Sex Hormones’ Regulation of Rodent Physical Activity: A Review

J. Timothy Lightfoot

In past studies physical activity was mostly thought of as a voluntary activity, but newer literature suggests that is regulated by biological factors. These factors include but are not limited to genetics and sex hormones.

In humans, females are generally less active than males. However, many rodent studies have shown that the female rodents are 20-50% more active each day compared to male rodents.

Some of the first studies showed that ovariectomies significantly decreased female rat activity, and this was similar to decreased running wheel activity in males following castration. The activity was restored when either ovarian tissue or testes were implanted in males or females, although the increase in activity was greater when the ovarian tissue was implanted. Therefore, future studies wanted to look at three specific sex hormones: estrogen, progesterone, and testosterone.

ESTROGEN: A study with female voles (who undergo induced estrus when exposed to males and who do not require progesterone for sexual receptivity), showed that the effect of estradiol on physical activity is linked to an increased number of estradiol receptors in the brain. These receptors can be in the alpha or beta isoform, and are located in the medial preoptic area and the anterior hypothalamus. Although it was determined that estrogenic activation of the ERalpha-pathway is the primary mediator in increased running wheel activity, the mechanism for it is unclear.

PROGESTERONE: When injected with estradiol, progesterone did not influence the activity of rats. However, when animals first received an injection of estrogen their activity increased. After progesterone was then injected, the activity sharply decreased. Once the progesterone injections were stopped, the activity increased once again. This suggested that the decrease in activity that progesterone causes was mediated through direct interference with estrogen. This would help explain why the variable activity pattern occurs in female rats.

TESTOSTERONE: When capsules of testosterone were implanted in castrated male rats, their locomoter activity increased. Other studies found that testosterone injections significantly increased running wheel activity in animals, however not as much as estradiol injections. In addition, testosterone implants in castrated males restored physical activity, but no increase in physical activity levels were seen when testosterone supplementation was given to intact animals.

In conclusion, female rodents are more active than male rodents due to sex hormones. This mechanism is mediated through an estrogen-alpha receptor pathway. This pathway requires the aromatization of testosterone into estrogen within the male animals.

 

~LNM

 

Signal transduction pathways and tyrosine hydroxylase regulation in the adrenal medulla following glucoprivation: An in vivo analysis

by Larisa Bobrovskaya, Hanafi A. Damanhuri, Lin Kooi Ong, Jennifer J. Schneider, Phillip W. Dickso, Peter R. Dunkley, and  Ann K. Goodchild

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Catecholamine synthesis in the adrenal medulla is dependent on the rate-limiting enzyme, Tyrosine Hydroxylase (TH). When chromaffin cells of the adrenal medulla release catecholamines such as epinephrine (Ad) and norepinephrine (NAd), an increase of catecholamine synthesis follows in order to keep intracellular catecholamine levels steady. An increase of catecholamine synthesis to maintain homestatic conditions suggests further regulation of the synthesis and activity of TH. TH activity is regulated by phosphorylation of residue Ser40 after feedback inhibition by catecholamines. Furthermore, Phos-Ser31 residue can double TH activity alone, while phosphorylated Ser31 and phosphorylated Ser19 is known to increase rate of Ser40 phosphorylation, which in turn increases TH activity.

The mechanisms behind TH regulation had only been experimented in vitro or in situ prior to this study.  Therefore, this study sought to identify various signaling pathways that regulate TH activity in conscious rats following a physiological stimuli which increases plasma levels of catecholamines. In this case, the physiological stimuli was a single episode of 2-deoxy-d-glucose (2DG), used to evoke glucoprivation to promote increases in plasma Ad and thereon increasing medullary TH levels. By increasing plasma catecholamine levels, the neurotransmitters or signaling systems involved in increasing medullary TH levels during glucoprivation could be analyzed. 


Materials and Methods: One day prior to the experiment, Sprague-Dawley rats (n=44 total) were housed in a temperature-controlled room with free access to food and water.  On the day of the study, rats were injected intraperitoneally  with either 2DG or saline as a control. Food and water were immediately removed from the cage following injection, and the rats were sacrificed after either 5, 20, 60 mins, or 24 hours. 

• Blood samples were taken for catecholamine analysis using a liquid-liquid extraction assay.
• In some cases, adrenal medullas were separated from cortices, and medullary membranes were immunoblotted with antibodies for analysis of phosphorylated Protein Kinase A (PKA), Protein Kinase C (PKC), MAPK (Mitogen-activated Protein Kinase), and MAPK/Cyclin-dependent kinase (CDK) substrates
• Whole adrenals were processed and run on SDS-PAGE to measure TH phosphorylation of Ser 19, 31, and 40, expressed as ratios to total TH. 

Results:

1. Catecholamine and BGL: The results revealed significant increases in Ad and Nad in the plasma 20m after 2DG injection, but not 24h after injection. There were also significant increases in blood glucose levels after 20 and 60m of 2DG injection 
2. Protein Kinase Activation: Phosphorylated PKA levels were significantly elevated  after 20 and 60m of 2DG injection, however no significant changes in PKC levels were observed. There was no significant difference of MAPK or MAPK/CDK phosphorylation at 20m, but there was  a significant increase at 60m.  
3. TH residues to total TH protein: Ser19 phosphorylation was not significantly changed at 5, 20, or 60m. Ser40 appeared to be activated after 20m and then declined with time, returning to baseline at 24h. Ser31 increased at 20m, reached maximum residue/protein at 60m, and returned to baseline at 24h. Total TH was only significantly increased 24h after 2DG injection.

Discussion: 

The data of this experiment suggested glucoprivation evokes increases in Ad and NAd plasma levels, which resulted in increase plasma glucose levels. Furthermore, activation of signalling pathways varied with respect to time, in which Ser40 was activated and declined sooner than Ser31 was, and changes in activation of Ser19 to total protein remained insignificant. Activation of PKA and MAPK/CDK substrate phosphorylation after 20 and 60m of 2DG injection but not PKC indicates effects of glucoprivation on specific protein kinases involved in TH activity. The significant increases in total TH protein after 24h of glucoprivation suggests a response that restores and also increases Ad in the adrenal medulla. 

This study is relevant to my project due to the importance of catecholamines in the cardiovascular response to exercise, which is a metabolic stressor. This study provided insight to the regulatory mechanisms that maintain TH and epinephrine levels within the adrenal medulla following a metabolic stressor involved in the production and release of catecholamines. Since the pathway of catecholamine synthesis is dependent on TH, activation of different protein kinases involved in TH phosphorylation (such as PKA or MAPK) may also have a notable role in the production and replenishment of catecholamines. Without the effects of kinases on TH phosphorylation, we would expect to see altered physiological responses and, more specific to our studies, altered catecholamine synthesis and secretion which in turn would change cardiovascular responses such as heart rate, contraction, conduction, and vasodilation during periods of physical activity. 

-NSS

Ventrolateral Medulla AT1 Receptors Support Arterial Pressure in Dahl Salt-Sensitive Rats

By Saturo Ito and Alan Sved

Angiotensin II acting in the brain has been implicated in the pathogenesis of hypertension. The site at which angiotensin acts to maintain increased AP in hypertensive rats is unknown (at the time this paper was written). It is thought that the RVLM could play a role for a number of reasons, one of which is the high concentration of angiotensin receptors (AT1 receptors). The RVLM may also play a role in the effects of changes in dietary salt intake on cardiovascular regulation.

This study tested the hypothesis that activation of RVLM AT1 receptors contributes to the increased AP in rats fed a diet high in sodium. The role of the PVN in the maintenance of resting AP was also examined.

All rats were initially fed a diet containing 0.3% NaCl for at least 4 weeks and then some were switched to a high salt diet (8% NaCl) 4 weeks prior to experiments. Rats were anesthetized and prepared for measuring AP and HR. The RVLM was identified with glutamate injections before ANG II injections followed by valsartan (binds to AT1 receptors and inhibits ANG II action) injections. For PVN experiments, rats were first tested with injections of bicuculline (GABA receptor antagonist) followed by injections of muscimol (GABA receptor agonist).

It was found that injection of valsartan into the RVLM or injection of muscimol into the PVN produced a significant decrease in MAP in the high-salt diet hypertensive rats, whereas these treatments had little effect on MAP in low-salt diet rats. These results suggest that RVLM AT1 receptors are tonically activated in hypertensive rats. Additionally, they suggest that the PVN may contribute to the maintenance of baseline AP by way of a tonically active angiotensin-mediated input to the RVLM.

-BH

Acute sympathoexcitatory action of angiotensin II in conscious baroreceptor-denervated rats

By Ling Xu and Alan F. Sved

Angiotensin II (ANG II) levels have two competing influences on sympathetic outflow. ANG II seems to increase SNA. However, it also acts as a vasoconstrictor to increases MAP, which in turn stimulates baroreceptors, thereby inhibiting SNA. This makes it hard to determine the direct actions of ANG II. In order to determine what it directly effects, this experiment administered ANG II to sinoaortic-denervated rats (no baroreflex). 

Baseline MAP was higher in SAD than control rats. In control rats, infusion of ANG II rapidly increased MAP, which was accompanied by bradychardia and sympathoinhibition. Over time, HR and LSNA slowly returned to normal by the end of the infusion period. In contrast, denervated rats had a larger initial increase in MAP upon ANG II injection. However, the increase in MAP was accompanied by increased HR and LSNA, rather than the decrease seen in the control group. This indicates that ANG II can produce rapid sympathoexcitation. There are several sites at which ANG II could act to increase SNA and HR. It has been shown to act directly on the heart in high concentrations. Alternatively, in might increase HR by increasing sympathetic neural activity to the heart and/or decreasing parasympathetic neural activity to the heart. ANG II may act on areas of the CNS lacking a blood brain barrier, such as the area postrema. I will expand on its effects on the CNS in future blog posts.

PNMT-containing neurons of C1 cell group express c-fos in response to changes in baroreceptor input

by Alan F. Sved, Dara L. Mancini, Jennifer C. Graham, Ann M. Schreihofer and Gloria E Hoffman

This study used the expression of c-fos as a marker of neuronal stimulation to determine whether decreased baroreceptor afferent activity could activate PNMT-containing (C1) neurons.

Baroreceptor afferents were decreased using two treatments: hydralazine injection (vasodilatoràhypotension) or surgical denervation of the carotid sinus and aortic baroreceptors (artificial hypotension). Control rats received saline injections or sham denervation, respectively. Some rats received Fluorogold injections to retrogradely label bulbospinal RVLM neurons. Finally, neurons were stained for Fos, the protein produced by the c-fos gene. Separate groups of rats had catheters placed in the right femoral artery to record MAP and heart rate in response to hydralazine or sinoaortic denervation.  Stained neurons were counted via light microscopy.

In hydralazine-injected rats, approximately 80% of the PNMT-positive neurons in the RVLM were immunoreactive for Fos. This was true of the entire population of PNMT-positive neurons as well as the specific group of PNMT-positive neurons that were also labeled with Fluorogold. Approximately 45% of the Fluorogold-positive neurons contained PNMT in the hydralazine- and saline-injected groups. Approximately 40% of the PNMT-positive neurons were also label with Fluorogold. If 80% of the spinally projecting C1 neurons are affected by baroreceptor afferent input, and C1 neurons comprise 40% of the bulbospinal RVLM neurons, then greater than one-third of the bulbospinal RVLM neurons that are sensitive to baroreceptor afferent input must be C1 neurons. Neurons in the RVLM express Fos in response to sinoaortic denervation as well as hydralazine injection.

These results are central to a lot of the work we do in our lab.

-BH

Organization of Central Adrenergic Pathways: I. Relationships of Ventrolateral Medullary Projections to the Hypothalamus and Spinal Cord

By Diane Tucker, Clifford Saper, David Ruggiero and Donald Reis

This experiment had multiple purposes including:

- Determining whether PNMT-positive C1 cells contain other marker of catecholamine biosynthesis

- To determine the extent to which C1 neurons in the VLM send collaterals to both the spinal cord and the hypothalamus

- To examine the collateralization of VLM neurons of the A1 cell group that project to PVH and median preoptic nucleus (MnPO)

This summary will only focus on objective #2, since the others are not the relevant to what I am doing.

Dye was injected into rat spinal cord and hypothalamus. Rats were perfused, sacrificed, and their brain tissue was obtained and immunohistochemically processed. Cell counts were performed of:

- Total TH and PNMT positive cells

- The number of cells retrogradely labeled with each fluorescent dye

- The number of cells retrogradely labeled with both fluorescent dyes

- The number of cells labeled with each fluorescent dye that showed immunoreactivity for TH or PNMT

- The number of cells labeled with both fluorescent dyes that showed immunoreactivity for both TH and PNMT

Results showed that about half of the spinal projection cells in the RVLM were PNMT-positive. Catecholaminergic neurons throughout the length of the VLM project to the hypothalamus. The PVH also receives catecholaminergic afferents from the entire VLM. While there is sometimes considerable spatial overlap between cells with different projections (spinal cord, hypothalamus, PVH), very few VLM neurons are found to innervate more than one region.

There was no evidence for noradrenergic projection from the MVLM or CVLM to the spinal cord.

It should be noted that about half of the neurons in the RVLM that project to the spinal cord did not stain for PNMT. These other cell populations may play a role in blood pressure modulation as well.

-BH

RVLM: Selective Projections to the Thoracic Autonomic Cell Column from the Region Containing C1 Adrenaline Neurons

By Christopher A. Ross and Donald J. Reis

The purpose of this study was to determine the organization of projections from the RVLM to the thoracic spinal cord and to compare these to the distribution of PNMT-containing terminals in the intermediolateral column (IML).  This was done by first staining for TH and PNMT. Next, some animals had horseradish peroxidase injected into their spinal cord or adrenal medulla to act as a retrograde tracer. Additionally, some animals had HRP injected into their RVLM to act as an anterograde tracer.

The results showed that a large proportion of the projections from the RVLM to the IML are made up of PNMT-containing C1 neurons. This finding, along with previous findings that demonstrated that stimulation of the RVLM results in pressor responses and that lesions of the C1 regions cause a drop in MAP, leads us to believe that the C1 neurons play an important role in blood pressure regulation.

-BH

PNMT-containing terminals synapse directly on sympathetic preganglionic neurons in the rat

by. Teresa A. Milner, Shaun F. Morrison, Cory Abate and Donald J. Reis

The purpose of this study was to determine whether PNMT-containing terminals in the intermediolateral (IML) nucleus influence sympathetic nerve discharge through synapses directly on sympathetic preganglionic neurons (SPNs). This was done by using the peroxidase anti-peroxidase method to localize PNMT-containing terminals. Additionally, horseradish peroxidase (HRP) retrograde identification of SPNs was used to determine whether or not PNMT-containing terminals made direct contact with SPNs. The results showed that PNMT-containing terminals synapse directly on SPNs. This allows us to classify C1s as premotor neurons of the sympathetic nervous system.

Another interesting finding from this study is that PNMT-containing terminals form both asymmetric (excitatory) and symmetric (inhibitory) synapses with dendrites in the IML.

-BH

Rostral ventrolateral medulla: a source of the glutamatergic innervation of the sympathetic intermediolateral nucleus

by Shaun F. Morrison, Janie Callaway, Teresa A. Milner and Donald J. Reis

The purpose of this study was to demonstrate whether or not neurons in the RVLM contribute to the glutamatergic innervation of the intermediolateral (IML) nucleus in the spinal cord. This was done by injecting PHA-L (anterograde label) into the RVLM and subsequently treating the IML with PAP to detect PHA-L. The results confirmed that the axon terminals of RVLM neurons that project to the IML contain glutamate-like immunoreactivity and make excitatory contacts upon local dendrites.

Short but sweet!

-BH

Lesions in Rostral Ventromedial or RVLM Block Neurogenic Hypertension

by Kurt J. Varner, Elisardo C. Vasquez and Michael J. Brody

This study used NMDA-induced lesions of the RVLM or RVMM to determine whether neurons in either region are involved in the acute hypertension and tachycardia produced by sinoaortic deafferentiation (SAD). SAD is a technique used to induce hypertension by surgically interrupting peripheral baroreceptor afferent nerves.

First, NMDA was injected into the RVLM and RVMM. As the name would suggest, NMDA binds to NMDA-receptors, which are the glutamatergic receptors on neurons in the RVLM. However, NMDA does not have the same excitatory effects that glutamate does. Instead, it is an excitotoxin which means that it kills neurons by overexciting them. Following NMDA-lesion, SAD was performed. There were 4 groups used in this experiment: sham lesion (saline injection into RVLM or RVMM), NMDA lesion (lesion of sites rostral to RVLM or RVMM), RVLM lesion and RVMM lesion.

MAP decreased in both RVLM and RVMM conditions. RVLM lesioned animals showed the lowest MAP in both post-lesion and post-lesion + SAD conditions. The lack of hypertensive response likely reflects the loss of baroreceptor-sensitive sympathoexcitatory neurons in these regions. This study shows that both the RVLM and RVMM are involved in the development of neurogenic hypertension under these conditions.

-BH

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.