Neurosteroid modulation of arterial baroreflex function in the rostral ventrolateral medulla

Heesch, C. M. Neurosteriod modulation of arterial baroreflex function in the rostral ventrolateral medulla. Auton Neurosci 161(1-2): 28-33, 2011

CVD and hypertension are leading causes of death in the United States and risks are lower in young women than aged matched men. Ovarian hormones have significant effects, both genomic and nongenomic, on the central nervous system and cardiovascular system. 3α-hydroxy-dihydroprogesterone (3α-OH-DHP), also termed allopregnanolone, is a positive modulator of gamma aminobutyric acid (GABA) receptors that facilitates chloride into the neurons possibly by increasing the opening time of channels in the rostral ventrolateral medulla (RVLM). In fluctuation with the ovarian cycle, levels of 3α-OH-DHP are elevated in pregnant animals. Heesch's experiment was performed to determine if arterial baroreflex activity and its control on lower SNA in pregnant females could be mimicked by microinjecting allopregnanolone into the RVLM to decrease mean arterial pressure (MAP) in non-pregnant females. SNA was recorded for 15 minutes after microinjections. Heesch's results suggested reduced firing of the baroreflex and less activation of RVLM cells, resulting in hypotension (decrease of blood pressure). Lower SNA was reported in pregnant females than in non-pregnant females with less sympathoexcitation of the arterial baroreflex. In addition, increased GABAergic effects were interpreted in the RVLM, lowering the excitability (action potential) of the cell and attenuating neurotransmission release of glutamate at the spinal cord. Mean arterial pressure (MAP) did not change following microinjection of either the neuroactive progesterone metabolite or inactive isomer. The effects of progesterone metabolite are most likely nongenomic (binds to membrane vs nuclear receptors) and enhance effects of GABA. In previous studies from our laboratory, the association between males and females has been further examined. The variability of ovarian hormones like estradiol and progesterone are possible explanations for decreased MAP, resulting in lower risks of CVD in premenopausal women.   

-GKG

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