Differential activation of adrenal, renal, and lumbar sympathetic nerves following stimulation of the rostral ventrolateral medulla of the rat

Patrick J. Mueller, Nicholas A. Mischel, Tadeusz J. Scislo.

American Journal of Physiology – Regulatory, Integrative and Comparative Physiology (2011)

The sympathetic nerves are part of the autonomic nervous system, which controls unconscious actions, and are important in the response to changes in blood pressure.  The rostral ventrolateral medulla (RVLM) has been shown to direct sympathetic nerve activity (SNA) to cardiovascular targets and may have a role in the increased SNA of cardiovascular diseases. This study specifically looks at three areas of SNA: preganglionic adrenal SNA (ASNA), renal SNA (RSNA), and lumbar SNA (LSNA). The goal of this study is to determine how stimulating the RVLM effects the adrenal, renal, and lumbar sympathetic nerves individually.

This study used seventeen Sprague-Dawley rats and they were anesthetized with a mixture of alpha-chloralose and urethane. Microinjections into the RVLM were done by using a triple-barrel micropipette at a 90 degree angle. Dye was injected after the experiments were done to later identify the RVLM as the injection point. All rats were then killed using Fatal-Plus euthanasia solution.

Different concentrations of glutamate, which is the major excitatory neurotransmitter, were injected to observe responses in nerve activity, blood pressure, and heart rate. When the different concentrations were given in a fixed volume, the blood pressure was shown to be significantly increased and while there was a small increase in heart rate, it was not significant. The preganglionic ASNA was shown to significantly increase at each concentration of glutamate. The 10mM and the 100mM concentrations of glutamate did not produce a significant difference in RSNA, but they were significantly increased from the 1mM concentration change in nerve activity. LSNA was significantly increased after the 100mM glutamate injection when compared to the 1mM or 10 mM concentrations. The preganglionic ASNA was shown to exhibit significant increased activity as the concentrations were increased. Overall, the preganglionic ASNA activity exhibited the largest increase out of the three nerves.

The next test was to keep the concentration fixed but change the volume of glutamate. Volumes of 15, 30, 60, or 90 nl were injected into the RVLM and then changes in nerve activity, heart rate, and blood pressure were observed. While heart rate was shown to not significantly change, blood pressure significantly increased at a volume of 90 nl when compared to 15 nl. Nerve activity did increase, but not as large of an increase as when volume was fixed and concentration increased. When the volume was changed from 15 nl to 30 nl, the preganglionic ASNA was the only one to show a significant increase. At 60 nl the RSNA was significantly increased compared to the activity at 15 nl. Nerve activity at 90 nl of glutamate exhibited increases in both RSNA and preganglionic ASNA, but not LSNA. While there did seem to be a small increase in LSNA, it was not significant.

Another study compared nerve activity after given a dose of sodium nitroprusside (SNP) to the nerve activity after a bicuculline (Bic) injection. SNP lowers blood pressure to cause an increase in nerve activity, which attempts to counteract the dilating effects of SNP. Bic is a GABA receptor blocker which leads to a large increase in nerve activity.  The increase in nerve activity cause by Bic was about four times greater than the increase in nerve acitivty caused by SNP lowering blood pressure. While all nerves exhibited an increased response, preganglionic ASNA had the largest increase in response to SNP and Bic.

In conclusion, all three nerves exhibited increased sympathetic nerve activity when the RVLM was stimulated, though preganglionic ASNA increased more RSNA and LSNA. When the RVLM inhibitor is blocked, the SNA increase is much higher that the natural increase during low blood pressure. What I found most interesting about this study was the difference in activation between the Bic and SNP injections. I had not realized how much greater the response would be if all GABA receptors are blocked. This data helps to reinforce that even if blood pressure is very low and nerve activity is increasing to respond, there are still inhibitory factors in play.

-Paul M

GABAB receptor-mediated mechanisms in the RVLM studied by microinjections of two GABAB receptor antagonists

GIAN LUIGI AVANZINO, PIER0 RUGGERI, DONATELLA BLANCHI, CARLA E. COGO, ROSA ERMIRIO, AND LYNNE C. WEAVER
Istituto di Fisiologia Umana, Universith degli Studi, Viale Benedetto XV 3, I-l 6132 Genoa, Italy; and The John P. Robarts Research Institute, London, Ontario N6A 5K8, Canada
Journal of American Physiology Society (1994)

The current study is one of the first to investigate the role of GABA_B receptors in the RVLM. While it is older than others, the study is important to understand the history of the microinjection technique and the previous investigations of the GABA receptors in the RVLM. At the time of the study, the GABA_B antagonists (2-OH-s and CGP-35348) had just been created, allowing the study to research whether or not the GABA_B receptors were present in the rostral ventrolateral medulla (RVLM). Additionally, Avanzino et al. wanted to determine whether or not GABA_B receptors contribute to the regulation of the sympathetic nervous system output (which the researchers call the central cardiovascular regulation).

The animals were paralyzed and artificially respirated after the first round of microinjections to control for any respiratory changes during the measurements. Heart rate and blood pressure were measured during the microinjection experiments. The researchers assumed that if the GABA_B receptors were present in the RVLM, the antagonists would produce an increase in the blood pressure and heart rate. Two different microinjections followed: 1. Rats received bilateral microinjections of one of the GABA_B receptor antagonists or; 2. Rats received unilateral microinjections of the antagonist followed by either a GABA_A or GABA_B receptor agonists following the electrolytic lesion of the contralateral RVLM.

The first experiments produced increases in blood pressure and heart rate after the microinjections of either of the GABA_B receptor antagonists. These results held true after the rats were paralyzed, as well. The researchers concluded that the GABA_B receptors could be present in the RVLM and may contribute to the regulation of the central cardiovascular regulation. To further investigate this, the researchers injected the antagonists with either a GABA_B agonist or a GABA_A agonist. During this second experiment, the microinjections were completed unilaterally after the electrolytic lesion of the opposite RVLM. After the injections of 2-OH-s and CGP-35348, either badlofen (the GABA_B agonist) or muscimol (the GABA_A agonist) followed. Both of these agonists produced depressor responses when injected alone. However, the antagonists prevented the decrease in blood pressure only when injected with badlofen. The muscimol + antagonist injections were not significantly different to the muscimol alone. The same results were obtained in the paralyzed animals.

The results from the study suggested that GABA_B receptors are present in the RVLM in rats. These results are useful for future studies looking to further investigate the regulation of the RVLM in the central cardiovascular regulation. Additionally, the protocol of the experiment shows the significance of the microinjections. This experiment type is useful for studying the presence of receptors, and for investigating the function of brain regions, specifically the RVLM.

-LivInLaVida

Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla

By Satoshi Koba, Eri Hanai, Nao Kumada,  Naoya Kataoka, Kazuhiro Nakamura, and Tatsuo Watanabe 

Division of Integrative Physiology, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago, Tottori 683-8503, JapanDepartment of Integrative Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan 
Journal of Physiology, 596.19 (2018) pp 4581–4595 

The rostral ventrolateral medulla (RVLM), a brain region involved in the baroreceptor reflex, is essential in the regulation of blood pressure through spinally projecting neurons that contribute to the sympathetic nervous output. The RVLM receives afferent input from the paraventricular nucleus (PVN), which is thought to regulate the outflow from the RVLM (PVN-RVLM neurons). However, the excitatory role of the PVN-RVLM neurons has never been researched prior to this study. Through the use of optogenetics, Koba et al. investigated the excitatory role of the PVN-RVLM neurons on the effects of renal sympathetic nerve activity (RSNA) and the coinciding changes in mean arterial pressure (MAP).

Three experiments were completed in order to answer the study’s question: 1. Photostimulation of PVN-RVLM axons on renal sympathetic nerve activity; 2. The effects of glutamate receptor blockade in the RVLM on the photostimulation of PVN and; 3. The effects of intermittent photostimulation of PVN-RVLM neuronal cell bodies on renal sympathetic nerve activity.  

The study used rats more than 7 weeks old to complete the experiments. To prepare the animals for the optogenetic studies, their PVN-RVLM neurons were first transfected with either a control virus vector (pAAV2-CMV-palGFP) or the channelrhodopsin-variant containing vector (pAAV2-CMV-ChIEF-tdTomato). The vectors were microinjected into either the PVN unilaterally or the RVLM bilaterally, depending on which experiment the animals were used for. Immunofluorescence staining determined which type of neuron was transfected after the experiment.

Experiment 1 animals received unilateral microinjections of the vectors unilaterally within the PVN. The experiment found that the active vector animals had significant increases in RSNA and MAP compared to baseline after photostimulation within the RVLM. Thus, the pre-synaptic PVN neurons expressed channelrhodopsin, which would activate the neurons at PNV-RVLM neurons, leading to excitation down to the renal sympathetic nerve. This did not happen in the control animals, signifying that they did not have channelrhodopsin expressed in the synapses of the PVN-RVLM animals. These neuron groups also expressed a large amount of VGLUT2 (vesicular glutamate transporter 2) in the PVN-derived axons, which lead the researchers to think that the glutamate transmission to the RVLM leads to excitation.

Experiment 2 investigated whether or not the PVN-RVLM neurons acted through glutamate to produce sympathoexcitation. Glutamate blockers AP5 and CNQX were unilaterally microinjected into the RVLM. After glutamate injection, the photostimulation did not produce the sympathoexcitation that was previously measured. Thus, the researchers concluded that glutamate was at least one of the neurotransmitters needed for sympathoexcitation with the PVN-RVLM neurons.

To further understand the how the PVN-RVLM neurons work, the cell bodies within the PVN received photostimulation, rather than the axons (Exp. 1). The cell bodies received 1-minute intervals of photostimulation at 10, 20, and 40 Hz, revealing a “synchronous” activation at the level of the renal sympathetic nerve. The level of activation correlated to the level of Hz used to stimulate the nerve.

The researchers concluded that the glutaminergic PVN-RVLM neurons help to drive sympathoexcitation. These neurons act on C1 neurons within the RVLM, which then project down the spinal cord to modulate blood pressure in the rats. While this study was interesting, it stuggled with transfecting all of the animals. Therefore,  it is difficult to say that all possible PVN-RVLM neurons were correctly transfected, which may produce inaccurate results otherwise. More research should be done of the efficacy of the optogenetic methods used within this paper. Furthermore, the role of non-C1 neurons, which also receive PVN input, may have been activated as well. Their activation may play a role in sympathoexcitation, and more research should be done. It would also be interesting to see the effects in rats younger than 7 weeks old. Additionally, female rats should be investigated, due to effects that estrogen may have on baroreceptor reflex. 

-LivInLaVida

Significance of Obstetrical History with Future Cardiovascular Disease Risk

By: Bassily et al. (American Journal of Medicine, 2018)

When a female is pregnant, her body undergoes major changes in the cardiovascular system. These changes are important for fetal development. This review article hopes to study the effects of the complications that a mother's health can come across during pregnancy. Cardiovascular disease (CVD) is one of the leading causes of female mortality in pregnant women.

One possible cause of CVD in pregnancy is gestational diabetes (GD), which is an intolerance to glucose during pregnancy (insulin resistance). Studies have shown that patients who had developed gestational diabetes had an increased risk of developing CVD later on in life. GD also showed to have affects on the vascularity of the mothers. Previous studies reported that there was decreased endothelium-dependent vasodilatation when compared to women who did not report to have GD. Arterial stiffness was also seen in women with GD. GD has also been seen to contribute to post-partum diabetes, hypertension, hyperlipidemia, and insulin resistance.

Another condition seen in pregnancies is preeclampsia, which is characterized by proteinuria and hypertension. "It is the leading cause of maternal and perinatal morbidity." The mechanisms of preeclampsia is not well understood, but is seen to contribute to CVD in both mothers and offspring.

 Women who deliver a new born with a low birth weight (less than 2.5 kg) have a 7-11 times the risk to die from CVD compared to mothers who did not deliver a baby with a low birth weight. There is a link between maternal risk factors for coronary heart disease.

Preterm delivery is premature birth, which is when the newborn is less than 37 weeks old when delivered. Studies show that when this occurs, women have twice the risk of developing coronary heart disease. Preterm labor seems to be an inflammatory process that leads to leukocyte infiltration at the cervix and uterine tissues. The coronary heart disease has been linked to this inflammatory response.

Healthcare providers must consider the history of pregnancies to prevent any future development of CVD. Preventable measures such as prescribing a low dose of aspirin or calcium supplementation will help. History documentation is very important to provide the proper care int he future for the mother.

This article was actually published a few days ago and I thought it was interesting because it relates the idea of how pregnancy can alter the women's future health. We are studying females more in the lab so I think it is important to consider all the different ways that CVD can be developed.


-Tsetse Fly

VOLUNTARY FREEWHEEL RUNNING SELECTIVELY MODULATES CATECHOLAMINE CONTENT IN PERIPHERAL TISSUE AND c-FOS EXPRESSION IN THE CENTRAL SYMPATHETIC CIRCUIT FOLLOWING EXPOSURE TO UNCONTROLLABLE STRESS IN RATS

By B. N. GREENWOOD, S. KENNEDY, T. P. SMITH, S. CAMPEAU, H. E. W. DAY, AND M. FLESHNER

Department of Kinesiology and Applied Physiology, University of Colorado, Boulder, CO 80309, USA
Department of Psychology, University of Colorado, Boulder, CO 80309, USA
Center for Neuroscience, University of Colorado, Boulder, CO 80309, USA 

Neuroscience 120 (2003) 269–281

Voluntary wheel running has been shown to modulate the stress responses in the from the peripheral sympathetic drive, which includes brain regions such as the locus coeruleus, the A5 cell group, and the rostral ventrolateral medulla. These brain regions project down to organs such as the adrenals and spleen. These are organs are important for the stress-induced response, releasing catecholamines into the blood system. Previous research suggests that exposure to exercise prevents the rise in norepinephrine in the blood of rats.

The current study put rats in either 6-weeks of voluntary wheel running or sedentary conditions and then had them undergo either a “control” condition (where they remained in their home cage) or began the 10, 50, or 100, 5-second inescapable tail shocks to induce a stress response. Rats were euthanized at either the baseline, 10, 50, or 100 shock exposure. Their spleens and adrenal glands were removed, flash frozen in liquid nitrogen and had their tissue catecholamine content measured. Additionally, the effects of exercise and sedentary conditions on the central sympathetic network was investigated. C-Fos protein, which is used as a neural-activation marker, was measured at baseline or after 100 tail shocks. The protein was then immunohistochemically labeled. Additionally, tyrosine hydroxylase was measured to ensure that the activity of neurons was measured only in the catecholaminergic cells in the brain regions of interest. This enzyme is essential in making norepinephrine, the neurotransmitter that the neurons use to activate the sympathetically innervated their organ targets.

In both the running and the sedentary rats, exposure to the tail-shock stressor depleted catecholamines, specifically norepinephrine, in the spleen. In the active animals, this effect was attenuated. The tail shocks showed a “dose-response,” meaning that the larger amount of tail shocks depleted more catecholamines. The same effects were measured in the adrenal glands.

c-Fos levels increased in TH-positive neurons in all brain areas after being exposed to stress. However, that increase was attenuated in exercise-exposed rats in specific brain regions that control the splenic sympathetic regulation: the LC, the intermediate to caudal A5 cell group, and the rostral ventrolateral medulla. These neurons project down as the adrenergic and noradrenergic spinally projecting sympathetic neurons to the preganglionic nerves. These then project to the organs.

These results suggest that the running condition may attenuate the splenic sympathetic activity after and during stress by reducing the input from the spinally projecting neurons. These neurons, after being exposed to exercise for 6-weeks, reduce the sympathetic excitatory drive during stress and thus there is less norepinephrine released from the spleen and adrenal nerves.

 -LivInLaVida