Transient receptor potential (TRP) proteins are non-selective cation channels that mediate sensory transduction. The neuroanatomical localization and the physiological roles of isoform TRPV2 in the rodent brain are largely unknown. We report here the neuroanatomical distribution of TRPV2 in the adult male rat brain focusing on hypothalamus and hindbrain regions involved in osmoregulation, autonomic function and energy metabolism. For this we utilized immunohistochemistry combined with brighfield microscopy. In the forebrain, the densest immunostaining was seen in both the supraoptic nucleus (SON) and the magnocellular division of the paraventricular nucleus (PVN) of the hypothalamus. TRPV2 immunoreactivity was also seen in the organum vasculosum of the lamina terminalis, the median preoptic nucleus and the subfornical organ, in addition to the arcuate nucleus of the hypothalamus (ARH), the medial forebrain bundle, the cingulate cortex and the globus pallidus to name a few. In the hindbrain, intense staining was seen in the nucleus of the solitary tract, hypoglossal nucleus, nucleus ambiguous, and the rostral division of the ventrolateral medulla (RVLM) and some mild staining in the area prostrema. To ascertain the specificity of the TRPV2 antibody used in this paper, we compared the TRPV2 immunoreactivity of wildtype (WT) and knockout (KO) mouse brain tissue. Double immunostaining with arginine vasopressin (AVP) using confocal microscopy showed a high degree of colocalization of TRPV2 in the magnocellular SON and PVN. Using laser capture microdissection (LCM) we also show that AVP neurons in the SON contain TRPV2 mRNA. TRPV2 was also co-localized with dopamine beta hydroxylase (DBH) in the NTS and the RVLM of the hindbrain. Based on our results, TRPV2 may play an important role in several CNS networks that regulate body fluid homeostasis, autonomic function, and metabolism.

The purpose of this study was to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurons 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, a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. This study tested the hypothesis that changes in TRPV channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilized laser capture microdissection of AVP neurons in the PVN and SON and Western blot analysis from brain punches. Laser capture microdissection and qRT-PCR demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL as 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 colocalization counts of TRPV2 and OXY in both the magnocellular regions evaluated. In the SON but not the PVN, transcription levels of TRPV4 was 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. Our data suggests that regionally specific changes in TRPV expression in the MNC AVP neurons could alter their osmosensing ability.

Noradrenergic A2 neurons in nucleus tractus solitarius (NTS) respond to stressors such as hypoxia. We hypothesize that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with green fluorescent protein (GFP) reporter having either short hairpin RNA (shRNA) for TH or scrambled virus (scRNA) were injected into caudal NTS. Virus-injected rats were exposed to 7 days of CIH (alternating periods of 10% O2 and of 21% O2 from 8 AM to 4 PM; from 4 PM to 8 AM rats were exposed to 21% O2). CIH increased mean arterial pressure (MAP) and heart rate (HR) during the day in both the scRNA (n = 14, P < 0.001 MAP and HR) and shRNA (n = 13, P < 0.001 MAP and HR) groups. During the night, MAP and HR remained elevated in the scRNA rats (P < 0.001 MAP and HR) but not in the shRNA group. TH immunoreactivity and protein were reduced in the shRNA group. FosB/ΔFosB immunoreactivity was decreased in paraventricular nucleus (PVN) of shRNA group (P < 0.001). However, the shRNA group did not show any change in the FosB/ΔFosB immunoreactivity in the rostral ventrolateral medulla. Exposure to CIH increased MAP which persisted beyond the period of exposure to CIH. Knockdown of TH in the NTS reduced this CIH-induced persistent increase in MAP and reduced the transcriptional activation of PVN. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH.

Koneru B, Bathina CS, Cherry BH, Mifflin SW. Mineralocorticoid receptor in the NTS stimulates saline intake during fourth ventricular infusions of aldosterone. Am J Physiol Regul Integr Comp Physiol 306: R61-R66, 2014. First published November 20, 2013 doi:10.1152/ajpregu.00434.2013.-The purpose of this study was to determine whether neurons within the nucleus tractus solitarius (NTS) that express the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Adult Wistar-Kyoto (WKY) rats received microinjections into the NTS of a short-hairpin RNA (shRNA) for the MR, to site specifically reduce levels of the MR by RNA interference (shRNA; n ϭ 9) or scrambled RNA as a control (scRNA; n ϭ 8). After injection of the viral construct, aldosteronefilled osmotic minipumps were implanted subcutaneously and connected to a cannula extending into the fourth ventricle to infuse aldosterone at a rate of 25 ng/h. Before and after surgeries, rats had ad libitum access to normal sodium (0.26%) rat chow and two graduated drinking bottles filled with either distilled water or 0.3 M NaCl. Before the surgeries, basal saline intake was 1.6 Ϯ 0.6 ml in the scRNA group and 1.56 Ϯ 0.6 ml in the shRNA group. Twenty-four days postsurgery, saline intake was elevated to a greater extent in the scRNA group (5.9 Ϯ 1.07 ml) than in the shRNA group (2.41 Ϯ 0.6 ml). Post mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for MR in shRNA-injected rats (23 Ϯ 1 cells/section) versus scRNAinjected rats (33 Ϯ 2 cells/section; P ϭ 0.008). shRNA did not alter the level of 11-␤-hydroxysteroid dehydrogenase type II (HSD2) protein in the NTS as judged by the number of HSD2 immunoreactive neurons. These results suggest that fourth ventricular infusions of aldosterone stimulate saline intake, and that this stimulation is at least in part mediated by hindbrain NTS neurons that express MR.

A2 catecholaminergic neurons located within caudal and sub‐postremal NTS play a critical role in modulating responses to physiological stress and project to multiple brain stem and forebrain hypothalamic regions. A2 neurons by definition express tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine synthesis. In vitro analyses of A2 neurons in the rat have been limited as identification of A2 neurons requires labeling of neuron and post‐mortem immunohistochemistry. As a result, one does not know the phenotype of the neuron at the time of study. To overcome this challenge we have used commercially available adeno ‐ associated virus (AAV) vector mediated delivery of green fluorescent protein (GFP) labeled TH promoter (AAV‐GFP‐TH), which will incorporate into the TH genome and express GFP with the TH expression. To verify that GFP expression was restricted to catecholaminergic neurons, conventional immunohistochemistry for TH was performed on sections from AAV‐GFP‐TH injected animals. We found that, 78.4 ± 3.07% TH immunoreactive neurons to be GFP labeled and 95 ± 2.40% (n=3 sections) of the GFP expressing neurons of caudal NTS to be TH‐immunoreactive, making our technique a reliable and accurate way to label A2 neurons prior to in vitro study. Brain slices from these rats can be used for direct visualization of TH‐immunoreactive neurons without any need for further processing.

We tested the hypothesis that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to CIH, a model of the arterial hypoxemia observed during sleep apnea. Adult male Sprague‐Dawley rats were implanted with radiotelemetry transmitters and AAV constructs with a GFP reporter having either short hairpin RNA for TH (shRNA) or scrambled virus (scrambled) were injected into caudal NTS. Virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). CIH increased MAP and HR during the day in both the scrambled (n= 14) and shRNA (n=13) groups (all p < 0.001). During the night MAP and HR remained elevated in the scrambled rats (both p<0.001) but not in the shRNA group. The number of TH‐immunoreactive neurons per section was reduced by 20% in sections with GFP fluorescence; shRNA 28±1 vs. scrambled 35±1 (p=.005) without altering the number of dopamine â‐hydroxylase (DBH) – immunoreactive neurons; shRNA 45±3 cells/section vs. scrambled 45±3 cells/section. Western blot analysis showed reductions in TH protein levels of 30% and 10% in caudal and sub‐postremal NTS respectively. Exposure to CIH increased MAP beyond the period of exposure to CIH. Knockdown of TH in the NTS reduces this persistent increase in MAP. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH. Supported by HL‐088052

The purpose of this study was to determine if neurons within the NTS that possess the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Adult WKY rats received microinjections into the NTS of a small, hairpin RNA for the MR (shRNA; n=9) or a scrambled RNA (scRNA; n=8). After the viral construct injections, aldosterone‐filled osmotic mini‐pumps were implanted subcutaneously and connected to tubing within the 4th ventricle to infuse aldosterone at a rate of 20ng/h. Prior to and after surgeries, rats had ad libitum access to food and two graduated drinking bottles filled with distilled water and 0.3M NaCl and salt intake expressed as 100 × the ratio of 0.3M NaCl intake to total fluid intake. Prior to surgeries, basal salt intake was 4.7% ± 2.9% in the scRNA group and 3.91% ± 2.6% in the shRNA group. 24 days post‐surgery salt intake was 20.6% ± 2.9% in the scRNA group and 4.3% ± 2.8% in the shRNA group. Post‐mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for the MR (scRNA 33 ± 2 cells/section; shRNA 23 ± 1 cells/section; p=.008). These results indicate 4th ventricular infusions of aldosterone stimulate salt intake and that at least part of the stimulation of salt intake during 4th ventricular infusions of aldosterone is mediated by hindbrain NTS neurons that possess the MR.

Patients with sleep apnea (SA) are at a risk of developing metabolic syndrome. In the rat, 7 days of Chronic Intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in SA patients, is associated with reduced body weight gain (p<0.005, n= 18), food intake (p<0.05, n=11), hyperinsulinemia (p<0.05, n=7), glucose intolerance and elevated fasting blood glucose levels (p<0.05, n=8). Brainstem melanocortins and other peptides has been well documented to be involved in metabolic homeostasis while tyrosine hydroxylase (TH) is known to be responsive to hypoxic stress; however the involvement of NTS TH neurons in regulating energy homeostasis following CIH has not yet been investigated. AAV‐TH shRNA virus was used to knockdown TH in the NTS of male rats, while virus with scrambled RNA was used as a control. Knockdown of TH in the NTS prevented the reduction in body weight following CIH (n=7) compared to scrambled virus injected rats and naïve rats subjected to CIH. Immunohistochemical staining for the neuronal activational marker FosB revealed elevated immunoreactivity following CIH (p<0.05, n=6) in the hypothalamic arcuate nucleus (ARH). Knockdown of TH resulted in reduced FosB staining in the ARH and dorsomedial hypothalamus while no change was observed in the lateral hypothalamic area. However, in the ventromedial hypothalamus, increased FosB staining was observed (p<0.05, n= 8). Our findings suggest that NTS TH neurons play an important role in regulating metabolic status during exposure to CIH and alter the neuronal activity in hypothalamic feeding centers, possibly via regulation of an anorexigenic signaling pathway. Grant Funding Source: P01 HL88052