Chronic intermittent hypoxia (CIH) models repetitive bouts of arterial hypoxemia that occur in humans suffering from obstructive sleep apnea. CIH has been linked to persistent activation of arterial chemoreceptors and the renin-angiotensin system, which have been linked to chronic elevations of sympathetic nerve activity (SNA) and mean arterial pressure (MAP). Because Fos and FosB are transcription factors involved in activator protein (AP)-1 driven central nervous system neuronal adaptations, this study determined if CIH causes increased Fos or FosB staining in brain regions that regulate SNA and autonomic function. Male Sprague Dawley rats were instrumented with telemetry transmitters for continuous recording of MAP and heart rate (HR). Rats were exposed to continuous normoxia (CON) or to CIH for 8 h/day for 7 days. CIH increased MAP by 7-10 mmHg without persistently affecting HR. A separate group of rats was killed 1 day after 7 days of CIH for immunohistochemistry. CIH did not increase Fos staining in any brain region examined. Staining for FosB/ΔFosB was increased in the organum vasculosum of the lamina terminalis (CON: 9 ± 1; CIH: 34 ± 3 cells/section), subfornical organ (CON: 7 ± 2; CIH: 31 ± 3), median preoptic nucleus (CON 15 ± 1; CIH: 38 ± 3), nucleus of the solitary tract (CON: 9 ± 2; CIH: 28 ± 4), A5 (CON: 3 ± 1; CIH: 10 ± 1), and rostral ventrolateral medulla (CON: 5 ± 1; CIH: 17 ± 2). In the paraventricular nucleus, FosB/ΔFosB staining was located mainly in the dorsal and medial parvocellular subnuclei. CIH did not increase FosB/ΔFosB staining in caudal ventrolateral medulla or supraoptic nucleus. These data indicate that CIH induces an increase in FosB/ΔFosB in autonomic nuclei and suggest that AP-1 transcriptional regulation may contribute to stable adaptive changes that support chronically elevated SNA.

Saxena A, Little JT, Nedungadi TP, Cunningham JT. Angiotensin II type 1a receptors in subfornical organ contribute towards chronic intermittent hypoxia-associated sustained increase in mean arterial pressure. Am J Physiol Heart Circ Physiol 308: H435-H446, 2015. First published December 24, 2014 doi:10.1152/ajpheart.00747.2014.-Sleep apnea is associated with hypertension. The mechanisms contributing to a sustained increase in mean arterial pressure (MAP) even during normoxic awake-state remain unknown. Rats exposed to chronic intermittent hypoxia for 7 days, a model of the hypoxemia associated with sleep apnea, exhibit sustained increases in MAP even during the normoxic dark phase. Activation of the renin-angiotensin system (RAS) has been implicated in chronic intermittent hypoxia (CIH) hypertension. Since the subfornical organ (SFO) serves as a primary target for the central actions of circulating ANG II, we tested the effects of ANG II type 1a receptor (AT1aR) knockdown in the SFO on the sustained increase in MAP in this CIH model. Adeno-associated virus carrying green fluorescent protein (GFP) and small-hairpin RNA against either AT1aR or a scrambled control sequence (SCM) was stereotaxically injected in the SFO of rats. After recovery, MAP, heart rate, respiratory rate, and activity were continuously recorded using radiotelemetry. In the normoxic groups, the recorded variables did not deviate from the baseline values. Both CIH groups exhibited significant increases in MAP during CIH exposures (P Ͻ 0.05). During the normoxic dark phase in the CIH groups, only the SCMinjected group exhibited a sustained increase in MAP (P Ͻ 0.05). The AT1aR-CIH group showed significant decreases in FosB/⌬FosB staining in the median preoptic nucleus and the paraventricular nuclei of the hypothalamus compared with the SCM-CIH group. Our data indicate that AT1aRs in the SFO are critical for the sustained elevation in MAP and increased FosB/⌬FosB expression in forebrain autonomic nuclei associated with CIH. angiotensin receptor; subfornical organ; chronic intermittent hypoxia; obstructive sleep apnea; AT1aR SLEEP APNEA (SA) is increasingly being recognized as a cause of neurogenic and treatment-resistant hypertension (12,19,32,37,52). SA is associated with a sustained increase in sympathetic nerve activity (SNA) and mean arterial pressure (MAP) even during periods of wakefulness and normoxia (4, 33). Animal models of chronic intermittent hypoxia (CIH), such as the one introduced by Fletcher at al. (17), produce cardiovascular sequelae similar to sleep apnea (11). Together, it appears that CIH episodes lead to pathophysiological adaptations that may generate and sustain a heightened basal MAP, which is partially dependent on increased SNA.The renin-angiotensin system (RAS) is activated during CIH (11,16,54), and it contributes to CIH hypertension (11, 16). For example, in rats exposed to CIH, peripheral administration of losartan, an angiotensin II (ANG II) type 1 receptor (AT1R) antagonist, has been shown to prevent the increase ...

High salt loading (SL) is associated with inappropriate arginine vasopressin (AVP) release and increased mean arterial pressure. Previous work has shown that chronic high salt intake impairs baroreceptor inhibition of rat AVP neurones through brain-derived neurotrophic factor (BDNF) dependent activation of tyrosine receptor kinase B (TrkB) and down-regulation of K+/Cl- co-transporter KCC2. This mechanism diminishes the GABA inhibition of AVP neurones in the supraoptic nucleus (SON) by increasing intracellular chloride. However, the source of BDNF leading to this ionic plasticity is unknown. In the present study, we used adeno-associated viral vectors with short hairpin RNA against BDNF to test whether SON is the source of BDNF contributing to increased AVP release and elevated mean arterial pressure in high salt loaded rats. Virally mediated BDNF knockdown (shBDNF) in the SON of salt loaded rats significantly blocked the increases in BDNF mRNA and AVP heterogeneous RNA expression. The observed increase in the activation of TrkB receptor during salt loading is consistent with previous studies. Western blot analysis of SON punches revealed that tyrosine phosphorylation of TrkB (pTrkBY515) was significantly decreased in salt shBDNF rats compared to the salt scrambled (SCR) rats. Injections of shBDNF in the SON also significantly prevented the increase in plasma AVP concentration associated with salt loading. However, the salt loading induced increase in mean arterial pressure was not decreased with BDNF knockdown in the SON. Average daily fluid intake and urine output were significantly elevated in both salt SCR and salt shBDNF rats compared to the euhydrated controls. Daily average urine sodium concentration was significantly higher in shBDNF injected salt rats than the other groups. These findings indicate that BDNF produced in the SON contributes to the increased AVP secretion during high salt loading but not with respect to the subsequent increase in mean arterial pressure.

Salt-loading (SL) impairs GABA A inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K + /Cl − co-transporter2 (KCC2) and up-regulating Na + /K + /Cl − co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl − ] i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl − ] i in response to focal application of the GABA A agonist muscimol (100 μmol L -1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39)or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L -1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L -1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl − influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 μmol L -1 ) and protein kinase inhibitor (K252a) (100 nmol L -1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl − ] i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats. K E Y W O R D S chloride imaging, SON and salt-loading, vasopressin S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Balapattabi K, Farmer GE, Knapp BA, et al. Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging.

The median preoptic nucleus (MnPO) is an integrative site involved in body fluid homeostasis, cardiovascular control, thermoregulation, and sleep homeostasis. Angiotensin II (ANG II), a neuropeptide shown to have excitatory effects on MnPO neurons, is of particular interest with regard to its role in body fluid homeostasis and cardiovascular control. The current study investigated the role of angiotensin type 1 (AT1a) receptor activation on neuronal excitability in the MnPO. Male Sprague-Dawley rats were infused with an adeno-associated virus with a shRNA against the AT1a receptor or a scrambled control. In vitro loose patch voltage clamp recordings of spontaneous action potential activity were made from labeled MnPO neurons in response to brief focal application of ANG II or the GABAA receptor agonist muscimol. Tissue punches from MnPO were taken to asses mRNA and protein expression. AT1a receptor knockdown neurons were insensitive to ANG II and showed a marked reduction in GABAA mediated inhibition. The reduction in GABAA mediated inhibition was not associated with reductions in mRNA or protein expression of GABAA β-subunits. Knockdown of the AT1a receptor was associated with a reduction in the potassium-chloride cotransporter KCC2 mRNA as well as a reduction in pS940 KCC2 protein. The impaired GABAA mediated inhibition in AT1a knockdown neurons was recovered by bath application of phospholipase C (PLC) and protein kinase C (PKC) activators. The following study indicates that AT1a receptor activation mediates the excitability of MnPO neurons, in part, through the regulation of KCC2.

Hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in liver failure patients. The mechanisms causing dysregulation of AVP secretion are unknown. Our hypothesis is that inappropriate AVP release associated with liver failure is due to increased brain-derived neurotrophic factor (BDNF) in the supraoptic nucleus (SON). BDNF diminishes GABA A inhibition in SON AVP neurons by increasing intracellular chloride through tyrosine receptor kinase B (TrkB) activation and downregulation of K+/Cl-cotransporter 2 (KCC2). This loss of inhibition could increase AVP secretion. This hypothesis was tested using shRNA against BDNF (shBDNF) in the SON in bile duct ligated (BDL) male rats. All BDL rats had significantly increased liver weight (p < 0.05; 6-9) compared to shams. BDL rats with control shRNA injections (BDL scrambled [SCR]) developed hyponatremia with increased plasma AVP and copeptin (CPP; all p < 0.05; 6-9) compared to sham groups. This is the first study to show that phosphorylation of TrkB is significantly increased along with significant decrease in phosphorylation of KCC2 in BDL SCR rats compared to the sham rats (p < 0.05; 6-8). Knockdown of BDNF in the SON of BDL rats (BDL shBDNF) significantly increased plasma osmolality and hematocrit compared to BDL SCR rats (p < 0.05; 6-9). The BDL shBDNF rats had significant (p < 0.05; 6-9) decreases in plasma AVP and CPP concentration compared to BDL SCR rats. The BDNF knockdown also significantly blocked the increase in TrkB phosphorylation and decrease in KCC2 phosphorylation (p < 0.05; 6-8). The results indicate that BDNF produced in the SON contributes to increased AVP secretion and hyponatremia during liver failure.

Background: Rodent Parkinson’s disease (PD) models are valuable to interrogate neurobiological mechanisms of exercise that mitigate motor impairment. Translating these mechanisms to human PD must account for physical capabilities of the patient. Objective: To establish cardiovascular parameters as a common metric for cross-species translation of aerobic exercise impact. Method: We evaluated aerobic exercise impact on heart rate (HR) in 21 early-stage PD subjects (Hoehn Yahr ≤1.5) exercising in non-contact boxing training for ≥3 months, ≥3x/week. In 4-month-old Pink1 knockout (KO) rats exercising in a progressively-increased treadmill speed regimen, we determined a specific treadmill speed that increased HR to an extent similar in human subjects. Results: After completing aerobic exercise for ∼30 min, PD subjects had increased HR∼35% above baseline (∼63% maximum HR). Motor and cognitive test results indicated the exercising subjects completed the timed up and go (TUG) and trail-making test (TMT-A) in significantly less time versus exercise-naïve PD subjects. In KO and age-matched wild-type (WT) rats, treadmill speeds of 8–10 m/min increased HR up to 25% above baseline (∼67% maximum HR), with no further increases up to 16 m/min. Exercised KO, but not WT, rats showed increased locomotor activity compared to an age-matched exercise-naïve cohort at 5 months old. Conclusion: These proof-of-concept results indicate HR is a cross-species translation parameter to evaluate aerobic exercise impact on specific motor or cognitive functions in human subjects and rat PD models. Moreover, a moderate intensity exercise regimen is within the physical abilities of early-stage PD patients and is therefore applicable for interrogating neurobiological mechanisms in rat PD models.

Obstructive Sleep Apnea (OSA) is characterized by cessations in respiration that leads to the development of chronic hypertension. Hypertension persists into the waking period even during normal respiratory patterns, and can increase a patient's risk factor for heart disease and stroke. Previous studies have shown that experimental models of chronic intermittent hypoxia (CIH) can produce a sustained hypertension similar to that associated with OSA. It has been proposed that the peripheral and CNS renin‐angiotensin systems contribute to hypertension associated with CIH. Our working hypothesis is that increased circulating angiotensin II feeds into the forebrain thereby increasing excitatory signaling through the hypothalamus and hindbrain, creating a vicious cycle. The median preoptic nucleus (MnPO) is an integrative forebrain region that contributes to blood pressure regulation. The MnPO has projections to the paraventricular nucleus (PVN) of the hypothalamus. The PVN contains pre‐autonomic centers that project to regions in the hindbrain that regulate sympathetic outflow. We hypothesized that by lesioning pathway specific projections from the MnPO to the PVN, we could attenuate the sustained component of CIH‐induced hypertension. For these experiments, adult male Sprague‐Dawley rats (250–300 g bw) were anesthetized with isoflurane and stereotaxically injected bilaterally in the PVN with a retrograde AAV containing Cre (AAV9.CMV.HI.eGFP‐Cre.WPRE.SV40) and with the caspase‐3 virus (AAV5‐flex‐taCasp3‐TEVp) or a control virus (AAV5‐hSyn‐DIO‐mCherry) in the MnPO. After 1 week recovery, rats were instrumented with aortic radio telemetry and allowed an additional week recovery following surgery. Rats were then moved to new homecages and underwent baseline recording for 7 days before undergoing our 7‐day CIH protocol. The control group exposed to CIH developed chronic hypertension, however, caspase lesions blunted the sustained hypertension developed during CIH. Brain tissue processed for FosB immunohistochemistry (IHC) showed decreased expression with caspase‐induced inhibition in the MnPO and downstream autonomic‐regulating nuclei in the PVN and rostral ventral lateral medulla (RVLM). CIH significantly increased plasma advanced oxidative protein products (AOPP) levels in controls. This increase in AOPP levels was blocked in caspase‐lesioned rats comparable to normoxic control concentrations. In situ hybridization experiments indicate a reduction in angiotensin type 1a receptors (AT1aR) expression in the caspase‐lesioned group exposed to CIH compared to CIH controls. The results indicate that MnPO neurons that project to the PVN play a significant role in blood pressure regulation and in the development of persistent CIH‐induced hypertension. Support or Funding Information P01 HL088052 T32 AG020494 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.