Access Type

Open Access Dissertation

Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Patrick J. Mueller

Abstract

Investigation of whether sedentary and physically active conditions lead to altered GABAergic signaling in the RVLM

MD Dombrowski, and PJ Mueller

Department of Physiology, Wayne State University, School of Medicine, Detroit, MI

A sedentary lifestyle is a major risk for cardiovascular disease. Cardiovascular disease has been associated with elevated basal sympathetic nerve activity. The rostral ventrolateral medulla is a bilateral brainstem region that is an important for the control of resting and reflex control of sympathetic nerve activity and blood pressure. The activity of these neurons in this region is tonically inhibited by the neurotransmitter γ-butyric amino acid (GABA). Interestingly, we have shown that following unilateral rvlm blockade, acute inhibition of the intact rvlm lead to decreases in splanchnic sympathetic nerve activity this response was pronounced under sedentary conditions when compared to active rats. These data suggests that there is a compensatory mechanism that develops under sedentary conditions in order to offset the enhanced sympathetic nerve activity that is present. In the current study we hypothesize that sedentary conditions will lead to increased GABAergic transmission when compared to active conditions. This dissertation had 3 specific aims in order to determine the mechanism(s) that were involved in the enhanced GABAergic transmission under sedentary conditions. AIM 1 was designed to determine in vivo whether sedentary conditions alter the responsiveness of the RVLM to GABA-mediated inhibition when compared to physically active conditions. We hypothesize that sedentary conditions enhance sympathoinhibitory responses to GABA inhibition of the RVLM. . In Inactin anesthetized, sedentary (SED) or physically active (WR) rats, mean arterial pressure (MAP), heart rate (HR) and splanchnic sympathetic nerve activity (sSNA) were recorded during unilateral microinjection of GABA (30 nl, 0.3-600 mM) into the RVLM. Following GABA injections, the contralateral RVLM was inhibited with 90 nl of 2mM Muscimol and the GABA injections were repeated. There were no significant differences between SED or EX conditions for BP, HR and sSNA responses to GABA before contralateral blockade of RVLM. However, after contralateral blockade of the RVLM, the SED had enhanced blood pressure responses compared to the EX. Based on our results our sedentary model leads to enhanced inhibition of BP only after blockade of the contralateral RVLM. These data suggest sedentary conditions lead to enhanced GABA sensitivity but only after removal of GABAergic input to the RVLM. The reason why we have to remove GABAergic input in order to see to the enhanced response to GABA may be due the arterial baroreceptors buffering the actions of GABA through the contralateral RVLM. Once we inhibit the contralateral RVLM then we are able to see differences. Since the arterial baroreceptor reflex provides tonic GABAergic input to RVLM, we wanted to know if this reflex was playing a role in sinoaortic denervated the sedentary and physically active rats and repeated the protocol from the intact study. We found no differences between sedentary and physically active before and after contralateral blockade of RVLM. These data suggests that the baroreceptor reflex may be playing a role in buffering decreases in sympathetic outflow under sedentary conditions. This may serve as a compensatory mechanism to offset the developing sympathoexcitation. AIM 2 was designed to determine whether sedentary conditions lead to altered GABAergic input to RVLM when compared to physically active conditions. We hypothesize that sedentary conditions will lead to enhanced GABAergic input. We tested the hypotheses that 1) ionotropic glutamate receptors (iGluRs) contribute to increases in AP and SSNA in response to blockade of GABAA receptors and 2) SED conditions enhance glutamatergic excitation. Prior to microinjections in Inactin-anesthetized SED (n=6) and WR (n=5) rats, SAD was performed and one RVLM was inhibited (muscimol 2mM, 90nl). In Inactin-anesthetized sedentary (n=7) and physically active (n=9) rats, SAD was performed and one RVLM was inhibited (muscimol 2mM, 90nl). In responses to GABAA receptor blockade alone (bicuculline 5mM, 90nl) alone in the intact RVLM there was a pressor and sympathoexcitatory response in sedentary and physically active rats that were not significantly different between groups for mean arterial pressure and percent change in splanchnic sympathetic nerve activity. However, the absolute change in splanchnic sympathetic nerve activity was significantly greater under sedentary when compared to physically active conditions. Following GABAA receptor blockade, we microinjected the ionotropic glutamate receptor blocker kynurenic acid (40mM, 90 nl) into the intact RVLM and then repeated the bicuculline microinjection. GABAA receptor blockade in the presence of iGluRs blockade produced pressor and sympathoexcitatory responses in sedentary and physically active rats that were not significantly different. However, the change in absolute splanchnic sympathetic nerve activity (mV.s) was significantly greater under sedentary when compared to physically active conditions. These data suggests that baroreceptor-independent GABAergic input to the RVLM does play a role in suppressing sympathoexcitation under both sedentary and physically active conditions. Once baro-independent GABAergic input to the RVLM is removed there is this excitation that remains that is partially being by glutamate receptors and this excitation is driving splanchnic sympathetic nerve activity. However, when ionotropic glutamate receptors are blocked prior to GABAA receptor blockade there is some excitation that remains, that is being driven by non-ionotropic glutamatergic input under sedentary and physically active conditions. Finally AIM 3 was designed to determine whether sedentary conditions alter GABAA receptor protein expression in the RVLM when compared to physically active conditions. We hypothesize that sedentary conditions enhance GABAA receptor protein expression in the RVLM. In order to investigate GABAA receptor α1 subunit expression on RVLM spinally projecting neurons, Sedentary (n=3) and physically active (n=4) male Sprague-Dawley rats were injected bilaterally with cholera toxin B (CTB) at spinal cord segment T9/T10. Double immunofluorescent labeling identified immunoreactivity for CTB (green, FITC) and the GABAA receptor α1 receptor subunit (red, Cy3) in RVLM. We found that there was no main effect of rostrocaudal CTB expression nor was there a difference found between sedentary and physically active rats. From this data we concluded that sedentary conditions do not alter the expression of GABAA receptor α1 subunit on spinally projecting neurons that control sympathetic outflow at T9-T10 region of the spinal cord. We also looked at over all expression of GABAA receptor α1 subunit in the RVLM using western blot technique. Sedentary and physically active male Sprague-Dawley rats’ fresh brainstem tissue was section at 80 µm on cryostat. Each section was collected and mounted onto a clean glass slide. Micropunches were taken from each section bilaterally and then placed into a lysis solution. The sections were than stained with cresyl violet, coverslipped and looked at under a brightfield microscope in order to determine which section contained RVLM. Samples were pooled so that they contain an area of 240 µm. A BCA was done to determine protein concentration. The samples were prepared and loaded on a Mini-Protean® TGX precast gel and electrophoresis was performed. The protein was transferred to a PVDF membrane. The membrane was exposed to the primary antibody for GABAA receptor α1 subunit (1:250 lot no. AGA001AN1002; Alomone Labs) overnight and then the secondary antibody the next day for 2 hours. The membrane was incubated in ECL and developed. The GABAA receptor α1 subunit was normalized to GAPDH. There was no main effect of rostrocaudal expression of GABAA receptor α1 subunit. Sedentary conditions did not enhance the protein expression of GABAA receptor α1 subunit when compared to physically active rats. These data suggest that sedentary conditions do not enhance GABAA receptor expression. Overall, the data from this study show that sedentary conditions do enhance GABAergic transmission in the RVLM which be due to changes in GABA sensitivity of release.

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