Impaired sodium-evoked paraventricular nucleus neuronal activation and blood pressure regulation in conscious Sprague-Dawley rats lacking central Gαi2 proteins.

AIM: We determined the role of brain Gαi2 proteins in mediating the neural and humoral responses of conscious male Sprague-Dawley rats to acute peripheral sodium challenge. METHODS: Rats pre-treated (24-h) intracerebroventricularly with a targeted oligodeoxynucleotide (ODN) (25 µg per 5 µL) to downregulate brain Gαi2 protein expression or a scrambled (SCR) control ODN were challenged with an acute sodium load (intravenous bolus 3 m NaCl; 0.14 mL per 100 g), and cardiovascular parameters were monitored for 120 min. In additional groups, hypothalamic paraventricular nucleus (PVN) Fos immunoreactivity was examined at baseline, 40, and 100 min post-sodium challenge. RESULTS: In response to intravenous hypertonic saline (HS), no difference was observed in peak change in mean arterial pressure between groups. In SCR ODN pre-treated rats, arterial pressure returned to baseline by 100 min, while it remained elevated in Gαi2 ODN pre-treated rats (P < 0.05). No difference between groups was observed in sodium-evoked increases in Fos-positive magnocellular neurons or vasopressin release. V1a receptor antagonism failed to block the prolonged elevation of arterial pressure in Gαi2 ODN pre-treated rats. A significantly greater number of Fos-positive ventrolateral parvocellular, lateral parvocellular, and medial parvocellular neurons were observed in SCR vs. Gαi2 ODN pre-treated rats at 40 and 100 min post-HS challenge (P < 0.05). In SCR, but not Gαi2 ODN pre-treated rats, HS evoked suppression of plasma norepinephrine (P < 0.05). CONCLUSION: This highlights Gαi2 protein signal transduction as a novel central mechanism acting to differentially influence PVN parvocellular neuronal activation, sympathetic outflow, and arterial pressure in response to acute HS, independently of actions on magnocellular neurons and vasopressin release.

Selective up-regulation of JunD transcript and protein expression in vasopressinergic supraoptic nucleus neurones in water-deprived rats.

The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the activator protein-1 gene family, namely c-fos, c-jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase-polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72 h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double-staining fluorescence immunohistochemistry with a neurone-specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin-containing neurones with markedly less staining seen in oxytocin-containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.

Region-specific changes in transient receptor potential vanilloid channel expression in the vasopressin magnocellular system in hepatic cirrhosis-induced hyponatraemia.

The present study aimed to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurones of the paraventricular (PVN) and supraoptic nucleus (SON) in an animal model of hepatic cirrhosis associated with inappropriate vasopressin (AVP) release. In these studies, we used chronic bile duct ligation (BDL) in the rat, which is a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. The present study tested the hypothesis that changes in TRP vanilloid (TRPV) channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilised laser capture microdissection of AVP neurones in the PVN and SON and western blot analysis from brain punches. Laser capture microdissection and quantitative reverse transcriptase-polymerase chain reaction demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL compared to sham-ligated controls. AVP transcription was also increased as determined using intron specific primers to measure heteronuclear RNA. Immunohistochemistry demonstrated increased AVP and TRPV2 positive cells in both the PVN and SON after BDL. Also, there was an increased co-expression of TRPV2 and AVP cells after BDL. However, there was no change in the colocalisation counts of TRPV2 and oxytocin in both the magnocellular regions evaluated. In the SON but not the PVN, the transcription levels of TRPV4 were also significantly increased in BDL rats. Western blot analysis of punches containing the PVN and SON revealed that TRPV2 protein content was significantly increased in these brain regions in BDL rats compared to sham rats. Our data suggest that regionally specific changes in TRPV expression in the magnocellular neurosecretory cell AVP neurones could alter their osmosensing ability.

Brain-derived neurotrophic factor-tyrosine kinase B pathway mediates NMDA receptor NR2B subunit phosphorylation in the supraoptic nuclei following progressive dehydration.

We studied the effects of water deprivation (WD) on the phosphorylation of tyrosine kinase B (TrkB) and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and quantitative reverse transcriptase polymerase chain reaction was used to demonstrate brain-derived neurotrophic factor (BDNF) and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, whereas staining for phosphorylated TrkB was increased following WD. Western blot analysis of brain punches containing the SON revealed that tyrosine phosphorylation of TrkB (pTrkBY(515)), serine phosphorylation of NR1 (pNR1S(866) or pNR1) and tyrosine phosphorylation of NR2B subunits (pNR2BY(1472) or pNR2B) were significantly increased in WD animals compared to controls. Access to water for 2 h reduced pTrkBY(515) content to control levels without affecting pNR1 or pNR2B. Four hours of rehydration was needed to reduce pNR1 and pNR2B to control levels. To test whether increased phosphorylation of TrkB in the present study is mediated by BDNF, a group of animals were instrumented with right SON cannula coupled to mini-osmotic pumps filled with vehicle or TrkB-Fc fusion protein, which prevents BDNF binding to TrkB. In the left SON contralateral to the cannula, TrkB phosphorylation was significantly enhanced following WD. Separate analysis of the right SON, which received TrkB-Fc, showed that the TrkB receptor phosphorylation following WD was significantly attenuated. Although increased pNR1S(866) following WD was not affected by local infusion of TrkB-Fc, pNR2BY(1472) was significantly reduced. Co-immunoprecipitation revealed an increased physical interaction between Fyn kinase and NR2B and TrkB in the SON following WD. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.

Chronic intermittent hypoxia sensitizes acute hypothalamic-pituitary-adrenal stress reactivity and Fos induction in the rat locus coeruleus in response to subsequent immobilization stress.

Obstructive sleep apnea (OSA) is associated with several pathophysiological conditions, including hypertension, obesity, insulin resistance, hypothalamic-pituitary-adrenal (HPA) dysregulation, and other endocrine and metabolic disturbances comprising the "metabolic syndrome." Repeated episodes of hypoxia in OSA may represent a chronic intermittent stress, leading to HPA dysregulation. Alterations in HPA reactivity could then contribute to or exacerbate other pathophysiological processes. We showed previously that another metabolic stressor, chronic intermittent cold stress, enhanced noradrenergic facilitation of acute HPA stress reactivity. In this study, we investigated whether chronic intermittent hypoxia (CIH), a rat model for the arterial hypoxemia that accompanies OSA, similarly sensitizes the HPA response to novel acute stress. Rats were exposed to CIH (alternating cycles of normoxia [3 min at 21% O(2)] and hypoxia [3 min at 10% O(2)], repeated continuously for 8 h/day during the light portion of the cycle for 7 days). On the day after the final CIH exposure, there were no differences in baseline plasma adrenocorticotropic hormone (ACTH), but the peak ACTH response to 30 min acute immobilization stress was greater in CIH-stressed rats than in controls. Induction of Fos expression by acute immobilization stress was comparable following CIH in several HPA-modulatory brain regions, including the paraventricular nucleus, bed nucleus of the stria terminalis, and amygdala. Fos induction was attenuated in lateral hypothalamus, an HPA-inhibitory region. By contrast, acute Fos induction was enhanced in noradrenergic neurons in the locus coeruleus following CIH exposure. Thus, similar to chronic cold stress, CIH sensitized acute HPA and noradrenergic stress reactivity. Plasticity in the acute stress response is important for long-term adaptation, but may also contribute to pathophysiological conditions associated with states of chronic or repeated stress, such as OSA. Determining the neural mechanisms underlying these adaptations may help us better understand the etiology of such disorders, and inform the development of more effective treatments.

Identification of central nervous system sites involved in the water diuresis response elicited by central microinjection of nociceptin/ Orphanin FQ in conscious rats via c-Fos and inducible cAMP early repressor immunocytochemistry.

Intracerebroventricular (i.c.v.) administration of the opioid-like peptide, nociceptin/Orphanin (nociceptin), in conscious rats produces diuretic and antinatriuretic effects. The present study utilised changes in Fos and inducible cAMP early repressor (ICER) immunocytochemistry expression to examine the central nervous (CNS) sites activated or inhibited, respectively, by central administration of nociceptin. Urine samples were collected during control (15 min) and after i.c.v. vehicle (5 microl, n = 12) or nociceptin (10 microg/5 microl; n = 12). Four additional urine samples (15-min) were collected after the i.c.v. injection. The brain was processed for Fos using a commercially available antibody (Oncogene AB-5) and for ICER using a polyclonal anti-ICER antibody raised in rabbits. In vehicle-injected conscious rats, renal excretion of water or sodium was not altered. However, nociceptin produced a rapid and marked increase in urine flow (V) and a decrease in urinary sodium excretion rate. In addition, i.c.v. nociceptin produced a significant increase in Fos staining in the dorsomedial nucleus of the hypothalamus, the perinuclear zone of the supraoptic nucleus, the organum vasculosum of the lamina terminalis (OVLT), the lateral preoptic area and the lateral hypothalamic area compared to control. By contrast, Fos expression decreased in the area postrema and locus coeruleus compared to controls. Furthermore, ICER staining was significantly increased in the perinuclear zone of the supraoptic nucleus, supraoptic nucleus, median preoptic nucleus, OVLT, medial preoptic area, central nucleus of the amygdala, and medial nucleus of the solitary tract. Together, central opioid receptor-like type 1 activation in these CNS regions may participate in the neural pathways involved in the diuretic and antinatriuretic effects of nociceptin.

Lesions of the diagonal band of broca enhance drinking in the rat.

This study examined the role of the diagonal band of Broca (DBB) in drinking behaviour and vasopressin release. Adult male rats were anaesthetized (pentobarbital 50 mg/kg) and received DBB injections of either ibotenic acid (0.5 microl of 5 micro g/ microl) or vehicle (0.5 microl of phosphate-buffered saline). Although baseline drinking and urine output were not affected, drinking to 30% polyethylene glycol (MW 8000; 1 ml/100 g s.c.) and angiotensin II (0, 1.5 and 3.0 mg/kg s.c.) were significantly increased in ibotenic acid in phosphate-buffered saline (DBBX) rats. Drinking to hypertonic saline (0.9, 4 and 6%; 1 ml/100 g), and water deprivation were not significantly affected. DBBX rats had significantly lower basal heart rates than controls but the cardiovascular responses to infusions of angiotensin II (100 ng/kg/min i.v. for 45 min) were not affected. DBBX rats had significantly higher basal vasopressin, but angiotensin-stimulated vasopressin release was not significantly different. Although the DBB is not involved in basal water intake, it is involved in dipsogenic responses to hypovolemic stimuli and possibly basal autonomic function and basal vasopressin release.

Proposed role of the paraventricular nucleus in cardiovascular deconditioning.

AIM: Cardiovascular deconditioning occurs in individuals exposed to prolonged spaceflight or bedrest and is associated with the development of orthostatic intolerance. Although the precise mechanisms remain to be fully elucidated, astronauts returning from space or bedrest patients returning to normal upright posture present with decreases in plasma volume and alterations in autonomic function. The hindlimb unloaded (HU) rat has been a useful model to study the effects of cardiovascular deconditioning as it mimics many of the changes that occur after spaceflight and bedrest. RESULTS: Experiments performed in HU rats suggest that cardiovascular deconditioning attenuates baroreflex mediated sympathoexcitation and enhances cardiopulmonary receptor mediated sympathoinhibition. These alterations appear to be due to changes in the central nervous system and may contribute to the pre disposition towards orthostatic intolerance associated with cardiovascular deconditioning. The paraventricular nucleus (PVN) of the hypothalamus is important in basal and reflex control of sympathetic outflow. Recent evidence suggests that nitric oxide (NO) is an important inhibitory neurotransmitter in the PVN and that alterations in nitroxidergic transmission in the PVN may be involved in elevated sympathetic tone in certain disease states. CONCLUSION: Based on evidence from other laboratories and published and preliminary data from our own laboratories, this review proposes a role for the PVN in cardiovascular deconditioning. In particular, we discuss the hypothesis that increased NO in the PVN contributes to the altered cardiovascular reflexes observed following deconditioning and how these reflexes may be related to the orthostatic intolerance observed after prolonged spaceflight or bedrest.

Evaluation of unexplained acute and acute recurrent pancreatitis using endoscopic retrograde cholangiopancreatography, sphincter of Oddi manometry and endoscopic ultrasound.

BACKGROUND AND STUDY AIMS: Unexplained pancreatitis represents a diagnostic challenge. The aim of this study was to determine the diagnostic utility of endoscopic retrograde cholangiopancreatography (ERCP) with sphincter of Oddi manometry (SOM), bile analysis, and endoscopic ultrasound (EUS) in evaluating such patients. PATIENTS AND METHODS: Of 162 patients referred for evaluation of pancreatitis, 72 with a known cause were excluded. The remainder ( n=90) was classified as having prior acute ( n=24) or recurrent acute pancreatitis ( n=66). Bile sampling and SOM were performed at the time of ERCP. EUS was used to assess for tumors and for chronic pancreatitis. Clinical outcomes were evaluated by questionnaire. RESULTS: ERCP was successful in 88/89 patients (99 %). Manometry was successful in 63/67 patients (94 %), and 56 patients underwent EUS. Findings were categorized into five distinct etiologies: sphincter of Oddi dysfunction (SOD) ( n=28; 31 %), pancreas divisum ( n=18; 20 %), biliary ( n=18; 20 %), idiopathic ( n=18; 20 %) and tumor-related ( n=8; 9 %). Features of moderate or severe chronic pancreatitis by EUS and ERCP criteria were found in 18 patients (21 %); an additional nine patients had chronic pancreatitis by EUS criteria alone. EUS identified all the tumors. The condition was improved in 96 % of all patients undergoing endoscopic therapy. CONCLUSION: An etiology was identified in the majority of patients with unexplained pancreatitis. SOD represented the most common finding. Moderate to severe chronic pancreatitis was found in over one-fifth of these patients. Bile analysis, SOM, and EUS are useful tools in the evaluation of unexplained acute pancreatitis.

Fos immunoreactivity in the diagonal band and the perinuclear zone of the supraoptic nucleus after hypertension and hypervolaemia in unanaesthetized rats.

We used Fos immunocytochemistry to study the effects of hypertension and hypervolaemia on neurones in the diagonal band of Broca and the perinuclear zone of the supraoptic nucleus, two nuclei that are both involved in the baroreceptor regulation of vasopressin neurones in the supraoptic nucleus. In addition, we used sino-aortic denervation to examine the role of arterial baroreceptors in the response to these haemodynamic changes. Sham-operated and sino-aortic denervated rats were infused with phenylephrine sufficient to increase blood pressure for 2 h. Control rats were infused with the same volume of isontonic saline. Only Sham sino-aortic denervated rats showed reflex bradycardia in response to the increased blood pressure. Volume expansion was produced by infusing the rats with isotonic saline equal to 10% of their body weight for 10 min, which significantly increased central venous pressure. In the diagonal band of Broca and the perinuclear zone, the number of Fos-positive neurones was significantly increased after phenylephrine infusion. Sino-aortic denervation blocked the significant increase in both regions. After volume expansion, a significant increase in Fos staining was observed only in the perinuclear zone of the supraoptic nucleus. This increase was not blocked by sino-aortic denervation. Our results indicate that both the diagonal band of Broca and the perinuclear zone of the supraoptic nucleus are activated by stimulating arterial baroreceptors; however, the perinuclear zone of the supraoptic nucleus is stimulated during volume expansion. Furthermore, the activation of perinuclear zone of the supraoptic nucleus after volume expansion is not dependent on intact arterial baroreceptors.

Endoscopic treatment of post-liver transplantation biliary leaks with stent placement across the leak site.

BACKGROUND: Biliary tract leaks occur in over 10% of patients undergoing liver transplantation and are the most common cause of biliary tract-related death in these patients. A number of treatment options are available, but a standard approach has not been established. METHODS: Twenty-six patients were retrospectively studied who had post-transplantation leaks develop with special reference to those who underwent endoscopic placement of a "leak-bridging" stent. RESULTS: Endoscopic retrograde cholangiography was performed in all cases with no procedure-related complications. Twenty-four patients had a leak-bridging stent, 1 a transpapillary stent, and 1 a nasobiliary drain. Leak resolution occurred in 23 cases (88%) after initial stent placement. The median time to stent removal was 8 weeks. Three patients did not respond to initial treatment; 2 were successfully treated with another stent and a single patient required surgical repair. Four deaths occurred during follow-up, all unrelated to the biliary leak. CONCLUSIONS: Placement of a leak-bridging stent is a safe and effective initial treatment for post-liver transplantation biliary leaks.

Cardiovascular regulation of vasopressin neurons in the supraoptic nucleus.

This paper reviews the regulation of hypothalamic vasopressin and oxytocin neurosecretory cells in the neural response to plasma volume expansion. Many questions remain unanswered regarding how an increase in volume affects neurohypophysial hormone secretion, what receptors are important in mediating this response, and which neural pathways are responsible for conveying the signal from those receptors to the hypothalamus. Plasma volume expansion activates regions of the central nervous system associated with inhibition of vasopressin release, oxytocin secretion, and inhibition of sympathetic nerve activity. Cardiac receptors, not arterial baroreceptors, are primarily responsible for activation of the regions associated with regulation of vasopressin secretion and sympathetic outflow. Other stimuli that as yet are undefined account for activation of oxytocin-secreting neurons. Electrophysiology experiments have measured the inhibition of vasopressin-secreting magnocellular neurons in the supraoptic nucleus by select stimulation of cardiac receptors in the caval-atrial junction. Further experiments suggest that the perinuclear zone, a population of neurons surrounding the supraoptic nucleus, is a necessary part of the pathway by which caval-atrial stretch decreases the excitability of vasopressin neurons. The perinuclear zone is also a necessary synapse for arterial baroreceptor-mediated inhibition of vasopressin neurons. This suggests that the neural pathways that inhibit vasopressin release in response to an increase in blood pressure and an increase in blood volume may overlap at the perinuclear zone of the supraoptic nucleus. Finally, the integration of various neural pathways activated by multiple receptors to ultimately determine the activity of magnocellular neurons and vasopressin secretion is discussed.

The decision-making value of magnetic resonance cholangiopancreatography in patients seen in a referral center for suspected biliary and pancreatic disease.

OBJECTIVE: To assess the ability of MRCP to alter the differential diagnosis and to prevent diagnostic and/or therapeutic ERCP. The diagnostic accuracy of magnetic resonance cholangiopancreatography (MRCP) for biliary and pancreatic disease is well documented. Some believe MRCP may prevent diagnostic ERCP or add useful information, however there are no reports of its impact on clinical management. METHODS: Consecutive patients referred for ERCP underwent clinic evaluation, then MRCP, and then ERCP. In Phase 1, the number of differential diagnoses and the perceived need for diagnostic ERCP were evaluated after each step by the endoscopist who performed the ERCP. In Phase 2, the process was repeated after presenting clinical information and MRCP results to different individual physicians: another endoscopist, a hepatologist, a radiologist, and a surgeon (all were blinded to ERCP results). RESULTS: Forty patients were enrolled. Clinical contexts were jaundice (19.7%), abnormal liver enzymes (42.6%), abdominal pain (11.5%), recurrent acute pancreatitis (11.5%), and suspected complications of chronic pancreatitis (14.7%). In Phase 1, adding MRCP information to diagnostic ERCP information did not change the mean number of differential diagnoses significantly and prevented no therapeutic ERCP. In Phase 2, adding MRCP to clinical information only (without ERCP) reduced the differential diagnosis significantly for the radiologist and the surgeon only and would have prevented < or =3% of diagnostic and therapeutic ERCP for all physicians. CONCLUSION: The value of MRCP information may be limited if patient selection is inappropriate and may differ in physicians depending on their speciality.

Lesion of the perinuclear zone attenuates cardiac sensitivity of vasopressinergic supraoptic neurons.

Discrete stretch of the caval-atrial junction decreases the activity of vasopressin-secreting neurons in the supraoptic nucleus (SON). The perinuclear zone (PNZ) of the SON is necessary for inhibition of vasopressin neurons following an increase in blood pressure. To determine whether the PNZ is necessary for cardiopulmonary regulation of vasopressin neurons, male rats received three unilateral injections of the excitotoxin ibotenic acid (n = 9) or phosphate-buffered saline vehicle (n = 10) into the PNZ. Extracellular activity of antidromically identified phasic vasopressin neurons in the ipsilateral SON was recorded. Of the 26 neurons recorded from vehicle-injected rats 26 were inhibited by an increase in blood pressure and 22 of those neurons were sensitive to caval-atrial distension. Of the neurons recorded from PNZ-lesion rats, only 12 of 29 were inhibited by an increase in blood pressure (P < 0.05), and only 11 neurons were sensitive to caval-atrial stretch (P < 0.05). Functional lesion of the PNZ significantly attenuates both arterial and cardiopulmonary baroreceptor-mediated inhibition of supraoptic vasopressin neurons, suggesting that the PNZ is a necessary component of both pathways.

Integration of neuroscience and endocrinology in hybrid PBL curriculum.

At the University of Missouri-Columbia, the medical school employs a problem-based learning curriculum that began in 1993. Since the curriculum was changed, student performance on step 1 of the United States Medical Licensing Examination has significantly increased from slightly below the national average to almost one-half a standard deviation above the national mean. In the first and second years, classes for students are organized in classes or blocks that are 8 wk long, followed by 1 wk for evaluation. Initially, basic science endocrinology was taught in the fourth block of the first year with immunology and molecular biology. Student and faculty evaluations of the curriculum indicated that endocrinology did not integrate well with the rest of the material taught in that block. To address these issues, basic science endocrinology was moved into another block with neurosciences. We integrate endocrinology with neurosciences by using the hypothalamus and its role in neuroendocrinology as a springboard for endocrinology. This is accomplished by using clinical cases with clear neuroscience and endocrinology aspects such as Cushing's disease and multiple endocrine neoplastic syndrome type 1.

Baroreceptor sensitivity of rat supraoptic vasopressin neurons involves noncholinergic neurons in the DBB.

Previous studies suggest that cholinergic neurons in the diagonal band of Broca (DBB) participate in the baroreceptor-mediated inhibition of phasic vasopressin neurons in the supraoptic nucleus (SON). To test this hypothesis, extracellular recordings were obtained from putative vasopressin SON neurons of anesthetized rats injected with the cholinergic immunotoxin 192 IgG-saporin (0.8 microg/microl) in the DBB. Baroreceptor sensitivity of neurons was tested with brief phenylephrine-induced (10 microg/10 microl iv) increases in blood pressure of at least 40 mmHg. In rats injected with vehicle or unconjugated saporin, 19 of 21 and 18 of 20 phasic neurons, respectively, were inhibited by increased blood pressure. In rats injected with 192 IgG-saporin, which significantly reduced the number of choline acetyltransferase (ChAT)-positive DBB neurons, 33 of 36 phasic neurons were inhibited. Normal rats and rats with DBB saporin injections received rhodamine bead injections into the perinuclear zone (PNZ) to retrogradely label DBB neurons, and their brains were stained for ChAT. ChAT-positive DBB neurons were not retrogradely labeled from the PNZ. Together, these results indicate that the pathway relaying baroreceptor information to the SON involves noncholinergic DBB neurons.