Medullary GABA Receptors and the Regulation of Blood Pressure in the Rat

by Robert N. Willette et al.

The purpose of this study was to determine the cardiovascular effect of a GABAergic agonist and antagonist upon vasopressor and vasodepressor motoneuron pools in the ventrolateral medulla. Rats underwent microinjection of muscimol (GABA receptor agonist) to vasopressor (VLDA) and vasodepressor (VLPA) sites in the ventrolateral medulla.

Muscimol injection to VLDA sites consistently caused an increase in BP, HR and pulse pressure. Pretreatment with alpha adrenergic blockers abolished the pressor effect of muscimol. It should be noted that increases in HR were resistant to this blockade. Muscimol injection to VLPA sites caused a fall in BP, HR and pulse pressure. Additionally, injection of biuculline (GABA receptor antagonist) reversed the effects of muscimol in both VLDA and VLPA sites.

These results indicate that VLPA and VLDA sites are tonically active in maintaining BP. The results also suggest that a GABAergic system may be involved with the modulation of neural activity in these areas.

-BH

Selective enhancement of glutamate-mediated pressor responses after GABAA receptor blockade in the RVLM of sedentary versus spontaneous wheel running rats

Patrick J. Mueller and Nicholas A. Mischel

Many previous studies have shown that sedentary conditions can result in enhanced nerve activity following activation of the rostral ventrolateral medulla (RVLM). This means that there is some type of mechanism that differs between active and sedentary animals in terms of the excitation and inhibition that occurs in the RVLM. The RVLM is primarily regulated by glutamate, an excitatory neurotransmitter, and GABA, an inhibitory neurotransmitter. This study was trying to determine how the glutamate could directly excite the RVLM, and the effects that blocking the GABA pathway may have on the response to glutamate. The hypothesis was that sedentary conditions would enhance sympthoexcitatory response to direct activation of the RVLM, with the responses increasing further by blocking tonic GABAergic transmission.

The methods of the experiment were similar to our microinjection protocols in terms of exposing the RVLM and recording nerve activity and blood pressure. The first protocol involved injection 1, 10, and 100mM concentrations of glutamate into the RVLM while recording the blood pressure, heart rate, and lumbar sympathetic nerve activity (LSNA). The second protocol administered a GABA receptor blocker called bicuculline before glutamate injections were performed. The glutamate was injected 5, 10, 15, 30, and 45 minutes after the bicuculline was administered to investigate the changes in nerve activity after certain periods of time. The third protocol was a control section in which glutamate response was tested in the presence and absence of artificial cerebral spinal fluid.

In protocol one, responses to varying concentrations of direct glutamate administration did not differ between the sedentary and active animals. This is because heart rate responses were small and increases in LSNA also did not differ significantly between the active and sedentary animals. In protocol two, the initial injections of bicuculline increased the baseline blood pressure, heart rate, and LSNA in both the active and sedentary animals prior to any glutamate being injected. The increases did not differ significantly between the two groups. The glutamate injection responses at 5 and 15 minutes after the bicuculline was administered were greatly enhanced in the sedentary animals compared to the physically active animals. These enhancements were observed as increases in mean arterial pressure, however, were no longer observed by the 30- and 45-minute marks. Increases in LSNA were enhanced at the 5-, and 15-minute marks as well, however there was not a significant difference between the sedentary and active groups.  Once again, these effects were no longer observed at 30 and 45 minutes. This suggests that there was full recovery from bicuculline within the time course mentioned. Protocol three showed that responses to glutamate injections at different time points after injection of the bicuculline vehicle (aCSF) did not differ significantly from the control microinjections. In addition, responses to the repetitive microinjections did not differ significantly between the active and sedentary groups.

Overall, there were results that both confirmed and opposed the hypothesis. The hypothesis was confirmed in that there was enhancement in the response to glutamate in sedentary animals after the GABA receptor block was administered in the RVLM.  However, the LSNA responses did not differ between groups under direct glutamate activation nor under GABA receptor blockade conditions.

Based on the results, the paper determined several new findings that helped distinguish the effects of sedentary verses active conditions on nerve activity and blood pressure. First, sedentary conditions enhance GABAergic control of glutamate-sensitive neurons in the RVLM that regulate blood pressure. Second, sedentary conditions increase nerve activity when glutamate is administered in the absence of GABAergic modulation. Lastly, LSNA does not control the responses recorded in animals after GABA receptor blockers were administered.

This study helps us to possibly think about what other protocols we may want to add to the microinjection experiments. We could find other drugs that block glutamate or GABA responses in the RVLM and then see what effects present themselves when glutamate and GABA doses are given in the active and sedentary animals.

 

-Lyndsey M.

 

Blockade of Rostral Ventrolateral Medulla Apelin Receptors Does Not Attenuate Arterial Pressure in SHR and L-NAME induced Hypertensive Rats

Philip R. Griffiths, Stephen J. Lolait, Louise E. Pearce, Fiona D. McBryde, Julian F.R. Paton, Anne-Marie O’Carroll. Frontiers in Physiology (October 2018)

Apelin is a neuropeptide found in many organs, including the brain. It is thought to regulate the intake of food, water, and the release of vasopressin. Previous studies have shown that apelin receptors play a role in heart failure, hypertension, and heart diseases.  Apelin has been shown to be increased in the Rostral Ventrolateral Medulla (RVLM) in hypertensive rats, which is the main control center for sympathetic activity. This study focuses on if the apelin receptor plays a role in the progression of hypertension in two different models and if it is also part of the development stage of hypertension.

Thirty-six Wistar Kyoto (WKY) rats, sixteen spontaneous hypertensive rats (SHRs), and twenty younger SHRs were used in these studies. To generate the L-NAME model of hypertension, six WKY rats were given a daily dose of a nitric oxide synthase inhibitor, which increased sympathoexcitation and leads to hypertension. Lentiviruses with an apelin receptor gene knockout were created and injected into the RVLM. Control lentiviruses without the knockout were also created and injected directly into the RVLM. Injections of an apelin receptor agonist were used to test the knockout of the gene and then glutamate injections confirmed RVLM location.

Researchers first looked at the micropunches of the RVLM and found that the protein levels of apelin and the receptor gene were significantly higher in SHRs, when compared to the control rats. When observing cardiovascular effects, researchers started by measuring the baseline blood pressure and observed it was significantly higher in the SHRs. Microinjections of apelin-13 were then given directly into the RVLM, which exhibited a significant increase in MABP and systolic blood pressure in both SHRs and the controls. When rats were injected with a saline solution in the place of the apelin-13, no increases were observed. The increases in blood pressure were much greater in the SHRs compared to the controls. When an apelin receptor antagonist was injected, only the increases from the apelin-13 cancelled out, but no further decrease occurred. Researchers then used a lentivirus vector to knockout the apelin receptor. The lentivirus delivery was confirmed using immunofluorescence and observing the expression within the RVLM 25 days after they were injected. The lentivirus was shown to decrease expression of the receptor by about 65% at day 25. Apelin-13 was then injected into the knockout and control rats. The control lentivirus rats exhibited a significant increase in blood pressure, while the knockout rats had no increase. Using injections of glutamate to also confirm the pipette was in the correct place, similar blood pressure increases in the lentivirus knockout and the control rats were observed. 

There were no significant differences in heart rate or blood pressure in the normotensive control rats given the lentivirus knockout or lentivirus control. Similarly, the blood pressure drops caused by an injection of hexamethonium exhibited no significant difference. The SHRs and the L-NAME treated rats exhibited higher blood pressures at the beginning of the experiments, which is expected with those models, though the respiration rate and the heart rate were similar to the normotensive rats. SHRs and the L-NAME treated models exhibited no significant difference in heart rate, blood pressure, hexamethonium- induced blood pressure drop, and body weight between the lentivirus knockout and lentivirus control rats. When the RVLM was injected with apelin-13 25 days after the lentivirus delivery, SHRs and L-NAME treated rats with the knockout exhibited a decrease in the apelin receptor gene and blood pressure, when compared to the rats treated with the lentivirus control.

Researchers then tested if a SHR was injected with apelin-13 when it was still young, would hypertension would develop? In SHRs that only received the lentivirus knockout or control, blood pressure remained very similar after 10 weeks. In SHRs that received the lentivirus and an injection of apelin-13, blood pressure was observed to slightly decrease in rats given the lentivirus knockout and increase in rats with the lentivirus control.

In conclusion, although the mechanisms are still not fully understood, it appears that the apelin receptor does not have a role in the development of high blood pressure or hypertension. These results also suggest that if the apelin receptor gene is knocked out in SHRs, the onset of hypertension is not slowed or prevented. Injections of the receptor agonist were shown to increase blood pressure and when the agonist and a receptor antagonist were given after one another, the agonist induced increases were eliminated. When just the antagonist was given to the SHRs, there was no change in blood pressure, which suggest these receptors do not play a role in the regulation of high blood pressure in hypertensive rat models.

-Paul M

Ablation of brainstem C1 neurons improves cardiac function in volume overload heart failure

David C. Andrade, Camilo Toledo, Hugo S. Diaz, Claudia Lucero, Alexis Arce-Alvarez,
Luiz M. Oliveira, Ana C. Takakura, Thiago S. Moreira, Harold D. Schultz, Noah J. Marcus, Julio Alcayaga, Rodrigo Del Rio. Clinical Science (2019).

Heart failure (HF) is a major problem among the older generations and is characterized by increased sympathetic activity. One of the most important regions in the brain which is in charge of sympathetic control is the Rostral Ventrolateral Medulla (RVLM). Recent research has shown that in cases of HF, the neurons in the RVLM are very active compared to normal. C1 neurons, catecholaminergic neurons, in the RVLM are known to control sympathetic outflow. The goal of this study was to determine the role of C1 neurons in the RVLM of HF rats.

This study used 45 adult male Sprague Dawley rats that were kept on a light dark cycle and had access to food and water. Heart failure was induced by creating an opening between the vena cava and the aorta using a needle. This causes volume overload heart failure. Sham rats were also given the same anesthesia and surgeries, but no anastomosis was created. four weeks after the heart failure surgery, rats were placed under anesthesia and given anti-dopamine-beta-hydroxylase saporin (DBH-SAP) injections bilaterally into the RVLM to destroy C1 neurons. Control rats were injected with a saline solution. Sympathetic and parasympathetic activity was tested by giving injections of Propranolol, a beta blocker, and atropine, which is used to increase heart rate. 

The injections of DBH-SAP resulted in a 1.9 fold decrease in C1 neurons in the RVLM for both the control and HF rats. When rats were given Propranolol, HF rats that were given a control injection exhibited a larger decrease in heart rate when compared to the control rats given the control injection. This suggests that the HF rats had higher sympathetic activity. HF rats that were given the DBH-SAP injection exhibited a larger decrease in heart rate compared to HF rates given a control. No significant difference was shown in the control rats given the DBH-SAP injection. Atropine injections were then given to compare parasympathetic activity. HF rats given a control injection showed significantly larger decrease in heart rate than the control rats. The DBH-SAP injection did not exhibit any significant results in the HF or control rats. HR rats also showed a significant reduction in baroreflex sensitivity (BRS). After the injection of DBH-SAP, BRS was significantly improved in HF rats. HF rats given a control injection showed a decrease in both diastolic and systolic cardiac function, when compared to the control rats. Injection of DBH-SAP was shown to significantly improve cardiac function in the HF rats. HF rats exhibited more cardiac arrhythmias when compared to the control rats, but this number was significantly reduced after the injections of DBH-SAP was given. Although arrhythmias were reduced, DBH-SAP did not exhibit a change in the tissue damage found in the HF rats.

In conclusion, targeted ablation of C1 neurons in rats with HF exhibited reduced sympathetic activity and improved baroreceptor sensitivity. Frequency of cardiac arrhythmias also decreased after the ablation of C1 neurons, but this did not lead to a change in the damaged tissue. This study shows that the progression of HF is dependent on C1 neurons in the RVLM. This past Thursday, the department hosted Dr. Osborn to discuss renal hypertension and inflammation. He discussed in detail how neuron ablation can affect sympathetic and parasympathetic responses. I have to be honest and say I did not know this was a possible treatment option. I then wondering how this would affect neurons in the in RVLM region, which brought me to this research. The researchers in this study discuss the possibility of targeted ablation becoming a possible hypertension or heart failure treatment, but stress that much more research must be done to determine if it is possible in humans. Dr. Osborn further discussed how neuromodulation may be the future treatment option due to the fact that completely ablating sets of neurons can lead to many side effects. 

-Paul M

Effects of HCN Channels in the Rostral Ventrolateral Medulla Contribute to the Cardiovascular Effects of Propofol

Zhiqiang Hu, Zhilin Wu, Jie Gao, Qi Jia, Na Li, Yeling Ouyang, Shanglong Yao, Xiangdong Chen.

Molecular Pharmacology (2018)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are intermembrane proteins that act as voltage gated channels expressed throughout the heart and central nervous system. HCN channels have been shown to be inhibited by the anesthetic Propofol. Propofol is a general anesthetic that has been shown to have side effects such as bradycardia and hypotension. Previous research has suggested that Propofol inhibits vasomotor pressor neurons in the rostral ventrolateral medulla (RVLM), which could lead to the side effects. In this study, researchers tested how HCN1 and HCN2 channels in the RVLM play a role in mediating the effects of Propofol.

Mice were kept under a 12 hour light/dark cycle and had free access to food and water. Mouse blood pressure was measured by a tail cuff while they were awake. All infusions of Propofol were given through the tail vein using a catheter. For microinjection trials, mice were anesthetized using sodium pentobarbital and warming pads were used to maintain body temperature. To access the RVLM, the two occipital bones were removed and then microinjections were given with multibarrelled micropipettes. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to test protein expression levels in different aged mice

While mice that were not under anesthetic, baseline blood pressure was not different between wild type and HCN1 KO mice, but heart rate in the HCN KO mice was significantly lower than the wild type. When Propofol was given continuously for 10 minutes, blood pressure exhibited a significantly larger decreased in wild type mice when compared to HCN KO mice. Heart rate was also shown to decrease more in the wild type mice during the Propofol infusions. Researchers then observed responses to RVLM microinjections of both Propofol and ZD-7288, the HCN channel blocker. Blood pressure and heart rate was shown to significantly decrease after the microinjections. When the RVLM was injected with ZD-7288 shortly before Propofol, the decrease in blood pressure and heart rate was not as severe as a solo Propofol injection. The inhibitory effects of HCN channels were then tested using voltage and current clamps. Propofol was shown to inhibit HCN channel current and cause an increase in resistance in the RVLM neurons, but had no effect on the resistance in the RVLM neurons after ZD-7288 eliminated the current. ZD-7288 was shown to decrease the firing rate of RVLM neurons. Researchers then used Real-time PCR and western blotting to see if aging has any effect on HCN1 and HCN2 protein channels in RVLM neurons. HCN1 expression was shown to significantly increase from 2-3 weeks of age to 8-10 weeks of age and then again from 8-10 weeks to 40-60 weeks of age. HCN2 expression was only shown to significantly increase from 2-3 weeks old age to 40-60 weeks.

In conclusion, blood pressure and heart rate were not different in wild type and HCN KO mice, but once Propofol was given, heart rate and blood pressure showed a larger decrease in wild type mice. ZD-7288 given before Propofol was shown to attenuate the decrease in blood pressure and heart rate. Propofol reduced the firing rate and caused hyperpolarization, but after ZD-7288 was given before Propofol, the firing rate was reduced and hyperpolarization did not occur. Lastly, HCN channel protein expression was shown to increase as age increases. The researchers discuss that these results could lead to the creation of new anesthetics that have fewer side effects on the cardiovascular system. Since most drugs have a long list of side effects, more research must be done to determine how these drugs are effecting the peripheral parts of the body and not just the singular location that the drug in designed to act on.

-Paul M

Sex Differences in Renal Inflammation and Injury in High-Fat Diet-Fed Dahl Salt-Sensitive Rats

Roxanne Fernandes, Hannah Garver, Jack R. Harkema, James J. Galligan, Gregory D. Fink, Hui Xu (Hypertension, 2018)

High fat diets (HFD) are known to lead to obesity, hypertension, and renal dysfunction. A HFD can also lead to an increase in sympathetic outflow, which is the main cause of hypertension. Hypertension is chronically high blood pressure and is one of the major causes of renal disease. Although sex differences in obesity and cardiovascular disease have been researched, it is not known how a HFD causing hypertension and renal injury differ between males and females. The goal of this study is to see if there are differences in blood pressure, renal injury, and fat accumulation between sexes.

This study used HFD Dahl SS rats that exhibit signs of HFD-induced hypertension and Sprague Dawley rats that are also fed HFD but are not hypertensive. Beginning at three weeks old, the rats were either given a control diet that had normal salt levels or a HFD. The Dahl SS rats were given the HFD for either 10, 17, or 24 weeks, while the Sprague Dawley rats were placed on the HFD for 24 weeks.

After 24 weeks HFD males and females exhibited an increase in adipose tissue and body weight than the control rats. Body weights remained similar in all groups through week 8 of the diet, but then the control and the HFD males body weight increased significantly more than the females. Leptin, which is produced by adipose cells, was also shown to increase in males and females that were fed the HFD. Rats given the HFD also exhibited increased blood pressure. After 10 weeks of the diets, there was no significant difference between HFD and control rats. At 15 weeks the blood pressure of HFD rats became significantly higher than the controls rats. From 15 weeks to 24 weeks blood pressure in the HFD rats continued to significantly increase compared to the control rats. The heart rates between the HFD and control rats did not differ throughout the 24 week experiment. 

At 24 weeks, rats were given an injection of hexamethonium, which is a ganglionic blocker for sympathetic nerve activity. The hex injection decreased blood pressure more in the HFD males than the control males. The female rats exhibited a larger blood pressure decrease in the control rats than the HFD rats. When comparing males and females, the HFD males exhibited a larger blood pressure decrease compared to the HFD females. 

Researchers also observed renal histological changes. At 10 weeks, male rats had higher renal injury scores than females in both the HFD and control groups. At 17 weeks, male rats given the HDF exhibited significantly higher renal injury scores than the HFD females or the control groups. At 24 weeks, the HFD males exhibited an increased injury score compared to 17 weeks. This time period was also when blood pressure in HFD males and females was significantly increased.

Researchers then looked at the renal inflammatory responses in HFD rats. At 10 weeks, cortical and medullary macrophage infiltration was higher in males than females. At 17 weeks, macrophage levels increased in females of both diets, while males stayed similar to 10 week males. At 24 weeks, female rats exhibited a decrease in macrophages when compared to 17 week females, but still higher than 10 week females. While macrophage levels in males at 24 weeks was similar to the males at 10 and 17 weeks, levels were overall higher than female macrophage levels. The levels of cortical and medullary T-cells were then measured. At 10 weeks, all male and females had lower levels of T-cells. T-cells increased at 17 weeks through 24 weeks in males but not females, which exhibited similar T-cells levels to 17 weeks. Levels of inflammatory cytokines did not exhibit a significant change between different diets.

In conclusion, the HFD males and females both exhibited an increase in adipose tissue compared to the controls, but males generally have a higher body weight. Male and female rats given the HFD exhibited increase blood pressure, while heart rate shoed no significant difference between groups. The ganglionic blocker hexamethonium lead to a larger decrease in blood pressure in male rats compared to female rats with HFDs. Renal injury increased over the course of 24 weeks in males, while renal injury in females remained lower over the 24 week experiment. Renal T-cell levels increased over 24 in both HFD and control male rats, while females did not. 

One of the major limitations of this study that researchers discussed is how sex hormones effect hypertension and organ damage. They discuss that future studies could observe how sex hormones and receptors play a role in sex differences in hypertension. Another limitation discussed by researchers was how the HFD caused fat accumulation. Future studies could show that other high calorie diets can dramatically change renal inflammation or hypertension. I chose this paper because Dr. Fink recently spoke to the department and although his research is different than what we are doing in lab, it is also very closely related. Dr. Fink’s lab is mostly focusing on the sedentary side of our research and is now beginning to look towards how sex hormones are affecting hypertension.

-Paul M.

Microglia in the RVLM of SHR have reduced P2Y12R and CX3CR1 expression, shorter processes, and lower cell density

E. Myfanwy Cohen, Suja Mohammed, Mary Kavurma, Polina E. Nedoboy, Sian Cartland, Melissa M.J. Farnham, Paul M. Pilowsky. Autonomic Neuroscience: Basic and Clinical (2019).

Glial cells are cells of the central and peripheral nervous system that are not neurons. One type of glial cell, called microglia, act as a clean-up crew for the central nervous system. Microglia are macrophages that maintain the brain by looking for damage to the neurons or infectious chemicals. Since microglia look for damaged neurons, they also regulate inflammation in the brain. This study focuses on the microglia within a part of the brainstem called the rostral ventrolateral medulla (RVLM), which contributes to the control of blood pressure. The goal of the study was to determine if chronic high blood pressure is associated with a decrease in microglia function in the RVLM.

Fifteen week old spontaneously hypertensive rats (SHRs) and wild type rats were used in this experiment. Blood pressure was taken using a tail cuff to determine if the rats were hypertensive. The brainstems were removed and then placed in the freezer. The frozen brainstems were sectioned and the RVLM and the facial nucleus were punched out. Anti-Iba1 and donkey anti-rabbit AlexaFluor488 were used in the fluorescent tests. At the end of the experiment, rats were euthanized with sodium pentobarbitone.

A significant difference in systolic blood pressure was measured to demonstrate the two distinct groups. The SHRs exhibited a significant higher blood pressure (195 ±8 mmHg) than the wild type rats (144 ±8 mmHg). Two G-coupled protein receptors involved in the normal function of microglia, P2Y12R and CX3CR1, were measured and compared between the two groups of rats.  In the RVLM, P2Y12R expression was significantly lower in SHRs by about 37% when compared to the wild type rats. Expression of P2Y12R was also measured in the facial nucleus and exhibited no significant difference between SHRs and the wild type rats. Expression of CX3CR1 was then measured in the RVLM and the facial nucleus. In the RVLM, CX3CR1 expression was shown to be 30.9% lower in the SHRs when compared to the wild type. Expression of CX3CR1 in the facial nucleus was not significantly different between SHRs and the wild type.

The enzyme phenylethanolamine N-methyltransferase (PNMT) is found in the adrenal medulla and plays a role in converting noradrenaline to adrenaline. This allows PNMT to be a marker for adrenergic neurons, which are found in the RVLM but not the facial nucleus. The expression of PNMT was then compared between SHRs and wild type rats. The RVLM exhibited a significant amount of PNMT mRNA, while the facial nucleus showed a very small amount, which verifies the tissue punches have the correct sites.

Researches then observed the differences in microglia cell density between SHRs and wild types rats. The SHRs exhibited 22.9% lower cell density than the wild type. To further observe how active the microglia were, branch length, endpoints, and branch number were also examined. While the number of endpoints and branch numbers did not show a significant difference, branch length was significantly lower in the SHRs when compared to the wild type.

In conclusion, the G-coupled receptors CX3CR1 and P2Y12R play a major role in the normal function of microglia. Spontaneously hypertensive rats exhibit decreased expression of P2Y12R which may lead to the decreased microglia cell density in the RVLM. The researchers do state in the article that more research needs to be done to determine if the change in microglia are the cause or effect of over-activation of the RVLM and exactly how the microglia are being affected. I found this article interesting to our lab work because we constantly discuss the increased activation of RVLM but may not think about the exact repair mechanisms involved that may also not be working properly.