Browsing by Author "Farmer, George"
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Item AT1R sniffer cells detects spontaneous and evoke release of AngII in the AP-NTS pathway(2021) Gusson Shimoura Almeida Lima, Caroline; Paundralingga, Obed; Farmer, George; Cunningham, JosephAlthough angiontesin II (AngII) has multiple actions in the brain, the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from subfornical organ projections to the median preoptic nucleus. In these studies, we examined another pathway involving the area postrema (AP) and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to the NTS, so the purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin 1a receptor (Origene Tech.) and R-GECO (Addgene #32462). These sniffer cells are sensitive to AngII and III but not angiotensin 1-7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. Sniffer cells were placed on coronal brainstem slices containing both AP and NTS from adult male Sprague – Dawley rats. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.Item AT1R sniffer cells detects spontaneous and evoke release of AngII in the AP-NTS pathway(2020) Paundralingga, Obed; Farmer, George; Cunningham, J. Thomas; Gusson Shimoura Almeida Lima, CarolineAlthough angiontesin II (AngII) has multiple actions in the brain, the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from subfornical organ projections to the median preoptic nucleus. In these studies, we examined another pathway involving the area postrema (AP) and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to the NTS, so the purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin 1a receptor (Origene Tech.) and R-GECO (Addgene #32462). These sniffer cells are sensitive to AngII and III but not angiotensin 1-7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. Sniffer cells were placed on coronal brainstem slices containing both AP and NTS from adult male Sprague and Dawley rats. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.Item Characterization of arterial pressure and carotid blood flow responses to pulsatile perfusion therapy in a rat model of hemorrhage(2022) Bhuiyan, Nasrul; Farmer, George; Anderson, Garen K.; Davis, Kenneth; Cunningham, Joseph; Rickards, CarolineIntroduction: In a human model of simulated blood loss, oscillatory patterns of arterial pressure and blood flow, or "pulsatile perfusion", can protect cerebral and peripheral tissue oxygenation, and prolong tolerance to this stress. In this pilot study, we investigate whether pulsatile perfusion therapy can protect arterial pressure and cerebral blood flow in a rat model of actual blood loss. We hypothesized that pulsatile perfusion therapy (PPT), applied via repeated thigh cuff inflations, would attenuate the reduction in arterial pressure and cerebral blood flow following hemorrhage. Methods: Sprague Dawley rats underwent the following protocols: hemorrhage alone (control: N=4; 2 male, 2 female), or hemorrhage plus PPT (N=3; 1 male, 2 female). PPT was applied via rapid 1 s inflations and deflations of a thigh cuff (0.5 Hz). A catheter was inserted in the femoral artery for continuous measurement of arterial pressure, and a perivascular flow probe was placed around the common carotid artery (CCA) for measurement of blood flow. Following instrumentation, each animal completed a baseline period (15 min), followed by a ~55% hemorrhage (25 min), PPT or control (30 min), and a recovery period (155 min or until death). Results: Decreases in mean arterial pressure (MAP) and CCA blood flow were observed in response to hemorrhage (P≤0.002). At the end of the PPT period, no differences were observed between the PPT and control groups for MAP (PPT: 46.7±27.3 mmHg vs. control: 30.2±13.5 mmHg; P=0.44) or CCA peak blood flow (PPT: 2.7±1.5 ml/min vs. control: 1.9±1.3 ml/min; P=0.92). Similarly, no differences were observed in the relative change from baseline to the end of the PPT period for MAP (PPT: -45±38% vs. control: -55±14%; P=0.65) or CCA peak blood flow (PPT: -65±21% vs. control: -66±12%; P=0.70). Conclusion: These results suggest that following a 55% hemorrhage in rats, PPT did not protect arterial pressure or carotid blood flow. However, the sample size was low in this pilot study, resulting in high variability in the observed responses. Accordingly, additional experiments are needed with an increased sample size to accurately determine the potential beneficial effects of PPT following hemorrhage.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 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 Hemodynamic Responses to Oscillatory Thigh Cuff Inflations(2023-05) McIntyre, Benjamin J.; Rickards, Caroline A.; Tune, Johnathan D.; Farmer, GeorgeExperimental generation of 0.1 Hz oscillations (~10-s cycle) in arterial pressure and cerebral blood flow (CBF) increases tolerance to simulated hemorrhage, and protects cerebral tissue oxygenation. In this study we evaluated a clinically applicable method of inducing 0.1 Hz oscillations in arterial pressure and CBF via repeated thigh cuff inflations. We also characterized the effect of common carotid artery (CCA) stiffness on the magnitude of cerebral blood flow oscillations, and evaluated the effects of intermittent thigh cuff inflation on several markers of cardiac function. We hypothesized that: 1) the amplitude of arterial pressure and CBF oscillations at 0.1 Hz would increase in response to repeated thigh cuff inflations at 0.1 Hz, 2) the magnitude of 0.1 Hz CBF oscillations would be positively correlated to the stiffness of the CCA, and 3) measurements of cardiac function would increase in response to thigh cuff induced oscillations of arterial pressure at 0.1 Hz. Thirteen healthy human participants were tested (6 male, 7 female; 27.1 ± 4.3 y). In response to 10-min of intermittent thigh cuff inflations at 0.1 Hz, the amplitude of 0.1 Hz oscillations increased for mean arterial pressure (MAP; 24.4 ± 20.1 mmHg2 vs. 932.0 ± 758.1 mmHg2; P<0.01) and middle cerebral artery velocity (MCAv; 17.5 ± 13.8 (cm/s)2 vs. 325.5 ± 279.9 (cm/s)2; P<0.01). There was also a large increase in MAP-MCAv coherence at 0.1 Hz (0.60 ± 0.24 a.u. vs. 0.90 ± 0.11 a.u.; P<0.01) during the oscillatory period compared to baseline. There was a moderate positive relationship between CCA stiffness and amplitude of MCAv power at 0.1 Hz during intermittent thigh cuff inflations (r=0.68, P=0.01), but not at rest (r=-0.08, P=0.80). When compared to baseline, no changes were observed during the oscillatory period for heart rate (P=0.47), stroke volume (P=0.87), cardiac output (P=0.55), MAP (P=0.20), or dP/dTmax (P=0.61). Future studies directly examining sympathetic nerve activity are needed to better elucidate the effects of induced 0.1 Hz hemodynamic oscillations on neural regulation of the cardiovascular system. In conclusion, we have shown that intermittent thigh cuff inflations can be used to increase hemodynamic variability at a target frequency, and therefore could be a therapy for treating tissue hypoperfusion following severe blood loss injuries.Item Impact of Endoscopic Vacuum-Assisted Closure on Quality of Life in Patients After Treatment of Gastrointestinal Leaks(2023-05) Rana, Rashmeen K.; Malaer, Joseph D.; Preskitt, John T.; Farmer, GeorgeEndoscopic Vacuum Assisted Wound Closure (EVAC) is an effective therapeutic option to treat Gastrointestinal (GI) leaks after the surgery. Prior to EVAC, conventional approaches to treat leaks included surgical intervention or endoscopic stents. Even though EVAC has been in use for more than a decade and has proven to be successful in treating GI leaks, the long-term quality of life impact of this treatment is uncertain. With the use of a short form (SF-36) survey, a validated questionnaire to assess both physical and mental health, the long-term impact of EVAC on the quality of life was evaluated. When assessing the long-term quality of life for patients who are at least 2 years out from their sentinel surgery, the EVAC group scored higher in all 8 quality of life domains with 4 domains reaching statistical significance as compared with conventional therapy group which received other treatments for leak management.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 A Modified Kidney Perfusion System for Improved Data Acquisition and Assessment of Renal Function and Metabolism(2023) Wade, Michael; Farmer, George; Yurvati, Albert; Mallet, Robert T.PURPOSE In the United States, there are over 88,000 candidates on the waitlist for kidney donation as of December 2022 with less than 42,0000 transplant procedures being recorded in the same year. Coinciding with this continued disparity has been a growing interest in the technology to preserve and improve the function of kidneys for transplantation, most notably machine perfusion (MP). Studies have shown that MP is superior to static cold storage, the most common method of kidney preservation used globally, which has allowed transplant programs to expand the current donor pool by use of marginal donor kidneys. Despite these advancements, machine perfusion technology used clinically is limited in its use for obtaining critical information regarding the real-time assessment of renal function, fluid dynamics, and metabolism. Here we discuss the development of a modified kidney perfusion system that allows for enhanced data acquisition of perfusion parameters, renal arterial and venous sampling, and organ monitoring within a controlled environment. METHODS The modified kidney perfusion system was assembled to house and monitor a singular organ. The organ is placed in a chamber that allows for maximal cooling to 2˚C - 6˚C or temperature regulation up to room temperature. Renal artery and vein cannulation allow for accurate perfusate sampling in different parts of the system without disruption of organ perfusion. Continuous monitoring of pressure, flow, and resistance is performed using pressure and flow transducers in line with the polyethylene tubing. In line with the system includes an air trap to prevent gas emboli, modifiable filter, and screen for large particulate matter. A specialized rotor allows for pulsatile perfusion into the kidney while a servomotor is used to set a desired pressure or flow into the system. Values for flow, pressure, resistance are recorded in real time using Spike2 software and the corresponding waveforms displayed in the program. RESULTS & CONCLUSION The limitations of machine perfusion technology currently used clinically hinder the ability to obtain data for interpretation of pre-transplant renal function. This modified kidney perfusion system improves upon many of these limitations and provides entirely new methods to gain insight on kidney function. Assessment of renal metabolite production and injury markers can be obtained from multiple sites throughout the perfusion circuit and instantaneous recording of both pressure and flow waveforms throughout the duration of machine perfusion.Item Pulsatile Perfusion Therapy at 0.1 Hz Improves Survival Following Severe Hemorrhage in Rats(2024-03-21) Dinh, Viet; Farmer, George; Rickards, CarolinePURPOSE: Oscillations in arterial pressure and blood flow at 0.1 Hz are associated with protection of tissue oxygenation during conditions of reduced tissue perfusion. Pulsatile Perfusion Therapy (PPT) is a method we have developed to induce these oscillations, and has been associated with increased tolerance to simulated hemorrhage via lower body negative pressure in humans. However, it is unknown how effective this therapy would be in an actual hemorrhage model. The aim for this study was to test the efficacy of PPT in a rat model of severe hemorrhage. We hypothesized that PPT at 0.1 Hz would protect arterial pressure and cerebral blood flow following severe hemorrhage in rats, subsequently improving survival. METHODS: Eleven adult Sprague-Dawley rats (six female, five male) were anesthetized via isoflurane, then underwent bilateral carotid artery catheterization for assessment of arterial pressure and carotid artery blood flow, while heart rate was assessed via lead II ECG. Following a 15-min baseline period, all animals were hemorrhaged to 50% of their estimated blood volume over 30-min. Rats were then randomly assigned to the PPT group (3 female, 3 male) or the control group (CON; 3 female, 2 male). PPT was administered via inflatable cuffs attached to both hind limbs, oscillating between 0 mmHg and 250 mmHg every 5-s (10-s cycles or 0.1 Hz) for 30-min immediately following hemorrhage. For the CON group, the leg cuffs were also attached but were not inflated for this 30-min period. All animals were then monitored for an additional 150-min recovery period post-hemorrhage, or until death – defined as the absence of ventricular function on the ECG. Survival time and peak mean arterial pressure (MAP), carotid blood flow, and heart rate were assessed to determine the effectiveness of PPT in protecting hemodynamic responses following hemorrhage. RESULTS: PPT increased survival time (P = 0.02), with 3 of 6 (50%) rats in the PPT group and 0 of 5 (0%) rats in the CON group surviving the entire 180-min recovery period following hemorrhage. During recovery, PPT protected MAP (PPT: −46.5 ± 12.9% vs. CON: −72.6 ± 19.5% from baseline; P = 0.07) and carotid blood flow (PPT: −61.5 ± 17.7% vs. CON: −83.7 ± 6.7% from baseline; P = 0.04), but did not affect heart rate (PPT: 0.11 ± 6.58% vs. CON: −18.70 ± 29.34% from baseline, P = 0.20), although responses were highly variable between subjects. CONCLUSIONS: PPT protected arterial pressure and carotid blood flow in rats following severe hemorrhage, subsequently improving survival. These data add further evidence for the use of 0.1 Hz hemodynamic oscillations as a therapeutic intervention for the treatment of hemorrhage.Item Sex Specific Effects of Salt Loading on GABAA Responses in Oxytocin Neurons from the Supraoptic Nucleus(2022) Brock, Courtney; Bachelor, Martha E.; Little, Joel; Farmer, George; Balapattabi, Kirthikaa; Cunningham, JosephArginine Vasopressin (AVP) and oxytocin (OXY) contribute to body fluid balance homeostasis. Salt loading (2% NaCl for 7 days) increases both AVP and OXY release in rats. The chronic increase in AVP release is associated with a change in the sensitivity of AVP neurons in the supraoptic nucleus (SON) to GABA so that GABAA receptor activation becomes excitatory. It is not clear if a similar mechanism is associated with chronic OXY release in this model. Our hypothesis is that changes in chloride homeostasis associated with salt loading occur in OXY neurons. To test this hypothesis, we used a chloride imaging approach with a ratio metric chloride sensitive dye, ClopHensorN (Addgene #50758) combine with an AAV with an oxytocin specific promoter (pFBOT563, Addgene # 40864). Adult, intact, Sprague Dawley rats of both sexes were anesthetized with isoflurane (2-3%) and were bilaterally injected with the AAV2-pFBOT--ClophensorN virus directly into the SON. Rats of both sexes were salt loaded by providing them with only 2% NaCl to drink for 7 days. Later, the animals were sacrificed and the brains were rapidly removed. The SON was dissected away from the brain and the cells were dissociated, plated on cover slips, and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging on an inverted microscope. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells and then allowed to wash off. Background fluorescence was subtracted. In cells from males, muscimol resulted in chloride influx in 70% OXY of the cells tested while chloride influx was observed in all OXY cells from females. The results suggest that salt loading may influence GABA responses of OXY neurons in males but not females.Item The Effect of Sex on GABAA Receptor Activation in Vasopressin Neurons from the Supraoptic Nucleus(2021) Brock, Courtney; Bachelor, Martha E.; Little, Joel; Farmer, George; Balapattabi, Kirthikaa; Cunningham, JosephPurpose: Arginine Vasopressin (AVP) is important in maintaining proper fluid balance and plasma osmolality. Disruption in its regulation occurs in patients with chronic heart failure (CHF) and liver failure, which leads to poorer patient outcomes. AVP neurons from the supraoptic nucleus (SON) receive input from GABA, yet it is unknown what effects GABAA receptor activation has on these neurons under pathophysiological conditions or whether the effects are sex-specific. What is known is that under pathophysiological conditions, AVP neurons are unaffected by negative feedback which leads to excessive AVP release. Understanding the role of the GABAA receptor in these conditions is important. Here, we investigate whether activation of the GABAA receptor leads to sex dependent effects. Methods: Adult, intact, Sprague Dawley rats were anesthetized and bilaterally injected with the AAV2-0VP1-ClophensorN virus directly into the SON. After a two-week recovery, the animals were sacrificed and the brains were rapidly removed. Cells from the SON were dissociated and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells. Results: In both males and females, application of muscimol resulted in chloride influx, which implies neuronal inhibition. Conclusion: Under normal physiological conditions, GABAA receptor activation does not show sex specific effects in neurons from the SON.Item The Effect of Sex on GABAA Receptor Activation on Vasopressin Neurons from the Supraoptic Nucleus(2020) Little, Joel; Cunningham, J. Thomas; Farmer, George; Bachelor, Martha; Balapattabi, Kirthikaa; Brock, CourtneyArginine Vasopressin (AVP), a hormone produced by the magnocellular neurosecretory cells (MNC) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus regulates fluid balance and plasma osmolality in healthy individuals. During congestive heart failure (CHF) and liver failure, however, AVP becomes dysregulated leading to increased renal water reabsorption and consequently persistent hyponatremia. Previous work done in the laboratory suggests that female rats may be protected from such dysregulation, perhaps by hormones such as estrogen. Our hypothesis is that under normal physiological conditions, activation of the GABAA receptor inhibits vasopressin neurons within the SON by causing chloride influx, but that under pathophyisiological conditions, activation of the GABAA receptor induces chloride efflux thereby causing activation of the vasopressin neurons in males. It is not known if AVP neurons from female rats behave in a similar manner to AVP neurons from males or are influenced by the estrous cycle. To test our hypothesis, the SON of adult, intact Sprague Dawley rats of both sexes were bilaterally injected with the ClophensorN virus which is a genetically modified virus used to detect Chloride flux in vasopressin neurons using fluorescence. Dissociated neurons from the SON were tested to assess chloride flux in response to the GABAA receptor agonist muscimol. Results show that under normal physiological conditions, muscimol induces Chloride influx in both males and females. Future experiments will assess the effect of GABAA activation in pathophysiological conditions by using the bile-duct ligation model of liver failure.