Browsing by Author "Little, Joel"
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Item ADENO-ASSOCIATED VIRUS CONSTRUCT ENABLES DIFFERENTIATION OF VASOPRESSIN AND OXYTOCIN NEUROPEPTIDE-EXPRESSING MAGNOCELLULAR NEURONS IN THE HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RAT(2014-03) Knapp, Blayne A.; Little, Joel; Cunningham, TomAdeno-associated viral (AAV) vectors are useful tools for transfecting specific cell populations through the use of cell-type specific promoters. Recently, promoters that are specific for either vasopressin (AVP) or oxytocin (OXT) magnocellular neurosecretory cells (MNCs) have been designed that can be used with AAVs to selectively drive gene expression in these cells. The goal of this study was to validate this approach and determine whether it can cause the selective transfection of AVP versus OXT MNCs in the supraoptic nucleus of the hypothalamus (SON). In these studies, an AAV2 vector with an AVP promoter and GFP (p2.OVPI.EGFP) was stereotaxically injected into the SON of adult male Sprague-Dawley rats (226 - 250g bw) during isoflurane anesthesia. After 14 days, the rats were each anesthetized with inactin (100 mg/kg ip) and their brains where prepared for immunofluorescence. Two separate sections of coronal sections containing the SON were processed for either AVP or OXT immunohistochemistry using a Cy3 conjugated secondary antibody. Colocalization of GFP with either AVP or OXT immunofluorescence was determined by light microscopy. Our results indicate the colocalization of GFP and AVP in MNCs of the SON (89% GFP-AVP double labeling, n=3), and not GFP and OXT (0.08% GFP-OXT double labeling, n=3). Given this demonstration of successful vector transduction, we can conclude that the AAV2 vector is selective to AVP expressing MNCs, enabling us to distinguish AVP versus OXT MNCs in the SON. This capability will permit differentiation of neuronal types and their respective properties during later electrophysiological studies. R56 HL62569. Purpose (a): The goal of this study was to validate this approach and determine whether it can cause the selective transfection of AVP versus OXT MNCs in the supraoptic nucleus of the hypothalamus (SON). Methods (b): In these studies, an AAV2 vector with an AVP promoter and GFP (p2.OVPI.EGFP) was stereotaxically injected into the SON of adult male Sprague-Dawley rats (226-250g bw) during isoflurane anesthesia. After 14 days, the rats were each anesthetized with inactin (100 mg/kg ip) and their brains where prepared for immunofluorescence. Two separate sections of coronal sections containing the SON were processed for either AVP or OXT immunohistochemistry using a Cy3 conjugated secondary antibody. Colocalization of GFP with either AVP or OXT immunofluorescence was determined by light microscopy. Results (c): Our results indicate the colocalization of GFP and AVP in MNCs of the SON (89% GFP-AVP double labeling, n=3), and not GFP and OXT (0.08% GFP-OXT double labeling, n=3). Conclusions (d): Given this demonstration of successful vector transduction, we can conclude that the AAV2 vector is selective to AVP expressing MNCs, enabling us to distinguish AVP versus OXT MNCs in the SON. This capability will permit differentiation of neuronal types and their respective properties during later electrophysiological studies.Item Assessment of Neuroinflammation in Cognitive and Motor Brain Regions in Female Rats Exposed to Chronic Intermittent Hypoxia(2024-03-21) Appiah, Cephas; Little, Joel; Kunwar, Kishor; Cunningham, TomSleep apnea increases the risk of neurodegenerative disorders in postmenopausal women. In this study, we tested whether chronic intermittent hypoxia (CIH), a preclinical model of sleep apnea-associated cyclical hypoxia, can be used to identify early changes in the brain that might contribute to impair neurological function in intact (INT) and ovariectomized (OVX) female rats. To test this hypothesis, we conducted immunohistochemistry studies using markers for glial activation and neuroinflammation. We hypothesize that CIH will increase glial activation in ovariectomized (OVX) relative to intact (INT) female rats. Adult female Sprague Dawley INT (n=4) or OVX (n=4) rats that were part of a larger study and underwent 7 days of CIH (10% O2 and 21% O2 cycle, every 6 mins, 8h/day during the light phase) or continuous normoxia (CON) were euthanized on day 8, and their brains were collected. Brains were processed for microglia (IBA1) and astrocytes (GFAP) activation markers in (CA1) hippocampus, medial prefrontal cortex (mPFC), and caudate putamen (CP) striatum. Confocal images from each region are being used to optimize a fractal analysis protocol to test for changes in glial morphology. Raw images will be linearly processed in Huygens Essentials software to limit noise and optimize quality for further analysis in ImageJ. Skeletal and fractal analyses will be performed on randomly selected cells in the photomicrographs to determine cell ramification (branching, junctions, endpoint voxels, branch lengths) and complexity (fractal dimension, cell span ratio, density) to complement cell counts of immunohistochemical markers of activation. Our preliminary analysis has allowed us to determine the appropriate parameters needed for image capture and subsequent analysis. We have observed some possible qualitative changes indicative of increased activation in the CA1, CP, and mPFC regions following CIH in OVX rats relative to intact rats. The results of this study aim to contribute to our understanding of the potential impact of CIH in female rats of differing ovarian function, providing insights into sleep apnea-associated CNS dysfunction in women.Item AT1aR dependent GABAa inhibition in the MnPO(2017-03-14) Little, Joel; Bachelor, Martha; Cunningham, Tom; Farmer, George Jr.Background: The median preoptic nucleus (MnPO) receives input from other circumventricular organs (e.g. SFO and the OVLT) sensitive to circulating Angiotensin II (Ang II) and plasma Na+ concentrations suggesting an involvement in hydromineral balance and blood pressure regulation. Additionally, evidence suggest the SFO synthesizes Ang II and releases it on the MnPO as a neurotransmitter suggesting the role of the MnPO in hydromineral balance and blood pressure regulation is mediated in part by Ang II. The Ang II activation of AT1aR has also been shown to influence the function of GABAaRs though the mechanisms are still unclear. Here we investigate the role of Ang II signaling via the AT1aR in the MnPO and its influence on excitatory/inhibitory balance. Methods: Male Sprague-Dawley rats received infusions of an AAV construct containing GFP reporter and shRNA against AT1aR (shAT1a) or a shRNA scramble (shScr) targeted to the MnPO. Two weeks following AAV infusion, slices containing the MnPO were cut using standard in vitro slice procedures followed by loose patch recordings obtained from GFP labeled neurons. Spontaneous action potential firing was recorded in response to focal application of Ang II or muscimol. Additionally, activity of MnPO neurons in response to muscimol was observed in the presence of a PLC or PKC activator. The GABAa mediated effects in AT1a KD were compared to acute blockade of AT1aRs in rats that did not receive AAV infusions. Western blot and RT-qPCR analyses were used to investigate the effect of AT1a KD on GABAa and KCC2 protein and mRNA expression. Results: Brief focal application of Ang II produced a time dependent increase in spontaneous firing of MnPO neurons. The Ang II dependent enhancement of spontaneous activity was blocked by bath application of the AT1aR antagonist Losartan. Additionally, Ang II failed to alter firing rate of MnPO neurons in shAT1a KD rats. In control animals, the GABAa agonist muscimol decreased action potential activity. In rats that received microinjections of the shAT1a muscimol failed to decrease action potential activity. In AT1a KD rats, RT-qPCR analysis shows a reduction in AT1a and KCC2 mRNA but no reduction in GABAa Beta subunit mRNA. Conclusions: The current findings demonstrate Ang II dependent increases in the excitability of MnPO neurons are mediated by activation of AT1aRs. Moreover, AT1aRs activation also mediates the inhibitory effects of GABAaR activation. The current study suggests the reduction in GABAa dependent inhibition following AT1a KD is mediated by a down regulation of KCC2 and subsequent disruption of intracellular Cl- homeostasis. AT1aR function can modulate the balance of excitatory and inhibitory activity within the MnPO and efferent nuclei involved in the regulation of blood pressure and hydromineral balance. However, mechanisms underlying the dual excitatory/inhibitory functions of AT1aR activation remain unclear.Item Cardiovascular Metrics Associated With Prevention of Aging-Related Parkinsonian Signs Following Exercise Intervention in Sedentary Older Rats(Frontiers Media S.A., 2021-12-15) Kasanga, Ella A.; Little, Joel; McInnis, Tamara R.; Bugnariu, Nicoleta; Cunningham, J. Thomas; Salvatore, Michael F.Preservation of motor capabilities is vital to maintaining independent daily living throughout a person's lifespan and may mitigate aging-related parkinsonism, a progressive and prevalent motor impairment. Physically active lifestyles can mitigate aging-related motor impairment. However, the metrics of physical activity necessary for mitigating parkinsonian signs are not established. Consistent moderate intensity (~10 m/min) treadmill exercise can reverse aging-related parkinsonian signs by 20 weeks in a 2-week on, 2-week off, regimen in previously sedentary advanced middle-aged rats. In this study, we initiated treadmill exercise in sedentary 18-month-old male rats to address two questions: (1) if a rest period not longer than 1-week off exercise, with 15 exercise sessions per month, could attenuate parkinsonian signs within 2 months after exercise initiation, and the associated impact on heart rate (HR) and mean arterial pressure (MAP) and (2) if continuation of this regimen, up to 20 weeks, will be associated with continual prevention of parkinsonian signs. The intensity and frequency of treadmill exercise attenuated aging-related parkinsonian signs by 8 weeks and were maintained till 23 months old. The exercise regimen increased HR by 25% above baseline and gradually reduced pre-intervention MAP. Together, these studies indicate that a practicable frequency and intensity of exercise reduces parkinsonian sign severity commensurate with a modest increase in HR after exercise. These cardiovascular changes provide a baseline of metrics, easily measured in humans, for predictive validity that practicable exercise intensity and schedule can be initiated in previously sedentary older adults to delay the onset of aging-related parkinsonian signs.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 Chronic Intermittent Hypoxia Alters the Chloride Gradient in Median Preoptic Nucleus (MnPO) Neurons of Rats(2021) Farmer, George E.; Little, Joel; Bachelor, Martha E.; Rybalchenko, Nataliya; Cunningham, JosephRats exposed to chronic intermittent hypoxia (CIH), an animal model simulating the hypoxemia associated with obstructive sleep apnea, exhibit persistent elevations in blood pressure during normoxic periods. In MnPO neurons, angiotensin II type 1 receptor function mediates reductions in GABAa inhibition that become excitatory following CIH. Here, we use the ratiometric Cl- sensor, ClopHensorN, to monitor the chloride flux of MnPO neurons in normoxic (Norm) and CIH treated rats following GABAa activation. Using isoflurane anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions of AAV9-Cre in PVN and DIO-ClopHensorN in MnPO. After recovery, rats underwent 7 consecutive days of CIH (6 min cycles of 3 min 21% O2, 3 min 10% O2 repeated 10x/hr for 8 hours) or Normoxia. For ClopHensorN imaging, rats were anesthetized with isoflurane and coronal slices containing MnPO were cut using standard in vitro slice procedures. Images were captured every 3 sec. Cl- flux was determined from ratiometric responses to 10 s focal application of muscimol (100 uM). Twelve rats (6 Norm, 6 CIH) were used for ClopHensorN studies. In MnPO CIH neurons, 20.1% showed decreased fluorescent ratios while 0.3% showed increased ratios indicative of Cl- efflux. In MnPO Norm neurons, 41.9% showed a muscimol dependent decrease in fluorescent ratio with 0 cells showing an increase. The magnitude of muscimol dependent decreases in fluorescent ratios were reduced in CIH treated rats suggesting reduced GABAa inhibition. Results demonstrate CIH alters Cl- flux of PVN projecting MnPO. These changes may contribute to hypertension associated with CIH.Item Chronic Intermittent Hypoxia Alters the Chloride Gradient in Median Preoptic Nucleus (MnPO) Neurons of Rats(2020) Cunningham, J. Thomas; Little, Joel; Bachelor, Martha; Rybalchenko, Nataliya; Yuan, Joseph; Farmer, GeorgeRats exposed to chronic intermittent hypoxia (CIH), an animal model simulating hypoxemia associated with obstructive sleep apnea, exhibit persistent elevations in blood pressure during normoxic periods. In MnPO neurons, angiotensin II type 1 receptor function mediates reduced GABAa inhibition that becomes excitatory following CIH. Here, we use the ratiometric Cl- sensor, ClopHensorN, to monitor the chloride flux of MnPO neurons in normoxic (Norm) and CIH treated rats following GABAa activation. Using isoflurane anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions of AAV9-Cre in the PVN and DIO-ClopHensorN in the MnPO. After recovery, rats underwent 7 consecutive days of CIH (6 min cycles of 3 min 21% O2, 3 min 10% O2 repeated 10x/h for 8 hours) or Normoxia. For ClopHensorN imaging, rats were anesthetized with isoflurane and coronal slices containing the MnPO were cut using standard in vitro slice procedures. Images were captured every 3s. Cl- flux was determined from the ratiometric response to 10s focal application of muscimol (100 uM). Twelve rats (6 Norm, 6 CIH) were used for ClopHensorN studies. In MnPO CIH neurons, 20.1% showed decreased fluorescent ratios while 0.3% showed increased ratios indicative of Cl- efflux. In MnPO Norm neurons, 41.9% showed a muscimol dependent decrease in fluorescent ratio with 0 cells showing an increase. The magnitude of muscimol dependent decreases in fluorescent ratios were reduced in CIH treated rats suggesting reduced GABAa inhibition. Results demonstrate CIH alters Cl- flux in PVN projecting MnPO. These changes may contribute to hypertension associated with CIH.Item Chronic Intermittent Hypoxia Increases Oxidative Stress and Impairs Spatial Memory in Male and Female Rats(2023) Gardner, Jennifer J.; Mabry, Steve; Bradshaw, Jessica L.; Wilson, E. Nicole; Little, Joel; Goulopoulou, Styliani; Cunningham, Rebecca L.Obstructive sleep apnea (OSA) is characterized by complex phenotypes and increased long-term risk of neurodegenerative disease. The impact of OSA in women is unknown due to sex differences in clinical presentation contributing to underdiagnosis. Using chronic intermittent hypoxia (CIH) to model OSA in rodents, our previous studies have shown CIH exposure increases oxidative stress and inflammation in male rats. However, the impact of CIH in female rats remains unclear. The objective of this study was to assess sex differences in CIH-mediated oxidative stress and rodent behaviors associated with neurodegenerative disease. Young adult male and female Long Evans and Sprague Dawley rats were exposed to CIH or normoxia for 14-15 days. Spatial memory and fine and gross motor skills were assessed. Plasma oxidative stress was measured and neuronal expression in the dorsal hippocampus was quantified. Female rats exhibited better spatial memory than males with increased neuronal expression in the CA1 region of the hippocampus. In both males and females, CIH impaired spatial memory and increased circulating oxidative stress. Yet, CIH increased CA1 neuronal expression in female rats only. CIH did not impact gross or fine motor skills, regardless of sex. Our preliminary findings indicate CIH increases oxidative stress and impairs spatial memory in males and females, but the impact of CIH on hippocampal neurons and region-specific contributions to spatial memory may be sexually dimorphic.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 Effects of Amyloid β on Recollective Memory: Sex and Hormone Differences(2023) Vera, Edward; Mabry, Steve; Wilson, Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Little, Joel; Rybalchenko, Nataliya; Cunningham, Rebecca L.PURPOSE: Alzheimer’s disease (AD) is linked with increased memory loss and inability to learn new topics. One of the defining neuropathological features of AD is amyloid beta (Aβ) plaques in brain regions, such as the hippocampus. The hippocampus brain region is important for memory and learning. AD risk is elevated in individuals older than 65 years old, especially menopausal women. Menopause is an aging associated endocrine event in which the ovaries stop producing estradiol but continue producing testosterone. Testosterone can be aromatized to estradiol, but aromatase is not functional in women with AD. Therefore, post-menopausal women with AD have more androgens than estrogens than pre-menopausal women and aged men. Androgens can be neuroprotective or neurotoxic depending on the cellular environment. It is unknown what the impact of androgens and sex are on amyloid beta’s effects on the brain, (e.g., hippocampus) and behavior (e.g., memory). We hypothesize that females with the hormonal condition of androgens in the absence of estrogens will exhibit increased recollective memory in response to hippocampal injection of Aβ. METHODS: To investigate the role of androgens and sex on Aβ associated memory impairments, adult male and female Sprague-Dawley rats were gonadectomized to remove circulating sex hormones. A subset of these rats was given either cholesterol or dihydrotestosterone (DHT), which cannot be converted into estrogen. To model AD, rats were injected with 5ug/ul of Aβ oligomer fibrils 1-40 or vehicle shams in the CA1 region of the hippocampus. One week after Aβ hippocampal injections, the rats were assayed for short term and long-term recollective memory via a 1-hour and 24-hour Novel Object behavioral test. The Novel Objective behavioral tests examines recollective memory by quantifying the time spent with a novel object versus the time spent with a known object. Data was quantified with a three-way ANOVA with sex, hormone, and Aβ as independent variables. Tukey’s was used as a post-hoc test. RESULTS: Sex differences were observed between hormone-deficient rats exposed to Aβ. Specifically, males exhibited worse short term recollective memory (1 hour novel object) compared to females. DHT had no effect on recollective memory, regardless of Aβ exposure. No effects were observed in the long-term recollective memory (24-hour novelty test). CONCLUSIONS: Our results indicate that Aβ 's effects on short term recollective memory is influenced by sex chromosomes, as we observed sex differences in the hormone deficient (cholesterol) treated animals. However, DHT did not impact these recollective memory. These results indicate that recollective memory in AD is impacted by the sex chromosomes and not androgens.Item Enhanced Excitability of Vasopressin Neurons in the Supraoptic Nucleus Following 48 hr. Water Deprivation(2016-03-23) Little, Joel; Knapp, Blayne; Cunningham, Tom; Farmer, George Jr.Purpose Arginine vasopressin (AVP) is a neurohypophyseal hormone released from the posterior pituitary by magnocellular neurosecretory cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. AVP is involved in the regulation of body fluid homeostasis and influences blood pressure, plasma osmolality, and blood volume. The regulation of AVP release is critical to maintaining body fluid homeostasis and is dependent on the activity of MNCs. The balance of excitatory and inhibitory inputs are important elements of activity, however the mechanisms leading to changes in AVP release are not fully understood. Water deprivation (WD), a physiological challenge, was used to examine changes in excitatory neurotransmission in MNCs using patch-clamp electrophysiology. Methods Male Sprague-Dawley rats weighing 250 – 350 g received bilateral SON infusions of an adeno-associated virus (AAV) construct containing mouse AVP gene promoter and EGFP reporter. Two weeks following AAV infusions, rats were water deprived for 24 hr. or 48 hrs. Controls were allowed ad libitum access to water. Following water deprivation, coronal brain slices (300 µm) containing the SON were cut using standard procedures. Whole-cell patch clamp recordings were obtained from slices superfused with aCSF containing tetrodotoxin (TTX; 0.5 µM) and bicuculine methbromide (Bic; 10 µM). Baseline mEPSCs were recorded then NMDAR mediated components were pharmacologically isolated with 5 minutes drug application of AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10uM). Parameters measured for mEPSCs were amplitude (pA), rise time (ms), decay time (ms), charge transfer, and mEPSC frequency. Results In EGFP labeled SON neurons (i.e. confirmed vasopressinergic neurons), water deprivation increased the frequency of mEPSCs in both the 24 hr. group (p Conclusion Although no changes in amplitude, rise time, decay time, or charge transfer were detected, there was an increase in the frequency of mEPSCs following both 24 hr. and 48 hr. WD. It remains unclear how water deprivation leads to enhanced mEPSC frequency in MNC of the SON. Future studies will investigate the mechanisms that underlie this water deprivation dependent plasticity.Item High Salt Loading Increases Brain Derived Neurotrophic Factor in Supraoptic Vasopressin Neurons(2018-03-14) Little, Joel; Cunningham, J. Thomas; Balapattabi, KirthikaaPurpose: Salt loading (SL) upregulates Brain Derived Neurotrophic Factor (BDNF) and causes increased arginine vasopressin (AVP) release from supraoptic nucleus of the hypothalamus (SON). BDNF diminishes or reverses the GABAA inhibition in the SON AVP neurons by increasing intracellular chloride ([Cl]i) through tyrosine receptor kinase B (TrKB) phosphorylation. This creates a feed forward loop that drives AVP release. However, the source of BDNF is not known. Hypothesis: We hypothesize that SON is the source of BDNF contributing to increased AVP release in SL rats. Methods: Adult male Sprague Dawley rats were anesthetized with isoflurane (2-3%) and bilaterally injected in the SON (300 nl/side) with an AAV2 vector with a U6 promoter containing either shRNA against BDNF or a control construct with an mCherry reporter. The vectors were injected at a titer of 1.0 X 1013 GC/ml (Vector Biolabs). Two weeks after the stereotaxic injections, the rats were provided with either water or 2%NaCl to drink for 7 days. At the end of the protocol, rats were anesthetized with inactin (100 mg/kg IP) and brains were collected and flash frozen. Fresh frozen brains were prepared for Laser Capture Microdissection (LCM) by cutting 10μm thick coronal sections through the hypothalamus at the level of the SON. Using LCM, we verified the accuracy of the injections by visualizing the mCherry reporter and collected the SON to measure changes in the BDNF mRNA and AVP hnRNA expression using quantitative Real Time PCR. Subset of brains from each group were used for Western blot analysis of punch samples containing the SON. Rats that did not have successful virus injections in the SON were separately analyzed. Plasma osmolality, hematocrit, and AVP concentration were measured. Data were analyzed by one-way ANOVA with Bonferroni comparisons. Results:SL was associated with significant increases in BDNF mRNA and AVP hnRNA in SON (P Conclusion: The results indicate that BDNF produced in the SON contributes to increased AVP secretion during SL. Supported by R01 HL119458Item Homer Mediates Vascular Store-Operated CA2+ Entry and is Required for Neointima Formation after Vascular Injury(2016-03-23) Jia, Shuping; Wu, Qiong; Williams, Arthur Jr.; Little, Joel; Cunningham, Joseph; Mifflin, Steve; Ma, Rong; Yuan, Joseph; Rodriguez, MiguelOcclusive arterial disease (OAD) refers to the pathological obstruction of arteries that become progressively narrowed over time and are eventually blocked due to various risk factors, such as hypertension, diabetes, and atherosclerosis. This chronic arterial damage results from vascular wall remodeling, leading to neointima formation. Store-operated Ca2+ channels (SOCs) and entry (SOCE) play a central role in the vascular smooth muscle cell (VSMC) phenotypic change from contractile to migratory and proliferative states. In the present work, we ask if Homer is a critical molecular component of VSMC SOCE and does Homer mediate VSMC migration/proliferation and neointima formation. Homer binds to transient receptor potential canonical (TRPC) channels and is required for gating of TRPCs, while stromal interacting molecule1 (STIM1) binds to and regulates TRPC and Orai channels as SOCs. We cultured rat aortic VSMCs to increase their SOCE and migration/proliferation, as seen in OAD. Studies were done using small-interfering RNA (siRNA) targeting Homer1, STIM1, and TRPCs. Scratch wound migration assays were performed, and VSMC proliferation was assessed by cell count. In our in vivo OAD model (rat carotid artery balloon injury), the arteries were treated with adeno-associated virus (AAV) encoding short-hairpin RNA (shRNA) targeting Homer1. We found that Homer1 protein expression levels increase in balloon-injured vs. intact VSMCs, similar to known increases in protein expression levels of STIM1, Orai1, and TRPCs. Furthermore, we show that Homer1 binds to Orai1 and that interactions between Homer1 & Orai1/TRPCs and between STIM1 & Orai1/TRPCs markedly increase in injured vs. intact VSMCs. Cultured VSMCs treated with siHomer1 exhibit significant reduction in SOCE (56 ± 4.0%) vs. control (scrambled siRNA), similar to the SOCE reduction seen in siSTIM1-/siTRPC-treated cells. SiHomer1-treated cells also migrate significantly less over the wound surface area (73.3 ± 5.9%), and proliferate significantly less (73.3 ± 4.2%) vs. control, similar to observations seen in siSTIM1-/siTRPC-treated cells. Finally, immunofluorescence staining shows that the increased Homer1, STIM1, and Orai1 protein expression levels are localized in the neointima of the injured carotid artery. Knockdown of Homer1 using AAV-shHomer1 reduces this neointima. These studies provide evidence that Homer is a critical component of VSMC SOCE and neointima formation.Item Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading(2019-03-05) Farmer, George; Little, Joel; Bachelor, Martha; Cunningham, J. Thomas; Balapattabi, KirthikaaPurpose Salt loading (SL) increases intracellular chloride concentration [Cl]i, impairing GABAA inhibition of arginine vasopressin (AVP) neurons in the supraoptic nucleus (SON) of hypothalamus. But the regulatory mechanisms leading to increased [Cl]i is not completely understood. Based on previous studies, we hypothesize that SL activates tyrosine receptor kinase B (TrkB) and downregulates K+/Cl- co-transporter 2 (KCC2) membrane expression. Downregulation of KCC2 decreases the efflux of chloride, Cl ion causing increase in [Cl]i in SON AVP neurons. In this study, we combined virally mediated ClopHensorN, a relatively new ratiometric Cl imaging technique with capillary based Simple Wes to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons. Methods Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2-0VP1-ClopHensorN. The ClopHensorN (Addgene Plasmid #50758) was packaged in an AAV2 vector with an AVP promotor (Addgene Plasmid #40868). After 2 weeks, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin and their SONs were dissociated. The cells were plated on coverslips and placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. ClopHensorN positive neurons were tested for decrease or increase in [Cl]i to focal application of GABAA agonist muscimol (100uM). After imaging, individual neurons were collected by aspirating into a patch pipette to verify KCC2 and ß-Actin protein expression. Protein Simple Wes (12-230kDa matrix) was used to identify and quantitate very low concentration of protein from single neuron. Data were analyzed by Chi-squared test and one-way ANOVA with Bonferroni comparisons. Results Muscimol application to SL SONs either significantly increased Cl efflux (p Conclusion Salt loading increases [Cl]i in SON AVP neurons through TrKB-KCC2 mechanism.Item Norepinephrine innervation of the supraoptic nucleus contributes to dilutional hyponatremia in male BDL rats(2022) Aikins, Ato; Little, Joel; Rybalchenko, Nataliya; Cunningham, JosephPurpose: Dilutional hyponatremia is a common complication associated with liver cirrhosis that is linked to inappropriate release of arginine vasopressin (AVP). Elevated plasma AVP causes water retention and hypoosmolality. In the cirrhotic liver, there is increase in resistance to blood flow resulting in portal hypertension, ascites formation, mesenteric vasodilation due to release of vasodilators and increased pooling of blood in the splanchnic circulation. The fluid redistribution decreases central vascular blood volume which is sensed by peripheral baroreceptors and volume receptors located in the aortic arch and heart. This could be relayed to A1 neurons in the caudal ventrolateral medulla (CVLM) and the A2 neurons in the nucleus tractus solitarius (NTS). The A1/A2 neurons stimulate the release of AVP from the supraoptic nucleus (SON). We propose that the A1 and A2 norepinephrine neurons in the hindbrain contribute to the activation of AVP-secreting neurons in the supraoptic nucleus (SON) leading to inappropriate AVP release and dilutional hyponatremia. Method: Anti-DBH saporin [IT-03] (Advanced Targeting Systems), a cytotoxin conjugated to an antibody against DBH was injected to the SON to lesion the norepinephrine innervation of SON including A1/A2 neurons. After two weeks, adult male rats received bile duct ligation surgery (BDL) which was used to model liver cirrhosis. In this model, the common duct that drains bile from the liver to the intestine is cauterized between two ligatures leading to obstructive cholestasis and liver cirrhosis. Four weeks after BDL surgery, rats were anesthetized with inactin (thiobutabarbital sodium salt hydrate; 100 mg/kg, i.p.). Blood samples were taken for plasma copeptin, osmolality, and hematocrit measurements. The rats were then perfused transcardially with 1M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (4% PFA) in 1M PBS. Plasma copeptin concentration was measured as a surrogate marker for AVP using commercially available copeptin ELISA kits. The brains were removed and processed for delta FosB (a marker of chronic activation), dopamine β-hydroxylase (DBH) and AVP immunohistochemistry. Results: Anti-DBH saporin (ADS) injection in the SON of BDL rats significantly decreased the number of cells positive for both delta FosB and DBH in the A1 and A2 cell groups as compared to vehicle injection (A1 neurons, ADS/BDL vs Vehicle/BDL P< 0.001; A2 neurons, ADS/BDL vs Vehicle/BDL P< 0.05). ADS treated BDL rats had fewer SON cells positive for both AVP and delta FosB as compared to vehicle injection (ADS/BDL vs Vehicle/BDL P< 0.05). Reduced colocalization of delta FosB and DBH in A1/A2 neurons was associated with a significantly lower plasma copeptin concentration in BDL rats. (ADS/BDL vs Vehicle/BDL P< 0.05). Similar effects were seen for plasma osmolality and hematocrit (ADS/BDL vs Vehicle/BDL P< 0.05). Conclusion: The result suggests that an increase in cells positive for both delta FosB and DBH in A1/A2 neurons is associated with an increase in plasma AVP and hypoosmolality in male BDL rats. Anti-DBH saporin lesions of SON prevented increases in plasma copeptin and neural activation of A1/A2 and AVP SON neurons associated with BDL.Item Role of A1/A2 Neurons in Increased Vasopressin Release in Male Bile Duct Ligated Rats(2020) Little, Joel; Cunningham, J. Thomas; Aikins, AtoBile duct ligation (BDL), a rat model of liver failure causes increased vasopressin release leading to water retention and a decrease in plasma osmolality. Water retained in ascites leads to a perceived decrease in plasma volume by the A1/A2 norepinephrine neurons in the caudal ventrolateral medulla (CVLM) and the nucleus tractus solitarius (NTS) respectively, increasing vasopressin release. We hypothesized that lesioning norepinephrine A1/A2 neurons will prevent increased plasma vasopressin (measured as a function of plasma copeptin concentrations), normalize hematocrit and plasma osmolality, and decrease the number of immunoreactive ΔFosB and DβH cells in the NTS and CVLM. To investigate the role of A1/A2 neurons in the increase in vasopressin release observed in male BDL rats, anti-DβH-Saporin [IT-03] (Advanced Targeting Systems), or vehicle was injected into the SON. BDL surgery or sham surgery was performed after two weeks. Four weeks following the BDL/Sham surgery, the rats were sacrificed, and blood samples were taken for copeptin, hematocrit and plasma osmolality measurements. Hindbrains were processed for immunohistochemistry. The results showed a significant decrease in CVLM ΔFosB reactive cells in the Saporin/BDL group (n=2) as compared to the Vehicle/BDL group (n=3) (p< 0.001). DβH immunoreactive cells in both the CVLM and NTS were significantly decreased in the Saporin/BDL group as compared to the vehicle/BDL group (p< 0.001). However, differences were not observed for the copeptin concentration, hematocrit and plasma osmolality. The results suggest that lesions of SON-projecting A1/A2 neurons may not be sufficient to prevent vasopressin release associated with BDL.Item Role of A1/A2 Neurons in the Dysregulation of Vasopressin Release and Dilutional Hyponatremia in Liver Disease(2021) Aikins, Ato; Little, Joel; Cunningham, JosephAbstract Purpose: Inappropriate release of arginine vasopressin (AVP) has been linked to dilutional hyponatremia in patients with cirrhosis. Elevated Plasma AVP causes water retention, hypoosmolality, ascites formation, and a perceived decrease in plasma volume. The perceived decrease in plasma volume is sensed by the A1/A2 norepinephrine neurons in the caudal ventrolateral medulla (CVLM) and the nucleus tractus solitarius (NTS) respectively. We propose that these neurons provide the initial stimuli that activates AVP-secreting neurons in the supraoptic nucleus (SON) leading to inappropriate AVP release and dilutional hyponatremia. Method: Adult male rats were bile duct ligated (BDL) to model cirrhosis. Selective lesioning of the SON-projecting A1/A2 norepinephrine neurons was achieved using anti-DβH-Saporin [IT-03] (Advanced Targeting Systems). Plasma copeptin concentration was measured as a surrogate marker for AVP using ELISA. Plasma osmolality and hematocrit measurements were also taken. Immunohistochemistry for delta FosB and dopamine beta-hydroxylase (DβH) was performed on brain slices. Results: Lesions of the A1/A2 neurons projecting to SON (Saporin/BDL n=9) was associated with decreased copeptin as compared to BDL controls (Vehicle/BDL, n=6, p< 0.05). However, the number of delta FosB immunoreactive A1/A2 cells was not significantly different. While A1/A2 lesions seemed to normalize osmolality and hematocrit in the BDL rats, the trends were not statistically significant. Conclusion: The result suggests that A1/A2 neurons could contribute to increased plasma AVP seen in male BDL rats, but there could be other contributing factors preventing a recovery of plasma osmolality.Item Role of Brain Derived Neurotrophic Factor in the Supraoptic nucleus on Response to Salt Loading(2017-03-14) Little, Joel; Bachelor, Martha; Cunningham, J. Thomas; Balapattabi, KirthikaaPurpose: Previous studies have shown that Brain Derived Neurotrophic Factor (BDNF) contributes to ionic plasticity of vasopressin neurons from the supraoptic nucleus of the hypothalamus (SON) in response to 7 days of salt loading. This ionic plasticity is mediated by BDNF dependent phosphorylation of TrkB receptors and downregulation of KCC2, which alter chloride homeostasis resulting in impairment of baroreceptor inhibition of vasopressin release and an increase in mean arterial pressure that is partially vasopressin dependent. However, the specific source of BDNF is yet to be elucidated. In this study, we used adeno-associated viral vectors with shRNA to test the hypothesis that the salt loading produces BDNF release from the SON which in turn contributes to changes in chloride homeostasis and increased blood pressure. Methods: Adult male Sprague Dawley rats (250-300 g b w) were anesthetized with isoflurane and bilaterally injected in the SON (300 nl) with either an AAV vector containing shRNA against BDNF or a control construct with an mCherry reporter. The vectors were injected at a titer of 1.0 X 1013 GC/ml (Vector Biolabs, Malvern, PA). Two weeks after the stereotaxic injections, some rats from each group were instrumented with radio telemetry transmitters using isoflurane anesthesia for recording mean arterial pressure and heart rate. Rats were then housed in commercial metabolism cages and given water or 2% NaCl to drink for 7 days. Volume of fluid intake, urine excretion, food intake, and body weight were measured during the study along with radio telemetry recording for heart rate, mean arterial pressure and activity. Brain regions were harvested for measuring gene expression and protein content at the end of the 7 days. Seven days of 2% salt loading increased TrKB phosphorylation in the SON of rats injected with the control vector. Results: Virally mediated BDNF knockdown in the SON of salt loaded rats decreased TrkB phosphorylation. However, the increases in blood pressure produced by salt loading were not different between these two groups. Both salt loaded groups also had comparable decreases in body weight and increases in fluid intake. Conclusions: The results suggest that, while locally produced BDNF contributes to TrKB phosphorylation in the SON during salt loading, it is not necessary for the increase in blood pressure.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 Role of Estrogen Receptors in a Model of Dilutional Hyponatremia(2020) Balapattabi, Kirthikaa; Little, Joel; Cunningham, J. Thomas; Nguyen, John-Bosco; Brock, Courtney; Nguyen, DiannaPurpose: Hyponatremia is the most frequently occurring electrolyte disorder and independent risk factor for increased patient mortality. Dilutional hyponatremia in liver failure due to inappropriate arginine vasopressin (AVP) release can be studied using rodent bile-duct ligation (BDL) model. Our previous sex differences studies in BDL rats show compared to males, female and ovariectomized (OVX) BDL rats did not develop hyponatremia, AVP neuron activation, or increased plasma copeptin (CPP; a marker for AVP), compared to sham ligated females. Due to increased adrenal and circulating estradiol (E2) in OVX BDL rats, the role of E2 was unclear. Intracerebroventricular infusion of estrogen receptor (ER) antagonist, ICI 182,780 (ICI) in female BDL rats increased CPP concentration compared to controls. These data suggest ER involvement in prevention of hyponatremia in female BDL rats. However, ICI is also a G protein-coupled estrogen receptor 1 (GPER) agonist. We tested GPER expression within hypothalamo-neurohypophyseal system of female rats. Methods: Immunohistochemistry was performed on three separate sets of forebrain sections from adult female Sprague-Dawley rats. All sets processed for GPER, and the separate sets stained for either AVP, oxytocin (OXY), or glia fibrillary acidic protein (GFAP). Results: Co-localization of GPER+AVP and GPER+OXY was observed in neurohypophyseal neurons (GPER+AVP, 64.8% and GPER+OXY, 64.0%). GPER+GFAP co-localization was not observed. Conclusion: GPER is expressed on subset of AVP and OXY neurons and not astrocytes in hypothalamo-neurohypophyseal system of female rats. Future studies in BDL rats will provide further insight about sex differences in neurohypophyseal function.