Browsing by Author "Marciante, Alexandria B."
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Item Caspase Lesions of PVN-Projecting MnPO Neurons Blocks the Sustained Component of CIH-Induced Hypertension in Adult Male Rats(2019-03-05) Wang, Lei; Little, Joel; Cunningham, J. Thomas; Marciante, Alexandria B.Purpose: Obstructive Sleep Apnea (OSA) is characterized by cessations in respiration that leads to development of hypertension and persists into the waking period even during normal respiratory patterns. Previous studies show that experimental models of chronic intermittent hypoxia (CIH) produces sustained hypertension similar to that with OSA. It has been proposed that 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, 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, which contains pre-autonomic centers that project to regions in the hindbrain and regulate sympathetic outflow. We hypothesize that lesioning pathway specific projections from the MnPO to the PVN could attenuate CIH hypertension. Methods: Adult male Sprague-Dawley rats (250-300g) 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 (AAV5-flex-taCasp3-TEVp) or 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. Rats were then moved to new homecages and underwent baseline recording before undergoing our 7-day CIH protocol. Results: The control group exposed to CIH developed chronic hypertension, however, caspase lesions blunted the sustained hypertension. Brain tissue processed for FosB immunohistochemistry showed decreased expression with caspase-induced inhibition in the MnPO and downstream autonomic regulating nuclei. 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. Conclusion: 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 hypertension.Item Contribution of K+/Cl- Cotransporters in AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia(2019-03-05) Little, Joel; Marciante, Alexandria B.; Cunningham, J. Thomas; Farmer, George Jr.Purpose: Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in blood pressure during periods of normoxia, similar to that observed in OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Here we investigate the role of AT1aR in the MnPO and the contribution of the K+/Cl- cotransporters KCC2 and NKCC1 on excitatory/inhibitory balance in rats subjected to CIH. Methods: Under isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions (0.4 µL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1aKD) or an AAV containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2-3%) and coronal slices (300 µm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using glass micropipettes containing aCSF as the internal solution (1-3 MΩ). Spontaneous action potentials (APs) were recorded in response to muscimol (100uM, 30s). Results: The GABAa agonist muscimol decreased AP activity of neurons from normoxic/Scr rats. GABAa inhibition was blunted in neurons from CIH/Scr and normoxic/AT1aKD rats. However, GABAa activation from neurons in the CIH/AT1aKD group produced an increase in spontaneous activity. KCC2 block reduced GABAa mediated excitation in CIH/AT1aKD but had no effect GABAa mediated inhibition in CIH/Scr. NKCC1 block reduced GABAa mediated excitation in CIH/AT1aKD and facilitated GABAa mediated inhibition in CIH/Scr. Conclusion: The current study shows AT1aKD mediated reduction in GABAa inhibition is exacerbated such that GABAa activation is excitatory following CIH. KCC2 and NKCC1 contribute to GABAa mediated excitability in CIH/AT1aKD but only NKCC1 contributes to attenuated GABAa function in CIH/Scr. Future studies will address the influence of reduced AT1a signaling and reduced GABAa mediated inhibition on downstream targets of the MnPO.Item DREADD-Induced cFos Expression in the Basal Forebrain of Male Rats(2017-03-14) Cunningham, J. Thomas; Marciante, Alexandria B.Purpose: Designer Receptors Exclusively Activated by Designer Drugs, or DREADDs, are genetically modified G-protein coupled receptors (GPCR) that are sensitive to an exogenous pharmacological agent, clozapine-N-oxide (CNO). DREADDs can be packaged in viral vectors with specific promoters or combined with CRE dependent platforms to express these receptors in specific neuronal phenotypes. This chemogenetic approach can be used to activate (Gq), inhibit (Gi), or stimulate cAMP (Gs) in neurons expressing DREADD receptors. In the present study, we tested the effects of a CRE independent Gq DREADD, using a CaM Kinase (CaMKIIa) promoter, and a mCherry reporter (rAAV5-CaMKIIa-hM3D(Gq)-mCherry) on Fos staining in the basal forebrain. Methods: Adult male Sprague-Dawely rats (250-300 g bw, Charles River) were anesthetized with isoflurane and stereotaxically injected with an AAV containing the hM3D(Gq) or a control virus (rAAV5-CaMKIIa-mCherry) in either the diagonal band of bregma (DBB) or the median preoptic nucleus (MnPO). Animals were monitored for recovery for two weeks and then administered CNO or vehicle. CNO was dissolved into dimethylsulfoxide (DMSO) and saline (ratio 20% to 80%) and given via intraperitoneal injection (IP) at a concentration of 10mg/kg. Rats were food and water deprived for 90 minutes following administration of CNO or vehicle and then perfused transcardially using 4% paraformaldehyde. Brains were harvested and placed into 30% sucrose until proper dehydration of the brain. Forebrains were then sliced into 40 micron segments using cryostat. Immunohistological techniques were performed as previously described using peroxidase staining for Fos to determine activation of the DREADD virus and fluorescent staining for mCherry to verify cells transfected with either the DREADD or control virus. Results: Overall, rats transfected with the Gq DREADD virus and treated with CNO showed significantly elevated Fos staining in the DBB or MnPO than groups transfected with either Gq DREADD and treated with vehicle or the control virus and treated with CNO or vehicle. Those transfected with the Gq DREADD virus in the DBB and treated with CNO, showed significantly more Fos staining than those transfected with either Gq DREADD and treated with vehicle (P Conclusions: These results indicate that Gq DREADD can be used to differentially activate neurons in either the DBB or the MnPO to influence activity in downstream regions that control autonomic and neuroendocrine function.Item DREADD-induced inhibition of the MnPO affects drinking behavior and neuroendocrine function in adult male rats(2018-03-14) Farmer, George; Wang, Lei; Cunningham, J. Thomas; Marciante, Alexandria B.Purpose: Angiotensin II (Ang II) is a peptide hormone that contributes to body fluid balance and hypertension. Forebrain circumventricular organs (CVOs) are sensitive to circulating Ang II and project to the median preoptic nucleus (MnPO). The MnPO projects to the paraventricular nucleus (PVN) and contributes to elevated sympathetic tone and thirst. Methods: We used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the MnPO in thirst and neuroendocrine responses to Ang II in adult male Sprague-Dawley rats (250-300g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory (Gi) DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After 2 weeks of recovery, each rat was administered 10 mg/kg of exogenous Clozapine-N-Oxide (CNO) ip to inhibit DREADD expressing cells or vehicle ip followed by 2 mg/kg Ang II sc twice per week for 4 weeks. Rats were anesthetized with inactin (10 mg/kg ip) and transcardially perfused 90 minutes after CNO and Ang II treatments. Brains were processed for cFos and mCherry immunohistochemistry. Results: DREADD-injected rats treated with CNO during Ang II exposure had a significantly attenuated drinking response compared to vehicle treatments or to control virus injected rats treated with CNO and Ang II (pIn vitro loose-cell voltage clamp recordings from DREADD-transfected MnPO slices indicated focal CNO (10 uM) application significantly reduces firing rates of these neurons. In situ hybridization experiments of DREADD-transfected MnPO neurons and vesicular glutamate transporter 2 indicated neurons transfected with the DREADD virus containing the CaMKIIa promotor are largely glutamatergic (89.17+1.32%). Conclusion: The results indicate CNO-induced inhibition of excitatory, CaMKIIa-expressing MnPO neurons influences drinking behavior and neuroendocrine function.Item Role of Chronic Intermittent Hypoxia and Hypercapnia Induced Hypertension in Regulation of Blood Pressure(2016-03-23) Cunningham, J. Thomas; Little, Joel; Marciante, Alexandria B.Role of Chronic Intermittent Hypoxia and Hypercapnia Induced Hypertension in Regulation of Blood Pressure Purpose Sleep apnea is a prevalent disease characterized by momentary cessations in respiration leading to sustained hypertension. The hypertension experienced can be mimicked by periodic decreases in oxygen or chronic intermittent hypoxia (CIH) in humans and animal models. More recently, CIH has been combined with hypercapnia (CIH-H) to determine if an increase in circulating carbon dioxide, which is also experienced by patients that suffer from sleep apnea, contributes to neural adaptations related to sustained hypertension. CIH has been shown to have a significant effect on increased blood pressure due to increased sympathetic outflow from initiation and maintenance of hypertension. However, it is not known if the additional hypercapnic component significantly affects blood pressure or central autonomic control. Methods Male Sprague-Dawley rats are instrumented with radio telemetry one week after arrival. The radio telemetry provides information regarding cardiovascular variables continuously over a specified period of time. Animals were monitored for recovery for one week and then acclimated to the CIH or CIH-H rooms for 6 days, and monitored during this period for baseline data before experiencing 7 days of CIH, CIH-H or normoxic conditions. The CIH exposure is applied for 8 hours during the light (nocturnal) period from 8:00 AM to 4:00 PM, during which time hypoxia is produced using 3 min on-3 min off cycles that reduces oxygen from 21% to 10% to then being flushed with room air, so the inspired oxygen rises back to 21% before the cycle repeats. During CIH-H, rats are exposed to the same conditions with the addition of carbon dioxide that is raised from 0% to 8% during hypoxia to also produce hypercapnia. Results Rodents exposed to hypoxic and hypercapnic conditions did exhibit a greater increase in blood pressure than rodents exposed to only hypoxic conditions in the light period. The difference was not sustained during the return to normoxic conditions. Conclusions The results are consistent with previous studies which showed periods where there were greater increases in blood pressure in CIH-H animals than those exposed to hypoxia alone. Both CIH and CIH-H produced a greater increase in blood pressure during the light period. The difference did not appear to be sustained when rats were breathing room air.Item Selectively Inhibiting the Median Preoptic Nucleus Attenuates Angiotensin II and Hyperosmotic-Induced Drinking Behavior and Vasopressin Release in Adult Male Rats(Society for Neuroscience, 2019-03-26) Marciante, Alexandria B.; Wang, Lei A.; Farmer, George E.; Cunningham, J. ThomasThe median preoptic nucleus (MnPO) is a putative integrative region that contributes to body fluid balance. Activation of the MnPO can influence thirst, but it is not clear how these responses are linked to body fluid homeostasis. We used designer receptors exclusively activated by designer drugs (DREADDs) to determine the role of the MnPO in drinking behavior and vasopressin release in response to peripheral angiotensin II (ANG II) or 3% hypertonic saline (3% HTN) in adult male Sprague Dawley rats (250-300 g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After two weeks' recovery, a subset of rats was used for extracellular recordings to verify functional effects of ANG II or hyperosmotic challenges in MnPO slice preparations. Remaining rats were used in drinking behavior studies. Each rat was administered either 10 mg/kg of exogenous clozapine-N-oxide (CNO) to inhibit DREADD-expressing cells or vehicle intraperitoneal followed by a test treatment with either 2-mg/kg ANG II or 3% HTN (1 ml/100-g bw, s.c.), twice per week for two separate treatment weeks. CNO-induced inhibition during either test treatment significantly attenuated drinking responses compared to vehicle treatments and controls. Brain tissue processed for cFos immunohistochemistry showed decreased expression with CNO-induced inhibition during either test treatment in the MnPO and downstream nuclei compared to controls. CNO-mediated inhibition significantly attenuated treatment-induced increases in plasma vasopressin compared to controls. The results indicate inhibition of CaMKIIa-expressing MnPO neurons significantly reduces drinking and vasopressin release in response to ANG II or hyperosmotic challenge.Item The Role of the MnPO in Body Fluid Balance and Blood Pressure Regulation(2019-05) Marciante, Alexandria B.; Cunningham, J. Thomas; Mifflin, Steve W.; Schreihofer, Ann M.; Goulopoulou, Styliani; Ma, Rong; Bugnariu, Nicoleta L.The median preoptic nucleus (MnPO) is situated on the anteroventral wall of the third ventricle (AV3V) between two circumventricular organs (CVOs) that lack a functional blood-brain barrier, the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). The SFO and OVLT project to the MnPO and together these regions regulate neuroendocrine and autonomic function, arousal, and fluid balance. Early studies demonstrated that the MnPO and other regions in the AV3V contribute to regulating thirst associated with body fluid homeostasis, as well as several forms of neurogenic hypertension. The MnPO is key in relaying signals from the SFO and OVLT to downstream regions that control fluid intake and autonomic function; however, pathway-specific and stimulus-dependent mechanisms are not fully understood. These studies investigate how the MnPO differentially responds to models of physiological challenges that induce thirst, as well as pathway-specific mechanisms of blood pressure in a known model of hypertension. To study the role of the MnPO in thirst, rats were tested with models of cellular (hyperosmolality) and extracellular (angiotensin II, ANG II) dehydration associated with hypovolemia. Previous studies have shown that different populations of MnPO neurons are osmo- or ANG II-sensitive; however, both stimuli lead to a converging behavioral outcome: water consumption. This led to the hypothesis that osmotic challenges and ANG II activate MnPO neurons that project to different regions in a stimulus-dependent manner. Results show that the MnPO signals to specific thirst-driving regions of the brain and the activation of these regions is dependent on the stimulus. To study the role of the MnPO in regulating blood pressure, an experimental model of chronic intermittent hypoxia (CIH) associated with obstructive sleep apnea (OSA) is used to successfully mimic the oxygen deprivation associated with apneic breathing patterns patients with mild to moderate forms of OSA experience. Both patients with OSA and rodents in the CIH model develop diurnal hypertension, which is a sustained increase in blood pressure that persists into the waking hours. Hypertension involves multiple organ systems, including the central nervous system, and can be a heterogenous disease state that manifests from a number of factors, including CIH, ANG II from renin-angiotensin system (RAS), and changes in body fluid osmolality. This led to the hypothesis that pathway-specific inhibition of MnPO neurons that project to pre-autonomic neurons in the paraventricular nucleus (PVN) of the hypothalamus would block persistent hypertension. Results indicate that lesioning PVN-projecting MnPO neurons can block CIH-induced hypertension, resulting in decreases in oxidative stress and improved cardiovascular health. These findings provide new information about how the MnPO differentially regulates behavioral and physiological outcomes in a stimulus-dependent manner. These outcomes also have broad clinical implications relating to the role of the central nervous system in disease states affecting body fluid balance and blood pressure regulation.