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

Sympathetic nervous system mediates cold stress-induced suppression of natural killer cytotoxicity in rats

Xing-Hong Jiang, Shi-Yu Guo, Shuang Xu, Qi-Zhang Yin, Yusuke Ohshita, Michiko Naitoh, Yuzo Horibe, Tadashi Hisamitsu. Neuroscience Letters (2004)

The hypothalamic-pituitary-adrenal (HPA) axis is a complex set of pathways and feedback systems that include the hypothalamus, the pituitary gland, and the adrenal glands. The HPA is a neuroendocrine system that controls reactions to stress and regulated the body. The corticotropin releasing hormone neurons in the paraventricular nucleus (PVN) are where the HPA axis starts. The locus coeruleus is an area in the pons clustered with noradrenaline containing neurons that regulates the sympathetic nervous system and the responses to stress. Previous studies have shown that a loop exists between the corticotropin releasing hormone neurons and the noradrenaline containing neurons which plays a big role in the response to stress. The goal of this study is to see how cold stress causes immunosuppression and using 6-OHDA, which is neurotoxic compound that destroys noradrenergic neurons, to cause chemical sympathectomy. The 6-OHDA is given by intracerebroventricular (ICV) injection or intraperitoneal (IP) injection to compare how cold stress effects the central noradrenergic system and the peripheral sympatho-noradrenergic system.

Adult male Wister rats weighting about 250 grams were used in this study. After allowing the rats to become acclimated to the conditions for three days, they were split into two main groups: a stress group and a non-stress group for a control. The stress group consisted of 4 subgroups: stress alone, ICV 6-OHDA and stress, IP 6-OHDA and stress, and IP propranolol and stress. To create the cold stress, the rats were placed in cages kept at 4 degrees Celsius, while the non-stress rats were kept at 22 degrees Celsius. After they were exposed to the cold, the rats were anesthetized and a blood sample was taken for the corticosterone radioimmunoassay. The spleen was then removed to be used in the natural killer cell assay. The toxicity of natural killer cells were measured by a chromium release assay. A concentration of 1E7 splenic lymphocytes/ml were used as the effector cells and chromium labeled YAC cells were used as target cells. Effector/ target cell ratios of 100:1, 50:1, 25:1, and 12.5:1 were used to titrate effector cells against target cells.

After the rats were exposed to the cold stressor of temperatures under 4 degrees Celsius, levels of plasma corticosterone levels were shown to be significantly elevated when compared to the group that did not receive the cold stressor (298.5 ± 78.03 ng/ml increased to 467.25 ± 74.8 ng/ml). When the rats were then given an injection of 6-OHDA, the plasma corticosterone levels were shown to significantly decrease (403.25 ± 54.3 ng.ml). When the 6-OHDA was injected into the group that did not receive the cold stress, there was no significant change in corticosterone levels, which suggests that the cold temperature stress leads to the hypothalamus activating the pituitary gland to release corticotropic releasing hormone. 

When observing the results of natural killer cytotoxicity between the two groups, the group that received cold stress exhibited a significantly lower level than the non-stressed group at each of the effector/target cell ratios. The results also show that when 6-OHDA was injected, the activity of natural killer cells significantly increased, which decreases the suppressive effect that the cold stressor presented. 

Researchers than studied the levels of Fos, which is associated with cell survival. When rats were exposed to the cold, Fos levels were increased in the PVN neruons compared to the non-stressed rats. The results showed 279.64 ± 33.53 Fos positive neurons in the stressed rats and only 9.17 ± 3.49 in the non-stressed rats. When the rats were given a intracerebroventricular injection with 6-OHDA, the stressed rats exhibited a significant decrease in Fos expression. When the same rats were given a intraperitoneal injection of 6-OHDA, there was no significant change in Fos expression, which suggests that activation of PVN neurons depend on the central noradrenergic system and not the peripheral noradrenergic system. Fos expression caused by cold stress followed a similar pattern in the LC and the effect of 6-OHDA was similar as well. 

Previous studies have shown that norepinephrine in the PVN is able to stimulate the release of corticotropin releasing hormone. Since 6-OHDA caused a decrease in Fos expression and there was an increase of plasma corticosterone because of the cold stress, the researchers suggest that the activation of the HPA axis due to cold stress might be mediated by central noradrenergic system.

Recently in one of my classes we discussed how the correct temperature for rats to feel comfortable is higher than originally thought. The thought is that until recently, many years of research on rats may have been done under cold induced stress, which could completely change outcomes and results. In our lab, we do in fact keep temperatures at the correct level, but now having this knowledge has caused me to look more closely at papers I am reading. I chose this paper to just read about a few of the effects cold induced stress can have on a rat.

-Paul M

Mu-opioid receptor inhibition decreases voluntary wheel running in a dopamine-dependent manner in rats bred for high voluntary running

Booth et. al.

Neuroscience

                Physical inactivity has been associated with many disease states including hypertension, cognitive dysfunction and other diseases. Rodent studies have shown that there is a genetic component in amount of voluntary physical activity and this laboratory has selectively bred rat models to study the difference between high and low physical activity. This paper tested the hypothesis that the dopamine and opioid systems interact to influence physical activity, specifically the motivation to run. The dopamine system in the midbrain is important for the motivation to run and this system is partly regulated by endogenous opioids. For all experiments High Voluntary Running (HVR) bred rats and Low Voluntary Running (LVR) rats were used. All rats were female.

                In experiment one inherent differences in opioid receptor expression and function were assessed in sedentary HVR and LVR rats. Rats were sacrificed at 8 weeks of age and opioid receptor mRNA levels were assessed in multiple areas of the midbrain. It was found the in HVR rats there was a significantly higher expression of opioid receptor mRNA and protein in the nucleus accumbens and a significantly higher expression of just the mRNA in the acruate nucleus.

                In experiment 2 the effects of the opioid antagonist naltrexone were assessed for neuronal activation in multiple areas of the midbrain. Fos was used as a measure of neuronal activation. This experiment showed that injection of naltrexone intraperitoneally significantly reduced Fos expression in the nucleus accumbens, acruate nucleus in the HVR rats but not the LVR rats.

In experiment 3 both HVR and LVR rats were given intraperitoneal injections of naltrexone and then wheel running and food intake were measured. All rats were given injection on the night of proestrus. This experiment showed that HVR rats were running more than LVR rats as expected, however, injection of naltrexone significantly decreased wheel running of HVR rats but not LVR rats. Food intake was also decreased in HVR rats but not LVR rats.

In experiment 4 nucleus accumbens dopamine nerve terminals were ablated and then running distance and food intake were measured. This experiment found that ablation of the dopamine nerve terminals did not have a significant effect on its own on running activity, however, when paired with injections of naltrexone, naltrexone no longer had the effect of decreasing running activity.

The major findings of this paper are that opioid receptor expression is higher in nucleus accumbens of sedentary HVR rats compared to LVR rats, opioid antagonism decreases mRNA’s associated with dopamine signaling in HVR but not LVR rats, naltrexone reduces wheel running in HVR but not LVR rats, and HVR rats with ablated dopaminergic terminals in nucleus accumbens are refractory to the decreases in wheel running associated with naltrexone. This paper postulates novel ideas for the interactions between the opioid and dopamine systems in the genesis of voluntary wheel running.

Ben R

Rethinking progesterone regulation of female reproductive cyclicity

Kubota et. al. 2016

Proceedings of the National Academy of Sciences, USA

                Control of the female reproductive cycle by the hypothalamic-pituitary-ovarian axis has been known for a long time. Hormones secreted by the hypothalamus cause the pituitary to produce hormones and release them into the bloodstream to affect the ovaries to release their hormones – progesterone and estrogen which in turn provide negative and positive feedback to the axis. This feedback is at the center of the regulation of female sex hormones. The current paper aimed to assess the role that progesterone has via the progesterone receptor on the cyclicity of the rat female reproductive cycle.

                Sprague-Dawley female rats were used in this experiment and maintained on a 14:10d light dark cycle. CRISPR/Cas-9 genome editing was used to create progesterone receptor mutations that rendered the receptors nonfunctional. This was confirmed with multiple measurements including ovulation, fertility of the rat, uterine responses to progesterone, mammary gland branching morphogenesis and others. All these measurements failed in the null progesterone receptor animals as expected from other studies. Vaginal lavages and cytology were performed to determine the stage and regularity of the reproductive cycle as well as analysis of blood samples that were taken during each of the stages. Rats were also allowed to run voluntarily on a wheel and this was recorded to determine daily running activity during the cycle.  

                It was found that despite the absence of a functional progesterone receptor, the progesterone receptor null rats demonstrated a highly regular estrous cycle. The paper states that this is in contrast to a similarly genetically modified mouse. Cyclic changes were observed in both groups of rats, progesterone null and wild type, in multiple measurements including morphology of the vaginal cell types, wheel running activity, hormone levels and uterine weights. However, it was found that there were some differences in the cyclicity and length of the estrous cycle between wild type and progesterone receptor null rats. Specifically, the estrous cycle was significantly longer in the wild type rats compared to the progesterone receptor null rats. It was also found that progesterone treatment abolished the estrous cycle in wild type rats but did not have an effect in the progesterone receptor null rats.

                Consistent with other papers the current study showed that progesterone is necessary for many aspects of female fertility, however, this paper provides evidence that while progesterone may be important for regulating the female reproductive cycle, it is not necessary for a rat to keep cycling. Progesterone does seem to be important for the feedback loops of the hypothalamus-pituitary-ovarian axis but is not necessary for the regular cycling of the estrous cycle. These findings were unexpected in the paper as previous studies have shown that in a progesterone receptor null mouse the cyclicity of the reproductive cycle is abolished. It seems that estrogen is the driving factor for the cycling of the cycle in rats, although, progesterone is important for many secondary sex characteristics and sexual behaviors.

Ben R

METHODS OF ANALYSIS AND PHYSIOLOGICAL RELEVANCE OF RHYTHMS IN SYMPATHETIC NERVE DISCHARGE

By: Susan Barman and Michael Kenney (Clinical and Experimental Pharmacology and Physiology (2007) 34, 350–355)

The basis of biology relies heavily on the idea of rhythmicity. More specifically,  the functions of every day life, such as a heart beat, breathing, flexion-extension, etc. The autonomic system is vital in a lot of these rhythm controls.

Three aspects of rhythm are discussed to be an important contributor. The first being the idea of synchronization. The second is the ability to predict repetitive events. Lastly, information encoded by frequency is more resistant to distortion by noise.

To quantify rhythmicity, one can perform a time domain analysis or a frequency domain analysis. Time domain will use time to detect periodicity by comparing a signal to a replica of itself. The lags in the signal will represent the peaks and valleys of the original signal. Fast Fourier transformation is an algorithms used to analysis the frequency of a signal. It matches the signal to sine waves over a range of frequencies. Frequency domain is more efficient to use, especially if a signal has multiple components. These analysis are also vital to assessing the correlation between two signals, or coherence. It defines the strength of the signal at each specific frequency. A value of 1.0 means the signal is undisturbed by noise.

Signals can vary depending on the physiological state of the animal, the type of nerve being recorded from, and what the species actually is. The characteristic of the signals also relate it to various events in the biological system, for instance, a heart beat or a respiratory cycle. These are called the cardiac-related rhythm and the respiratory-related rhythm. A range of frequencies will correspond to these events. The alteration of these physiological events will alter the signal. For example, smooth muscle can be induced at frequencies below 0.5 Hz, while arterial pressure in cats is controlled at a frequency of 10 Hz. One observation pointed out was that in an aperiodic setting, the signal was not as strong, but when signals were compared to each other, the coherence was high. This observation was crucial to validating the importance of synchronization of activities. The inputs that are driving each of these signals are strongly related. They also pointed out the prevalence in change when the body attempts to maintain homeostasis. For example, when acute heat or hypothermia is introduced, synchronization of sympathetic nerve discharged was gone.

The signals are vital to the responses in the biological system. It keeps processes in line and in sync with each other. When one thing is effected, the signaling changes, to address this change.


-Tsetse Fly