Chronic intermittent hypoxia (CIH), a hallmark of obstructive sleep apnea, produces a sustained sympathetically mediated increase in blood pressure. The median preoptic nucleus (MnPO) contributes to CIH-induced sympathoexcitation via projections to the paraventricular nucleus of the hypothalamus (PVN). However, the synaptic mechanisms underlying CIH-induced increased activity in this pathway remain to be determined. In addition, the impact of sex on MnPO synaptic changes in CIH exposure is unknown. We hypothesized that CIH affects MnPO synaptic activity differently between sexes and that brain-derived neurotrophic factor-tropomyosin receptor kinase B (BDNF-TrkB) signaling plays a role in CIH-mediated changes in the MnPO-PVN pathway. We performed whole cell voltage-clamp recordings from MnPO neurons in brain slices from male and female rats exposed to either normoxia or 7 days of CIH. In PVN-projecting MnPO neurons, CIH increased mEPSC frequency only in males, while females showed a reduction in mEPSC amplitude to CIH. CIH also increased BDNF expression in the MnPO of males exposed to CIH but not in females. Pharmacological studies in male rats using K252a, K252b, and ANA-12 attenuated the CIH-induced increase in mEPSC frequency, indicating that BDNF-TrkB signaling is essential for CIH. These findings highlight a sex-specific, TrkB-dependent mechanism that enhances excitatory signaling between the MnPO and PVN. In conclusion, our study shows that PVN-projecting MnPO neurons demonstrate sex-based changes in mEPSCs and that BDNF-TrkB signaling contributes to the CIH-induced increase in mEPSC frequency in neurons from males.NEW & NOTEWORTHY Chronic intermittent hypoxia (CIH) mimics arterial hypoxemia associated with sleep apnea. These studies demonstrate that CIH produces sex-dependent changes in mEPSC properties in paraventricular nucleus (PVN) projecting median preoptic nucleus (MnPO) neurons. In neurons from male rats, BDNF contributes to CIH-induced changes in mEPSC frequency. As the PVN directly innervates the sympathetic system, this could be a possible mechanism contributing to sustained sympathoexcitation in CIH.

Women affected by obstructive sleep apnea (OSA) face an increased risk of cognitive impairment and mood disorders, with emerging evidence suggesting that neuroinflammation plays a significant role in the pathophysiology. OSA causes intermittent hypoxia and activates the immune response in the brain. Additionally, menopause is a separate risk factor that can put women with OSA at an even higher risk of cognitive decline. Although neuroinflammation occurs in OSA, the underlying mechanisms of the neuroinflammatory response are not well understood, and it remains unknown whether ovarian hormones modify these mechanisms. To examine the impact of OSA and hormone status on neuroinflammation, we used a 7-day chronic intermittent hypoxia (CIH) and ovariectomy (OVX) to model OSA and hormone status in female rats, respectively. To examine neuroinflammation, we investigated changes in brain microglia and astrocyte morphology and the number of reactive cells. We used a comprehensive three-dimensional reconstruction and analysis of microglia and astrocytes to study the changes in the morphology of these cells. We focused on brain regions associated with cognitive function (CA1 of the dorsal hippocampus, medial prefrontal cortex - mPFC, caudate and putamen - CP). Specifically, immunofluorescence of Iba1 (microglia marker) and GFAP (astrocyte marker) was conducted, along with measuring indicators of glial reactivity (branching, complexity, cell size, and number of reactive cells). Our results found that there were CIH and hormone effects on neuroinflammation in these brain regions. Microglia activation was impacted by an interaction between CIH and hormone status in all regions examined. In contrast, astrocytes showed no reactivity in all regions examined, regardless of CIH or hormone status. These findings suggest that menopause and OSA may impact microglia remodeling in brain areas associated with cognitive function. Microglia-specific neuroinflammation may be part of early mechanisms that lead to the cognitive impairments observed in CIH and hormone loss in females.

Median preoptic nucleus (MnPO) neurons projecting to the hypothalamic paraventricular nucleus (PVN) are linked to hypertension induced by chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea. The modulation of MnPO-driven synaptic activity in PVN magnocellular neurons (MNCs) by CIH remains unexamined. We hypothesized that single and repetitive activation of MnPO-PVN projections causes differential synaptic plasticity in MnPO-PVN synapses with and without CIH exposure. Adult male rats were prepared using an intersectional viral approach to induce Cre-dependent channelrhodopsin expression in PVN-projecting MnPO neurons. Two weeks after stereotaxic surgery, some rats were exposed to 7 days of CIH. All rats were anesthetized and their brains were prepared for in vitro electrophysiological recording from PVN MNCs and optogenetic stimulation of the MnPO. We observed distinct EPSC and IPSC response patterns to the optogenetic stimulation of the MnPO. Low-frequency optogenetic stimulation (15 Hz) resulted in short-term potentiation manifested in increased poststimulatory spontaneous EPSC (sEPSC) frequency without altering amplitude while gradually increasing poststimulatory sIPSC frequency and amplitude, shifting some neurons to a more inhibitory state. CIH increased the amplitude of both sEPSCs and stimulation-evoked EPSCs while reducing their frequency. In contrast, CIH enhanced both the amplitude and frequency of sIPSCs and stimulation-evoked IPSC. Stimulation-evoked currents recorded during train protocols reflected a mixture of spontaneous and evoked events. Optogenetic stimulation increased the intrinsic excitability of MNCs in rats exposed to CIH. Activation of the MnPO-PVN pathway recruits both excitatory and inhibitory synaptic circuits converging onto PVN MNCs. CIH induces metaplasticity within this pathway, manifested as strengthened excitatory synaptic drive and heightened intrinsic excitability of PVN MNCs, which is counterbalanced by an adaptive increase in inhibitory tone. These parallel changes could explain why CIH is not associated with increased neurohypophysial hormone release.

Chronic intermittent hypoxia (CIH) is a key feature of obstructive sleep apnea (OSA) and leads to physiological changes that can cause cardiovascular and neurological issues. This review explores the role of nitric oxide (NO) and inflammation in the development of CIH-induced health problems, specifically focusing on hypertension and cognitive dysfunction. We synthesize current evidence regarding how CIH modulates inflammatory processes and NO signaling in different brain regions, especially autonomic control centers crucial for cardiovascular regulation. We also discuss the activation of proinflammatory transcription factors, the generation of reactive oxygen species, and the involvement of pattern recognition receptors in CIH-induced neuroinflammation. Regarding cardiovascular changes associated with CIH, we focus on the effects of NO and inflammation in central autonomic regions such as the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median preoptic nucleus (MnPO), and paraventricular nucleus (PVN), shedding light on their contributions to sustained hypertension in CIH. The review delves into the latest findings on sex differences in CIH-induced neuroinflammation. In examining the current knowledge, we have pinpointed significant gaps in understanding, especially concerning the specific mechanisms of NO and inflammation interactions in different brain regions during CIH. This review provides insights into potential therapeutic targets and emphasizes the need for further research to develop more effective treatments for OSA-related cardiovascular and neurological complications.

Chronic intermittent hypoxia (CIH), a preclinical model of hypoxemia associated with sleep apnea, causes hypertension associated with increased sympathetic activity linked to arterial chemoreflex and renin-angiotensin system activation. In some models of CIH, ovarian hormones have a protective effect against the change in blood pressure. We tested the effects of CIH and ovariectomy (OVX) on blood pressure and ΔFosB expression in female Sprague-Dawley rats. We hypothesized that CIH and OVX would increase ΔFosB in central autonomic regions associated with increases in blood pressure. Intact (INT) and OVX adult female Sprague-Dawley rats were exposed to 7 days of CIH only during the light (sleep) phase or continuous normoxia (CON). Mean arterial pressure (MAP) was continuously monitored by radiotelemetry. Rats were anesthetized with Inactin and euthanized, and brains were collected and processed for immunohistochemistry. CIH increased MAP relative to CON during the light phase, regardless of gonadal status. During the dark (active) phase, an increase in MAP was observed in OVX but not in INT rats, irrespective of CIH. CIH significantly increased ΔFosB immunoreactive cells in the caudal nucleus of the solitary tract, A5 region, and rostral ventrolateral medulla regardless of OVX. Independent of CIH, OVX significantly increased ΔFosB immunoreactive cells in the organum vasculosum of the lamina terminalis, median preoptic nucleus, rostral nucleus tractus solitarius, and the dorsal, medial, and lateral parvocellular subdivisions of the paraventricular nucleus. Our data indicate that CIH and OVX increase blood pressure independently and exert region-specific effects on ΔFosB immunoreactivity in central autonomic regions.NEW & NOTEWORTHY We investigated the effects of ovarian hormone depletion and chronic intermittent hypoxia (CIH) on blood pressure and ΔFosB staining in central autonomic regions. Our findings indicate that ovariectomy (OVX) and CIH have independent effects on blood pressure and produce region-specific changes in ΔFosB staining in the central autonomic regions associated with hypertension. This suggests that the effects of CIH and OVX on ΔFosB staining and hypertension are not additive.

BACKGROUND:Obstructive sleep apnea (OSA) is an intermittent hypoxia disorder associated with cognitive dysfunction, including learning and memory impairments. There is evidence that alterations in protease activity and neuronal activation are associated with cognitive dysfunction, are dependent on sex, and may be brain region-specific. However, the mechanisms mediating OSA-induced cognitive impairments are unclear. Therefore, we used a rat model of OSA, chronic intermittent hypoxia (CIH) to investigate protease activity (e.g., calpain and caspase-3) on spectrin, a cytoskeletal protein associated with neurotransmitter release, and neuronal activation (early growth response protein 1, EGR-1) in brain regions associated with learning and memory. METHODS:Male and female Sprague Dawley rats were exposed to CIH or room air (normoxic) for 14 days. We quantified protease activity and cleaved spectrin products, along with EGR-1 protein expression in hippocampal subregions (CA1, CA3), cortical regions [entorhinal cortex (ETC), retrosplenial cortex (RSC), cerebellar cortex (CC)], and subcortical regions [raphe nucleus (RN), locus coeruleus (LC)] associated with learning and memory. Within each group, Pearson correlations of calpain activity, caspase-3 activity, and EGR-1 expression were performed between brain regions. Sex differences within normoxic and CIH correlations were examined. RESULTS:CIH dysregulated calpain activity in male ETC, and female CA1 and RSC. CIH dysregulated caspase-3 activity in male RN, and female CA1 and RSC. CIH decreased calpain and caspase-3 cleavage products in male ETC. CIH decreased calpain-cleaved spectrin in male RSC but increased these products in female RSC. EGR-1 expression was decreased in male and female RN. Correlational analysis revealed CIH increased excitatory connections in males and increased inhibitory connections in females. EGR-1 expression in males shifted from negative to positive correlations. CONCLUSIONS:Overall, these data indicate CIH dysregulates protease activity and impairs neuronal function in a brain region- and sex-dependent manner. This indicates that males and females exhibit sex-specific vulnerabilities to mild OSA. These findings concur with our previous behavioral studies that demonstrated memory impairment in CIH-exposed rats.

Chronic intermittent hypoxia (CIH) is an animal model simulating the hypoxemia associated with obstructive sleep apnea (OSA) in humans. Male rats exposed to CIH exhibit increased sympathetic tone and persistent elevations in blood pressure similar to that observed in patients with. In MnPO neurons, CIH reduces GABAa inhibition and, in some neurons, GABAa activation produces excitation. Here, loose patch and perforated patch recordings were conducted in MnPO neurons in response to optogenetic stimulation of GABAergic SFO (efferent) and MnPO (local) neurons to assess the role of GABAergic signaling in these nuclei on MnPO excitability. Our working hypothesis was that CIH would diminish the inhibitory effects of optogenetic stimulation of GABA neurons. This hypothesis was tested using an AAV vector that selectively expresses channel rhodopsin in GABA neurons Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received infusions (0.2 μL) of pAAV-mDlx-ChR2-mCherry-Fishell-3 in the SFO or MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hr) or normoxia (Norm). CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For in vitro recordings, rats were anesthetized with isoflurane (2-3%), and sagittal or coronal slices (300 μm) containing MnPO were cut using standard slice procedures. Voltage-clamp loose patch or perforated patch recordings using gramicidin (100 ug/ml in 140 mM K-gluconate) were collected in MnPO neurons in response to optogenetic stimulation of GABAergic neurons (2 ms pulse, 20 Hz, 1 sec). MnPO neurons from CIH-treated rats showed differing responses to GABAergic stimulation while MnPO neurons from Norm-treated rats predominantly exhibited GABAergic inhibition. MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH-treated rats show variable mIPSC frequencies and heterogeneous responses to GABAergic neuronal activation in the SFO and MnPO. The results demonstrate that CIH alters the Cl− homeostasis in some MnPO neurons altering their response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH. Supported by R01 HL155977. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Obstructive sleep apnea, a common form of sleep-disordered breathing, is characterized by intermittent cessations of breathing that reduce blood oxygen levels and contribute to the development of hypertension. Hypertension is a major complication of obstructive sleep apnea that elevates the risk of end-organ damage. Premenopausal women have a lower prevalence of obstructive sleep apnea and cardiovascular disease than men and postmenopausal women, suggesting that sex hormones play a role in the pathophysiology of sleep apnea-related hypertension. The lack of protection in men and postmenopausal women implicates estrogen and progesterone as protective agents but testosterone as a permissive agent in sleep apnea-induced hypertension. A better understanding of how sex hormones contribute to the pathophysiology of sleep apnea-induced hypertension is important for future research and possible hormone-based interventions. The effect of sex on the pathophysiology of sleep apnea and associated intermittent hypoxia-induced hypertension is of important consideration in the screening, diagnosis, and treatment of the disease and its cardiovascular complications. This review summarizes our current understanding of the impact of sex hormones on blood pressure regulation in sleep apnea with a focus on sex differences.

Chronic intermittent hypoxia (CIH) experimentally mimics the recurrent hypoxia characteristic of sleep apnea, and it induces sustained hypertension in male and ovariectomized (OVX) female rats but not intact females. While central autonomic control regions are known to mediate CIH-induced hypertension in males, their contribution to CIH hypertension in OVX females remains unclear. We tested the hypothesis that 7 days of CIH increases mean arterial pressure (MAP) and FosB staining in central autonomic regions in OVX but not intact female rats. Adult female Sprague Dawley gonadally intact (INT) or OVX rats were exposed to 7 days of CIH (10% O2 and 21% O2 cycle, every 6 mins, 8h/day) or continuous normoxia (CON). MAP was monitored by radiotelemetry. On day 8, the rats were sacrificed, and their brains were collected and processed for FosB immunohistochemistry. During the light/sleep phase, CIH increased MAP in INT females (n=7; 3.4 ± 0.4 mmHg) as compared to CON (n=6, ΔMAP 1.4 ± 0.3 mmHg; P=0.01 CON vs CIH). This difference was not observed during the dark/active phase (CON ΔMAP -1.7 ± 0.4 mmHg, CIH 0.5 ± 0.3 mmHg, P=0.07). In OVX females, CIH was not associated with significant increases in MAP during the light/sleep phase (CON (n=6) ΔMAP 0.3 ± 0.4 mmHg, CIH (n = 6) 2.1 ± 0.3 mmHg, P=0.10). During the dark phase, MAP was significantly increased in CON OVX and CIH OVX compared to their respective baselines but there were no differences between groups (CON ΔMAP 2.4 ± 0.3 mmHg, CIH 2.2 ± 0.4 mmHg, P=0.0002 vs baseline, P=0.8 CON vs CIH). OVX rats had higher baseline FosB staining in the median preoptic nucleus compared to intact female rats independent of CIH exposure (INT: CON (n = 6) 13 ± 3 cells/section, CIH (n = 6) 14 ± 2; OVX: CON (n = 6) 21 ± 3, CIH (n = 6) 23 ± 2, INT CON vs OVX CON, P < 0.05, INT CIH vs OVX CIH P < 0.05). Compared to CON, CIH significantly increased the number of FosB immunoreactive cells in the caudal nucleus of the solitary tract in INT but not OVX females (INT: CON, 7 ± 1 cells/section, CIH 11 ± 1; OVX CON 6 ± 1, CIH n=6, 9 ± 1; INT CON vs CIH, P=0.02; OVX CON vs CIH, P = 0.09). In the rostral ventrolateral medulla, there were no significant differences among any of the groups (INT: CON n=4, 19 ± 2 cells/section, CIH n=4, 20 ± 3; OVX: CON n=6, 18 ± 2, CIH n=6, 21 ± 2, all P>0.05). In OVX females, CIH tended to increase MAP during both the light and dark phases. CON OVX females showed a trend for increased MAP during the dark phase, independent of CIH. CIH was not associated with increased FosB staining in either the median preoptic nucleus or the rostral ventrolateral medulla of OVX females This suggests that changes in MAP observed in CIH OVX females may not involve the same autonomic control regions as male rats exposed to CIH. Supported by RO1 HL155977. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiotensin II (ANG II) has been shown to have central nervous system effects. Although tissue renin-angiotensin systems (RAS) have been demonstrated in multiple tissues, the existence of a brain RAS is still a matter of debate. These studies test for angiotensin release from brain slices prepared from adult male Sprague-Dawley rats and male and female renin knock-out rats using Chinese hamster ovary cells modified to express both the angiotensin II type 1 receptor and a fluorescent calcium indicator. Sniffer cells were placed on the slices and calcium transients were measured from those located on or adjacent to the median preoptic nucleus with and without stimulation of the subfornical organ. Bath application of tetrodotoxin (1 µM) significantly attenuated spontaneous events while abolishing evoked sniffer cell activity. Bath application of dl-AP4 (10 µM, glutamatergic antagonist) did not affect either spontaneous or evoked release. Incubating the slices with fluorocitrate to inactive astrocytes did not influence sniffer cell activity in the MnPO. Pharmacological experiments indicate that ANG II release is largely both renin (aliskiren 10 µM) and ACE-1 (captopril 100 µM) dependent. However, experiments with brain slices prepared from male and female Renin knock-out rats suggest that alternative synthetic pathways may exist. Finally, these studies demonstrate that increases in ANG II release are observed following 7 days of chronic intermittent hypoxia. These studies suggest the existence of a tissue-specific RAS in the brain that involves canonical and alternative ANG II synthetic pathways and is upregulated in an animal model of sleep apnea.NEW & NOTEWORTHY These studies used Chinese hamster ovary cells that were cloned to express an angiotensin receptor (At1ra) and a calcium indicator (R-GECO) to detect the release of angiotensin from brain slices containing the lamina terminalis of rats. Some of the experiments use tissue from renin knockout rats. The results support the existence of an angiotensin system in the brain that may involve alternative synthetic pathways and is upregulated by intermittent hypoxia.

The brain is sensitive to Angiotensin II (ANG II) and we have previously shown that ANG II is released by brain slices containing the median preoptic nucleus (MnPO) and subfornical organ (SFO) in an activity dependent manner. In addition, angiotensin II type 1 receptors and angiotensin converting enzyme 1 are upregulated in the MnPO following chronic intermittent hypoxia (CIH), an animal model of obstructive sleep apnea. Here, we investigate changes in ANG II release in the SFO to MnPO pathway following 7-day CIH treatment.Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions (0.4 μL) of AAV2-hSyn-ChR2(E123A)-eYFP-WPRE in the SFO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hrs) or Normoxia. The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For sniffer cell measurements, rats were anesthetized with isoflurane (2-3%) and sagittal slices (300 μm) containing the MnPO and SFO were cut using standard in vitro slice procedures. Sniffer cells consisted of Chinese Hamster Ovary cells transfected with a commercially available plasmid for the angiotensin type 1a (AT1a) receptor (Origene Tech.) and R-GECO (Addgene #32462) as previously described. Sniffer cells were placed on the median preoptic nucleus (MnPO) of sagittal in vitro brain slices. Both spontaneous and evoked (SFO stimulation) changes in fluorescent intensity of sniffer cells on the MnPO were measured.Sniffer cell recordings were made from 5 normoxia and 8 CIH treated rats. The frequency of spontaneous sniffer cell activity in slices from normoxic rats (n = 92) was comparable to rates previously reported in Sprague-Dawley rats. Spontaneous sniffer cell activity in slices from CIH treated rats (n = 156) was increased compared to normoxic treated rats (p < 0.05). Furthermore, bath application of TTX (1 uM) reduced, but did not abolish, spontaneous sniffer cell activity in slices from both normoxic (n = 95, p < 0.001) and CIH (n = 108, p < 0.001) treated rats. Incidence of optogenetic evoked responses in sniffer cells were limited in slices from both the normoxic and CIH treated rats and there were no differences detected in these evoked responses.The current study indicates that spontaneous ANG II release in the MnPO 1) is not entirely action potential dependent, 2) is enhanced following 7-days CIH treatment, and 3) the CIH induced increase is action potential dependent. While optogenetic stimulation of SFO increased sniffer cell activity, CIH had no effect evoked release of ANG II in these preliminary studies. Work supported by: P01 HL088052, R01 HL155977 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Obstructive sleep apnea (OSA) results in sustained daytime hypertension. Chronic intermittent hypoxia (CIH) mimics the repetitive bouts of arterial hypoxemia associated with OSA. Male Sprague Dawley rats treated with CIH develop sustained hypertension and increased activation of central autonomic regions that regulate mean arterial pressure (MAP). However, gonadally intact female rats exposed to modest CIH treatment are not hypertensive. In male rats, lesions of the median preoptic nucleus (MnPO) prevent CIH-induced increase of MAP. We hypothesize that activation of MnPO and other autonomic control regions may contribute to the sex differences in the MAP response to CIH. To test this hypothesis, adult gonadally intact male and female rats (250-300 g bw) were exposed to either continuously normoxic (CON) or treated with CIH (10% O2 every 3 mins alternating with 21% O2 every 3 mins, 8 h/day) for 7 days. One week before the experiment started, some rats were instrumented with radiotelemetry transmitters to measure MAP and heart rate (HR). After one week of baseline recording, the rats were exposed to either normoxia or CIH and were euthanized (inactin 100 mg/kg ip) on the 8th day for immunohistochemistry. Forebrain and brainstem sections were stained for FosB/ΔFosB. Forebrain sections were also stained for neuronal nitric oxide synthase (NOS1) while brainstem sections were processed for dopamine-β-hydroxylase (DBH). The numbers of cells positive for NOS1 and FosB neurons in MnPO were counted and DBH and FosB positive neurons were counted in the hindbrain autonomic regions. CIH was associated with increased FosB staining in males but not females. Male exhibited an increase in the average number of FosB positive neurons (CON male 20 ± 2 cells/section, CIH male 35 ± 3; CON female 11 ± 1, CIH female 12 ± 2,) and colocalization of FosB and NOS1 (CON male 10 ± 1 cells/section, CIH male 18 ± 4; CON female 5 ± 1, CIH female 6 ± 1) in the MnPO. CIH females (n = 3) did not demonstrate increases in the numbers of FosB positive cells or DBH positive neurons in the commissural nucleus tractus solitarius (CON 7 ± 2, CIH 8 ± 2), rostral ventrolateral medulla (CON 2 ± 1, CIH 3 ± 1), caudal ventrolateral medulla (CON 5 ± 1, CIH 6 ± 2), and area postrema (CON 2 ± 1, CIH 2 ± 1) compared to CON females (n = 2). These preliminary results suggests that CIH is associated with increased FosB staining in the autonomic regions of male rats as opposed to female rats which is consistent with our working hypothesis. In addition, CIH was associated with increased FosB staining in NOS1 positive MnPO neurons suggesting that they may play a role in the sustained hypertension reported in male rats. The research is funded by NIH grant RO1 HL155977 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Our previous results indicate that Chronic intermittent hypoxia (CIH) enhances glutamatergic neurotransmitter release in PVN-projecting MnPO neurons. Very little information exists regarding how CIH affects the excitability of PVN-projecting MnPO neurons. MnPO neurons exhibit a wide diversity of spontaneous and evoked firing patterns. Initial studies indicate CIH modulates resting membrane properties and AP firing patterns which may be associated with alterations in hyperpolarization-activated cyclic nucleotide-gated currents (Ih). The Ih current is an inward current activated by hyperpolarization from the resting potential and is an important modulator of action potential firing frequency. Data were collected in PVN-projecting MnPO neurons which play an important role in CIH-induced hypertension. Adult male Sprague Dawley rats (250-350g) received bilateral PVN injections of 200 nL retrograde adeno-associated virus pAAV-CAG-tdTomato. Three weeks following injections, rats were either exposed to a 7-day intermittent hypoxia protocol consisting of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) or room air (Norm). Following CIH/Norm treatment, rats were deeply anesthetized and sagittal brain slices of MnPO were prepared using standard slice procedures. Spontaneous activity and responses to current injections were recorded from PVN projecting MnPO neurons using whole cell current-clamp. Data was analyzed off-line using Clampfit. There was a trend for spontaneous activity to be higher in PVN-projecting MnPO neurons from rats exposed to 7 days CIH when compared to normoxic controls (Norm: 2.29 ± 0.51 vs CIH: 3.3 ± 0.84) that was not due to a change in resting membrane potential. The threshold to evoke AP potentials was significantly lower in CIH neurons (5 pA injecting current, 17/19 CIH neurons vs 9/13 Norm neurons, CIH 8.6 ± 0.79 mv vs Norm 13.06±1.53 mv, p<0.01; 10 pA injecting current, 19 CIH neurons vs 12/13 Norm neurons CIH 14.74±0.92 mv vs Norm 19.72 ±1.64 mv, p<0.01). However, evoked action potentials were less sensitive to increasing current injection amplitudes in CIH vs Norm treated rats. Following bath application of the specific Ih blocker ZD7288 (ZD, 30 μm) spontaneous action potential activity was reduced by 24% in Norm neurons and 47% in CIH neurons (p=0.0313). These data demonstrate that CIH facilitates action potential generation in PVN projecting MnPO neurons and changes in Ih currents may contribute to this increase in excitability. R01 HL155977 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The paraventricular nucleus of the hypothalamus (PVN) is an important autonomic control center that receives afferent inputs from the median preoptic nucleus (MnPO). The link between the MnPO and the PVN is essential in generating chronic intermittent hypoxia hypertension. An optogenetic intersectional viral approach was used to gain more insight into the contribution of MnPO inputs to changes in PVN function that could contribute to hypertension. Adult male Sprague-Dawley rats were anesthetized with isoflurane and injected with 100 nL of AAV9.hSyn.HI.eGFP-cre.WPRE.SV40 in the PVN bilaterally and AAV1-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-HGHpA in the MnPO. This method induced a CRE-dependent expression of channel rhodopsin in PVN-projecting MnPO neurons. Three weeks after the injections, the rats were sacrificed as oblique brain slices of PVN were made. Postsynaptic currents (PSCs) were recorded from PVN neurons (VHold = -70 mV) with an aCSF (2-3 ml/min) bath solution. MnPO axon terminals in the PVN were stimulated with 15 Hz LED-generated blue light (470nM) pulses of 20-ms duration for a total of 1 min with an optical fiber directed at PVN. The stimulation train was repeated 5 times at 5 minutes intervals. PSCs were recorded for 40 minutes, including 5 min baseline periods before stimuli and 10 min post-stimulation period. Cells were characterized as type I, type II, or type III PVN neurons based on the presence of transient outward rectification. Amplitude and frequency data were analyzed offline using Easy Electrophysiology v2.5.0 software from a total of 17 Type I PVN neurons. Optogenetic stimulation evoked EPSC or mixed EPSC/IPSCs in 13 Type I neurons. From the 13 photo-evoked neurons, 10 neurons showed a significant increase in EPSC frequency following stimulation train as compared to the preceding baseline (1st: 6.149±1.438 Hz vs 27.377±5.474 Hz, p=0.0187; 2nd 7.532±1.670 Hz vs 27.562±5.514 Hz, p=0.0161; 3rd 7.395±1.659 Hz vs 25.415±5.028 Hz, p=0.0170; 4th 7.343±1.466 Hz vs 24.225±4.750 Hz, p=0.0183; 5th 6.709±1.375 Hz vs 21.837±4.464 Hz, p=0.0220). In the same neurons, spontaneous EPSC frequency in the first minute after stimulation trains 1 to 4 also was significantly higher than the first pre-stimulus baseline but the effect gradually decreased over time (baseline 6.149±1.438 Hz vs 14.615±3.260 Hz, p=0.0236; vs 12.055±2.519 Hz, p=0.0213; vs 10.904±2.340 Hz, p=0.0247; vs 9.801±2.027 Hz, p=0.0331). There were no changes in EPSC amplitude. Repetitive optogenetic stimulation of MnPO inputs to Type I PVN neurons increased EPSC frequency in a time-dependent manner. Additional experiments will be needed to specify the mechanism behind the increase in frequency and whether this phenomenon occurs in other PVN cell types. P01 HL088052; R01 HL155977 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Osmosenstion is a fundamental homeostatic process that controls the release of vasopressin (AVP). Hypothalamic neurons that make and release AVP are intrinsically osmosensitive and receive input from extrinsic osmoreceptors. This review examines the role of osmoreceptors in sodium-sensitive hypertension and how this important regulatory input interacts with non-osmotic systems such as angiotensin II to support hypertension. Sustained activation of AVP neurons by salt loading can lead to changes in chloride homeostasis that reduces or reverses the inhibitory effects of taurine or GABA. This would mitigate the inhibitory effects of hypoosmolality and baroreceptor stimulation. These changes have been observed in several models of hypertension. In sodium-dependent hypertension and salt loading, the intrinsic osmosensitivity of AVP neurons is amplified. Angiotensin II, which contributes to several models of hypertension, increases excitation from extrinsic osmoreceptors. Together, these observations suggest that changes in osmosensation may contribute to alterations in AVP release involved in the pathogenesis of salt-sensitive hypertension.

The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that secrete the hormones vasopressin and oxytocin. Sex differences in SON gene expression have been relatively unexplored. Our study used spatially resolved transcriptomics to visualize gene expression profiles in the SON of adult male (n = 4) and female (n = 4) Sprague-Dawley rats using Visium Spatial Gene Expression (10x Genomics). Briefly, 10-μm coronal sections ( 4 × 4 mm) containing the SON were collected from each rat and processed using Visium slides and recommended protocols. Data were analyzed using 10x Genomics’ Space Ranger and Loupe Browser applications and other bioinformatic tools. Two unique differential expression (DE) analysis methods, Loupe Browser and DESeq2, were used. Loupe Browser DE analysis of the SON identified 116 significant differentially expressed genes (DEGs) common to both sexes (e.g., Avp and Oxt), 31 significant DEGs unique to the males, and 73 significant DEGs unique to the females. DESeq2 analysis revealed 183 significant DEGs between the two groups. Gene Ontology (GO) enrichment and pathway analyses using significant genes identified via Loupe Browser revealed GO terms and pathways related to (1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females, (2) Gi signaling/G-protein mediated events for the significant genes unique to males, and (3) potassium ion transport/voltage-gated potassium channels for the significant genes unique to females, as some examples. GO/pathway analyses using significant genes identified via DESeq2 comparing female vs. male groups revealed GO terms/pathways related to ribosomal structure/function. Ingenuity Pathway Analysis (IPA) identified additional sex differences in canonical pathways (e.g., ‘Mitochondrial Dysfunction’, ‘Oxidative Phosphorylation’) and upstream regulators (e.g., CSF3, NFKB complex, TNF, GRIN3A). There was little overlap in the IPA results for the two different DE methods. These results suggest sex differences in SON gene expression that are associated with cell signaling and ribosomal pathways. The brain releases the hormones oxytocin and vasopressin from the supraoptic nucleus. Oxytocin is involved in maternal behaviors, lactation, and childbirth. Vasopressin is involved in sex-based differences in social behavior and body fluid regulation. However, how the brain contributes to sex-based differences in vasopressin and oxytocin release is poorly understood. This study aimed to address this knowledge gap using spatial transcriptomics to test for sex-based differences in gene expression in the supraoptic nucleus. Spatial transcriptomics combines anatomy with gene sequencing technology, allowing us to identify groups of genes that are expressed in specific locations in the brain. We applied this approach to brain sections containing the supraoptic nucleus from four adult male and four adult female rats. Using a data analysis workflow specifically for spatial transcriptomics, we identified genes that are significantly expressed in the supraoptic nuclei of both males and females (116 genes), primarily males (31 genes), and primarily females (73 genes). Genes enriched in the supraoptic nucleus of both males and females are related to the synthesis and release of peptides like vasopressin and oxytocin. Genes specific to the male supraoptic nucleus are broadly related to cell signaling, while the female-specific genes are related to ion transporters/channels. Results from a more traditional data analysis workflow identified sex-based differences in the expression of genes related to cell metabolism and protein synthesis. Together these results may provide a mechanistic foundation that can be used to better understand how differences in gene expression related to biological sex influence brain function.

Dilutional hyponatremia due to increased plasma arginine vasopressin (AVP) is associated with liver cirrhosis. However, plasma AVP remains elevated despite progressive hypoosmolality. This study investigated changes to inhibitory control of supraoptic nucleus (SON) AVP neurons during liver cirrhosis. Experiments were conducted with adult male Sprague-Dawley rats. Bile duct ligation was used as a model of chronic liver cirrhosis. An adeno-associated virus containing a construct with an AVP promoter and either green fluorescent protein (GFP) or a ratiometric chloride indicator, ClopHensorN, was bilaterally injected into the SON of rats. After 2 weeks, rats received either BDL or sham surgery, and liver cirrhosis was allowed to develop for 4 weeks. In vitro, loose patch recordings of action potentials were obtained from GFP- labeled and unlabeled SON neurons in response to a brief focal application of the GABA(A) agonist muscimol (100 mu M). Changes to intracellular chloride ([Cl]i) following muscimol application were determined by changes to the fluorescence ratio of ClopHensorN. The contribution of cation chloride cotransporters NKCC1 and KCC2 to changes in intracellular chloride was investigated using their respective antagonists, bumetanide (BU, 10 mu M) and VU0240551 (10 mu M). Plasma osmolality and hematocrit were measured as a marker of dilutional hyponatremia. The results showed reduced or absent GABA(A)-mediated inhibition in a greater proportion of AVP neurons from BDL rats as compared to sham rats ( 100% inhibition in sham vs. 47% in BDL, p = .001). Muscimol application was associated with increased [Cl]i in most cells from BDL as compared to cells from sham rats (chi(2) = 30.24, p < .001). NKCC1 contributed to the impaired inhibition observed in BDL rats (p < .001 BDL - BU vs. BDL + BU). The results show that impaired inhibition of SON AVP neurons and increased intracellular chloride contribute to the sustained dilutional hyponatremia in liver cirrhosis.

Chronic intermittent hypoxia (CIH) has been used to mimic the hypoxemia associated with sleep apnea and determine how these hypoxemias influence neural function. The nucleus of the solitary tract is the main site for chemoreceptor input to the CNS, but how CIH influences NTS inhibition has not been determined. These studies show that CIH increases glycine-mediated miniature IPSCs through mechanisms that depend on protein trafficking and astrocyte activation.

Chronic intermittent hypoxia (CIH) has been shown to alter the response of neurons in Median Preoptic nucleus (MnPO) to activation of cardiovascular inputs. Our previous results indicate that CIH enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. In other systems, BNDF/TrkB signaling has been shown to facilitate excitatory neurotransmission, so we tested its role in the increased MnPO excitation triggered by CIH. Data were collected from PVN-projecting MnPO neurons which play an important role in CIH-induced hypertension. Adult 250-350g male rats were injected bilaterally in the PVN with 200 nL retrograde adeno-associated virus (pAAV-CAG-tdTomato). Three weeks after the injections, the rats were exposed to a 7-day intermittent hypoxia protocol (6 min cycles; 3 minutes of 21% oxygen, 3 min of 10% oxygen maintenance at 10% oxygen for 8 h during the light phase). Normoxic controls were exposed to room air. At the end of the protocol, the rats were anesthetized with isoflurane (2-3%) and horizontal brain slices of MnPO were prepared. Miniature Excitatory Postsynaptic Currents (mEPSCs) were recorded using whole cell voltage clamp configuration from PVN-projecting MnPO neurons. The mEPSCs were recorded with the holding potential of -60 mV in the presence of a GABAa receptor antagonist (SR 95531 hydrobromide, 25 µM), tetrodotoxin (1µM), and a glycine receptor antagonist (strychnine, 3 µM) in aCSF. Data were analyzed off-line using Synaptosoft software. Differences in mEPSCs properties, such as peak amplitude, frequency, decay time, area, and cell capacitance were analyzed by ANOVA and Tukey multiple comparison tests. The results showed that CIH significantly increased mEPSCs frequency (Hz: CIH 1.32 ± 0.09 vs Norm 0.83 ± 0.09, p=5E-04), decay time (ms: CIH 8.01 ± 0.28 vs Norm 6.64 ± 0.36, p=0.004), area (pA.ms: CIH 63.85± 3.65 vs Norm 50.27 ± 3.6, p=0.01), and cell capacitance (pF: CIH 31.55 ± 1.8 vs Norm 25.34 ± 1.95, p=0.02). The contribution of BDNF/TrkB signaling to these CIH effects were tested using K252a and K252b, two structurally related kinase receptor inhibitors. MnPO slices were pre-incubated with either K252a or K252b (200nM) for 30 min before recording. Neither K252a nor K252b influenced mEPSCs of MnPO neurons from normoxic slices. Both K252a and K252b significantly affected CIH-induced changes mEPSCs but at different magnitudes. The K252a significantly reduced the effects of CIH on frequency (Hz: K252a 0.46 ± 0.05, p=1E-08), decay time (ms: K252a 6.07 ± 0.34, p=1E-04), area (pA.ms: K252a 43.26 ± 3.55, p=5E-04), and cell capacitance (pF: K252a 19.59 ± 1.61, p=1E-04;). In addition, K252a also significantly decreased mEPSCs peak amplitude in CIH neurons (pA: CIH 14.87 ± 0.41 vs K252a 13.27 ± 0.39, p=0.03). While K252b pretreatment also affected the same parameters, the changes produced by K252a were significantly greater. These studies suggest a role for BDNF/TrkB signaling in CIH induced changes in MnPO but additional experiments will be required to validate the specificity of our results.