsive neurons in the dorsal cap of the organum vasculosum lamina terminalis. These cells, by virtue of their response to infusions of hypertonic saline and their axonal connections to regions of the hypothalamus responsible for vasopressin production, are likely candidates for cerebral osmoreceptors.