Browsing by Author "Mifflin, Steve"
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Item Central nuclei activated during long-term facilitation of blood pressure following acute exposures to intermittent hypoxia(2015-03) Batliwala, Shehzad; Wu, Qiong; Yamamoto, Kenta; Mifflin, SteveIntroduction: Acute intermittent hypoxia (AIH) is a protocol used to mimic the arterial hypoxemia that occurs during sleep apnea. AIH involves brief (1 min) exposures to systemic hypoxia (10% FIO2) repeated at 6 min intervals for an hour. Such exposures to AIH induce a phenomenon termed long-term facilitation (LTF). LTF is a long-lasting (at least 3 hr) increase in mean arterial pressure (MAP), heart rate (HR), sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). As LTF represents a form of neuronal plasticity, we were interested in determining what sites within the CNS might be involved in the generation of LTF induced by AIH. Our hypothesis is that AIH will induce activity in central sites typically associated with cardiovascular regulation in the brain. Methods: After rats underwent tracheal intubation and were artificially ventilated, they had femoral artery and venous catheters implanted for measurement of arterial pressure and administration of drugs, respectively. AIH was induced as previously described (Yamamoto et al., 2015). The test rats (n=3) were maintained for 1 hour after the last hypoxic exposure. The control group (n=2) was surgically prepared exactly as the experimental group but was not exposed to hypoxia during the 2-hour experimental period. Following the AIH protocol, rats were sacrificed, transcardially perfused with paraformaldehyde and their brains sectioned on a cryostat and processed for immunohistochemical detection of c-Fos in alternate sections. Cardiovascular parameters were statistically analyzed using 2-way ANOVA. Brain sections containing the central nuclei of interest were examined for the presence of c-Fos. Results: Our protocol of AIH induced a significant increase in all measured cardiovascular parameters (MAP, HR, renal SND and PND) measured 1 hr after cessation of exposure to AIH. This LTF was associated with c-Fos immunoreactivity in neurons located with the NTS and the RVLM but not within the hypothalamic PVN. Conclusions & Future Studies: The generation of LTF appears to be dependent upon medullary sites involved in cardiorespiratory regulation (NTS) and sympathetic outflow (RVLM) but not within hypothalamic cardiorespiratory and sympathetic regulatory nuclei such as the PVN. Future experiments can also use neuronal inhibitors to determine the specific role of each area in AIH-induced LTF, and further clarify the statistical significance of this result. This could provide insights into central areas involved in the persistent sympatho-excitation and hypertension observed in sleep apnea patients.Item Corticotropin-Releasing Hormone Receptor 2 in the Nucleus of the Solitary Tract Contributes to Sleep Apnea Induced Hypertension(2018-03-14) Nguyen, Dianna; Cross, Stephanie; Mifflin, Steve; Wang, LeiPurpose: This study tested the hypothesis that corticotropin-releasing hormone (CRH) derived from the paraventricular nucleus of the hypothalamus (PVN) acts in the nucleus of the solitary tract (NTS) to facilitate sleep apnea induced hypertension. Materials and Methods: To model sleep apnea induced hypertension, we exposed rats to intermittent hypoxia (IH) for 7 days. To detect CRH type 1 and type 2 receptors (CRHR1 and CRHR2) in the NTS, we conducted in situ hybridization. To study the signaling pathway of CRH, we performed calcium imaging on NTS slice preparation using Fura-2-acetoxymethyl ester. To test whether central CRH contributes to IH-induced hypertension, we implanted telemetry transmitters and osmotic mini pumps to infuse CRH receptor agonist/antagonist into the 4th ventricle of rats. To selectively excite CRH-producing pathways from the PVN to the NTS, we optogenetically stimulated the PVN and the NTS of CRH-Cre mice that received intra-PVN injections of Cre-inducible viral constructs expressing Channelrhodopsin 2 (ChR2). Results: The mRNA level of CRHR2 was significantly higher than CRHR1 in the NTS. CRH induced a transient increase of intracellular calcium level in NTS neurons that was abolished by the voltage-dependent calcium channel blocker nifedipine. CRH-induced calcium influx was attenuated by the CRHR2 antagonist K41498 but not by NBI-35965, an antagonist for CRHR1. Calcium influx was induced by the CRHR2 agonist Urocortin II but not by the CRHR1 agonist Stressin I. More importantly, IH decreased the CRHR2 mRNA level and attenuated the CRH-induced calcium influx in the NTS. Further in vivo studies revealed that IH-induced hypertension was significantly attenuated by chronic intra-4th ventricle infusion of the CRHR2 antagonist K41498, but was significantly exacerbated by chronic intra-4th ventricle infusion of the CRHR2 agonist Urocortin II. Optogenetic stimulation of either CRH somas in the PVN or CRH fibers in the NTS that originated from the PVN significantly increased blood pressure (somas, 12.3 ± 1.13 mmHg; fibers, 3.54 ± 0.69 mmHg), suggesting that activation of CRH projections from the PVN to the NTS increases blood pressure. Conclusions: These results suggest that CRH derived from the PVN activates CRHR2 in the NTS, which may contribute to sleep apnea induced hypertension; down-regulation of CRHR2 and CRHR2-mediated calcium influx in the NTS may serve as compensatory responses to protect against sleep apnea induced hypertension.Item Estrogen Receptor Alpha on Catecholaminergic Neurons in the Nucleus Tractus Solitarius(2018-03-14) Horn, Christopher; Wang, Lei; Mifflin, Steve; Nguyen, DiannaPurpose: Catecholaminergic neurons in the Nucleus Tractus Solitarius (NTS) are involved in Hypothalamic-Pituitary-Adrenal (HPA) axis and cardiovascular response to stress. While many studies have shown that pre-menopausal females are protected against the hypertensive and sympatho-excitatory effects of stress, very little is known about the location of neurons expressing estrogen receptors within the NTS. Our goal was to elucidate whether estrogen receptor alpha (ERα) is expressed on catecholaminergic neurons in the NTS in male and female rats. Methods: Adult male and female Sprague-Dawley rats were transcardially perfused with 4% paraformaldehyde and hindbrains harvested. In coronal sections containing the NTS (40um thick) immunohistochemistry was performed to determine whether NTS catecholaminergic neurons express ERα using monoclonal anti-tyrosine hydroxylase (TH) antibody (1:1000, Millipore) and secondary antibody Alexa Fluor 488 donkey anti-mouse (1:500, Jackson ImmunoResearch) and polyclonal anti-ERα antibody (1:2000-5000, Millipore) and secondary antibody Cy3 donkey anti-rabbit (1:400, Jackson ImmunoResearch). Sections were captured using an Olympus BX41 Fluorescence Microscope and analyzed using ImageJ. The NTS was divided into 2 regions: sections caudal to the area postrema (caudal CAUD) and sections lying below the area postrema (sub-postrema SP), and the number of immunoreactive neurons in each region counted and expressed as an average number of labeled neurons per section±SEM. The number of sections analyzed ranged from 7-11 in CAUD and 3-7 in SP. Results: In male rats, TH in CAUD NTS (n=6) was observed in 26±4 and ERα in 24±4 neurons/section. Co-localization of ERα and TH was observed in 13±2 neurons/section. TH in SP NTS (n=5) was observed in 54±3 and ERα in 34±2 neurons/section. Co-localization of ERα and TH was observed in 15±2 neurons/section. In female rats, TH in CAUD NTS (n=6) was observed in 27±2 and ERα in 30±3 neurons/section. Co-localization of ERα and TH was observed in 17±2 neurons/section. TH in SP NTS (n=6) was observed in 50±4 and ERα in 52±5 neurons/section. Co-localization of ERα and TH was observed in 27±2 neurons/section. At sacrifice, females were in estrus (1), diestrus (2) or proestrus (3). Conclusions: In both males and females, ERα is expressed on a subset of catecholaminergic NTS neurons, as well as non-catecholaminergic neurons. This could provide a substrate for estrogen-mediated cardiovascular protection in females.Item 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 Neurons in the Nucleus Tractus Solitarius show no discernable change in intracellular calcium during acute application of 17β-estradiol(2018-03-14) Paundralingga, Obed; Wang, Lei; Mifflin, StevePurpose: Neurons in the Nucleus Tractus Solitarius (NTS) are important regulators of cardiovascular and hormonal responses to stress and a potential site for estrogen modulation of stress responses. Our goal was to elucidate whether acute estrogen receptor activation is associated changes in intracellular calcium in NTS neurons in male and female rats. Methods: Adult male and female Sprague-Dawley rats were anesthetized with isoflurane and the hindbrain removed and cut into 300um thick sections. The NTS slices were then incubated for 50min with 10μM of Fura-2AM, 0.1% F-127 at 40°C and then washed for 20min in aCSF gassed with 95%O2+5%CO2. A single slice was transferred to the recording chamber on an upright epi-fluorescent microscope. The slice was held in place with a nylon mesh and superfused with normal aCSF at a rate of 2.5 ml/min. Application of 100 nM 17β-estradiol or 63 mM KCl dissolved in aCSF solution was done using multi barrel patch pipette positioned close to the neuron so that injection (volume) occurred in the same direction as the flow of aCSF being perfused. Fluorescence of Fura-2AM was excited by epi-illumination with light provided by a 75 W Xenon lamp band-pass filtered alternatively at 340 or 380 nm. Emission light pass through a barrier filter (510 nm). Pairs of 340 and 380 nm images were acquired at intervals of 5s and analyzed off-line with NIS-Elements AR 3.2 software. All images were captured with a charge-coupled device (CCD) camera. Results: A total of 19 sections were examined from 4 female rats and an average of 7 cells/slice were analyzed in each section. In response to superfusion of the slice with 100 nM 17β-estradiol no discernable change in the 340/380 ratio was observed in any cell. In 58 neurons from 10 slices in an additional 2 rats, a higher concentration of 1mM 17β-estradiol still failed to alter the 340/380 ratio. Cell viability was confirmed by application of 63 mM KCl which induced a 14 ± 5.4% increase in the 340/380 ratio relative to baseline in the cells studied. Conclusions: Acute application of 17β-estradiol did not alter intracellular calcium in NTS neurons. This could reflect a true lack of coupling between estrogen receptors and calcium signaling mechanisms in NTS neurons. However, if estrogen receptors are on a subset of NTS neurons, perhaps the fura-2 did not label the relevant population of NTS neurons. Further, perhaps estrogen does not have an acute (non-genomic, membrane bound receptor) effect in NTS and a more prolonged application of 17β-estradiol.Item Preliminary Findings on Sex Differences in Response to Various Acute Stressors in Male and Female Mice(2019-03-05) Cross, Sissy; Wang, Lei; Mifflin, Steve; Nguyen, DiannaPurpose: Studies in both humans and animals have shown that pre-menopausal females are protected against the hypertensive and sympatho-excitatory effects of stress. Our goal was to identify whether sex difference exists between male and female mice in response to various acute stressors. Methods: Adult male (n=4) and female (n=4) C57BL/6J mice underwent telemetry implantation (HD-X10, DSI) surgery and allowed 1-week recovery. Each day the mice were exposed to 1 of 5 acute stressors (acute restraint, hypoxia, new cage, cold, or forced swim). Mice were allowed 1-2 days of recovery between stressors. Acute restraint: placing the mouse in a conical tube for 30 min; hypoxia: exposing the mouse to 20 min of 8% O2; new cage: placing the mouse in an empty cage with no bedding for 30 min; cold: exposing the mouse to 1-4C for 30 min; forced swim: placing the mouse in a water-filled beaker for 10 min. Mean arterial pressure (MAP), heart rate (HR), and activity were recorded and data analysis (2-way repeated measures ANOVA followed by Holm-Sidak) was performed. Results: Acute restraint: male mice responded with peak MAP of 135±4, peak HR of 768±21, and peak activity of 0.00±0.0; whereas female mice responded with peak MAP of 131±2, peak HR of 749±21, and peak activity of 0±0.0. Hypoxia: male mice responded with peak MAP of 122±4, peak HR of 780±6, and peak activity of 0.50±0.3; whereas female mice responded with peak MAP of 131±1, peak HR of 784±18, and peak activity of 0.50±0.3. New cage: male mice responded with peak MAP of 137±7, peak HR of 789±8, and peak activity of 1.75±0.5; whereas female mice responded with peak MAP of 137±4, peak HR of 790±3, and peak activity of 1.50±0.3. Cold: male mice responded with peak MAP of 133±4, peak HR of 800±9, and peak activity of 1.00±0.4; whereas female mice responded with peak MAP of 137±7, peak HR of 797±14, and peak activity of 1.50±0.3. Forced swim: male mice responded with peak MAP of 136±5, peak HR of 729±30, and peak activity of 1.50±0.3; whereas female mice responded with peak MAP of 134±5, peak HR of 694±7, and peak activity of 1.25±0.5. Conclusions: In this preliminary study, no significant sex difference was observed in male and female mice in response to the various acute stressors, however there was a trend for sex difference in MAP during acute restraint stress. This study needs to be repeated to increase sample size before further conclusions can be made.Item Quantification of Nucleus Tractus Solitarius neurons projecting to Hypothalamic Paraventricular Nucleus(2017-03-14) Beig, Mirza; Ellazar, Sharon; Mifflin, Steve; Nguyen, DiannaBackground: Nucleus tractus solitarius (NTS) neurons integrate and relay visceral afferent inputs to various sites in the brain, notably the hypothalamic paraventricular nucleus (PVN). Understanding connectivity between the NTS and PVN will provide insight into possible interactions between the two areas in normal and pathophysiology. Purpose: To quantify the number of NTS neurons that project to the PVN using the retrograde transport agent cholera toxin B (CTB). Methods: Adult male Sprague-Dawley rats (n=3) were anesthetized intraperitoneally with ketamine (75mg/kg) and Dexdomitor (0.5mg/kg) then placed in a stereotaxic frame. Under aseptic conditions, the cranium overlying the PVN was exposed. Using a glass electrode, 100nL of 0.25% CTB in isotonic saline was slowly injected into the PVN bilaterally and the electrode withdrawn after 5 minutes. The skin was sutured and animals were allowed to recover. Three weeks later, the rats were transcardially perfused with 4% paraformaldehyde and their brains were harvested. PVN sections (40um thick) were used to verify the injection site. NTS sections (40um thick) were processed using immunohistochemistry. Polyclonal anti-CTB antibody (1:2000, Millipore) and secondary antibody Cy3 Donkey Anti-Goat (1:800, Jackson ImmunoResaerch Laboratories, Inc.) were used to visualize NTS neurons with axonal projections to PVN. Monoclonal anti-tyrosine hydroxylase (TH) antibody (1:1000, Millipore) and secondary antibody Alexa Fluor 488 Donkey Anti-Mouse (1:800, Jackson ImmunoResearch Laboratories, Inc.) were used to visualize catecholaminergic neurons. The number of CTB-immunoreactive, TH-immunoreactive, and co-labeled CTB and TH neurons were counted manually using Image J. Analysis was restricted to those NTS sections that had CTB-immunoreactivity and ranged from 15-21 sections each in the 3 rats. CTB injection sites were within the boundaries of the PVN. Results: We found 110±6 CTB immunoreactive and 346±33 TH immunoreactive neurons in NTS. 29±5 neurons were dual labeled with CTB and TH immunoreactivity indicating catecholaminergic NTS neurons projected to PVN. Conclusions: The majority of NTS neurons that project to PVN appear to be non-catecholaminergic. Approximately 10% of catecholaminergic NTS neurons project to PVN. Results will be useful in future studies using laser capture microdissection to examine gene expression in NTS neurons that project to PVN under a variety of conditions (e.g., hypoxia, hypertension).Item Sex Differences in Responses of Nucleus Tractus Solitarii Neurons to Acute Tissue Hypoxia(2019-03-05) Mifflin, Steve; Paundralingga, ObedObstructive sleep apnea (OSA) is a risk factor associated with cardiovascular diseases, such as hypertension. Females are protected from the hypertensive and tachycardia effects of chronic intermittent hypoxia model of OSA. The present study examined the change in nucleus tractus solitarius (nTS) intracellular calcium level response to exposure to acute hypoxia in male rats and in female rats and the role of voltage gated calcium channel (VGCC) in mediating the response. Adult rat brainstem slices (250 μm thick) containing commissural and caudal nTS were incubated for 45 min with 10 μM Fura-2AM and 30 μL of F127 at room temperature and then washed for 20 min in artificial cerebrospinal fluid (aCSF) bubbled with 95% O2/5%CO2. A single slice was transferred to the recording chamber on an upright epifluorescent microscope and superfused with normal aCSF bubbled with 20% O2/5%CO2 balanced with N2 at a rate of 2.5 ml/min. Acute hypoxia was established by exposing hindbrain slices to aCSF bubbled with 95% N2/5% CO2. In slices where the role of VGCC was being studied, the slice was preincubated with 20 µM nifedipine in aCSF before the recording and was exposed to hypoxia in the presence of the same drug during the recording period. Fluorescence of Fura-2AM was excited by epi-illumination with light filtered alternatively at 340 or 380 nm while the emitted light passed through a barrier filter (510 nm). Pairs of 340 and 380 nm images were acquired at intervals of 5 s and analyzed off-line with NIS-Elements AR 3.2 software to yield 340/380 ratio. A total of 22 sections were examined from 3 male and 3 female rats and an average of 6 cells/slice were analyzed in each section. Five minutes of hypoxic aCSF triggered a greater increase from baseline in slices from male rats compared to female rats (males: 3±0.16%, n=49 vs females: 2.2+0.19%, n=40; P2+]i as a response to acute tissue hypoxia in caudal NTS is differentially regulated between male and female rats and is also dependent, at least in part, upon Ca2+ influx through L-type voltage gated calcium channel in the males. As hypoxia-induced elevations in intracellular Ca2+ are likely to alter caudal NTS neuronal function under hypoxic conditions, its differential regulation in males and females might mediate some of the protection seen at organismal level.Item TIME COURSE OF CHANGES IN GLUTAMATERGIC TRANSMISSION WITHIN NTS DURING CIH EXPOSURE AND THE ROLE OF ΔFOSB(2014-03) Wu, Qiong; Mifflin, SteveΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant-negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p<.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post-synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement. ΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant-negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs from second order NTS neurons by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p<.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both 1 day and 7 days CIH groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post-synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement.