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

Characterizing the reduction of stimulation artifact noise in a tripolar nerve cuff electrode by application of a conductive shield layer

Parisa Sabetian, Bita Sadeghlo, Chengran Harvey Zhang, Paul B Yoo. 

Medical Engineering and Physics (2017)

Nerve cuff electrodes are designed to record activity from a bundle of axons and are able to create excellent nerve recordings, but they are limited by a high signal to noise ratio. This study uses a conductive shield layer (CSL) to reduce the noise contamination of nerve recordings. 

All experiments were non-survival and involved six Spraque Dawley rats anesthetized using isoflurane. The nerve cuff electrode was placed on the sciatic nerve. Simulated external noise was created by introducing a change in resistance with the nerve cuff electrode. The electrode imbalance ratio was increased to further increase the noise levels. The CSL was then added and simulations were conducted with it and without. Results showed that the simulated noise amplitude was significantly reduced by an average of 80% when the CSL was added. They then tested the electrical conductivity of the CSL to see if it had an effect on the magnitude of the noise. The electrode imbalance ratio was set at 10 and the conductivity of the CSL was increased. The results exhibited that the amplitude of the noise was reduced by about 85% when the conductivity of the CSL had values between 1E-1 S/m (siemens per meter) and 1E4 S/m. 

Changing the geometry of the CSL was also tested. When the length of the CSL was increased there was a significant noise reduction. When the CSL was 77% of the cuff, there was a noise reduction of about 34%. When the CSL was made to match the size of the cuff, the noise was reduced by 86%. When observing how the CSL reduced the noise, it was shown that the CSL causes a more uniform potential gradient over the cuff. The noise generated a large potential gradient along the nerve cuff (8.6 mV), but that was significantly reduced when the CSL was added (1.8 mV).

In conclusion, external electrical noise can cause errors in recorded neural activity when using nerve cuff electrodes. The conductive shield layer can offer a solution to the limitations of the cuff electrode and help to create a more accurate animal nerve recording. While we do not use a nerve cuff electrode in our lab, I am interesting in learning about the solutions to external noise reduction in nerve recordings. This past week I have begun to dig deeper into some abnormalities I came across within the nerve recordings and have been curious about the different methods of reduction.

Paul 

The protective effects of voluntary exercise against the behavioral consequences of uncontrollable stress persist despite an increase in anxiety following forced cessation of exercise

The protective effects of voluntary exercise against the behavioral consequences of uncontrollable stress persist despite an increase in anxiety following forced cessation of exercise 

Behav Brain Res. 2012 August 1; 233(2): 314–321. doi:10.1016/j.bbr.2012.05.017. 

Benjamin N. Greenwood1,3,*, Alice B. Loughridge1, Nouara Sadaoui1, John P. Christianson2,3, and Monika Fleshner1,3
1Department of Integrative Physiology, University of Colorado-Boulder 2Department of Psychology and Neuroscience, University of Colorado-Boulder 3Center for Neuroscience, University of Colorado-Boulder 

Physical activity is protective against stress-induced effects and reduces the chance of developing anxiety- and depression-like behaviors. These stress-induced behaviors often include many sequelae. The current study focused primarily on three: social avoidance; shock-elicited fear, and; deficits in escape learning. Additionally, these behaviors and the stress that causes them are shown to coincide with an increase in corticosterone. However, it is unknown how long the protective effects of exercise persist after ending exercise.

The first goal of the study investigated whether or not six weeks of running inhibited the anxiety-linked behaviors. The second goal sought to determine whether or not the protective factors lasted up to 25 days after the forced cessation of exercise. Two experiments were completed to explore these questions. The animals in each of the studies were 6 to 7-week-old male, Fischer F344 rats. They were group houses in cages of 6. The rats each underwent 6 weeks of either running or sedentary conditions. However, each rat’s individual running distance was not accounted for during this time. It would be interesting to see whether the distance ran each day for each rat impacted their results. Additionally, it would be interesting to see if the stressor of being singly-housed would impact the rat running behavior or if running itself would be protective over their development of stress.

Rats either remained inactive or began the “uncontrollable stress” protocol, outlined below, at different times post cessation of exercise—the next day, 4 days, 14 days, or 24 days later. Social exploration began 24 hours after the uncontrollable stress protocol was administered. Shock-elicited fear and shuttle box escape—both of which were used to measure fear conditioning and escape learning—began immediately after the social exploration protocol.

Uncontrollable stress was induced through tail shocks due to its success in producing behavioral results that are similar to the stress-related behaviors observed in humans. The rats were either a part of the “No Stress” group or the “Stress” group that would receive 100, 5s, 1.5 mA tail shocks while restrained in a Plexiglass tube. “No Stress” rats would not experience this protocol at all.

Social exploration was tested at baseline (post-5 weeks of running or sedentary environment) and 1 week afterward (at 6 weeks of running or sedentary environment). Each individual rat would be placed in a clean, separate cage that would have a juvenile male rat introduced 1 hour after habituation. Exploratory behaviors were observed for 3 minutes. After the baseline measurements, the rats would be returned to their home cage. 1 week after baseline measurements, the same procedure would occur 24 hours after the rats underwent the uncontrollable “Stress” protocol. After the final social exploration, the rats either underwent the second experiment (the shock-elicited freezing and escape behavior protocol) or were sacrificed right away to have their corticosterone levels measured. Their corticosterone levels were measured against naïve rats—rats that had not undergone the social exploration protocol.

After the social exploration experiment, the rats were placed in a shuttle box that gave foot shocks on one half and was “safe” on the other. Shock-elicited freezing and escape behavior were used to assess escape learning. Additionally, exaggerated fear—presented as freezing rather than escaping when presented with shock cues—was used as a measure of anxiety-like behavior. While in the shuttle box, the rats underwent a fixed ratio of foot shocks that could be escaped by crossing to the other side of the box. This was followed by a 20-minute post-shock “freezing observation.” The rats then underwent a second fixed ratio of foot shocks. Their “escape latency” was measured before the end of the trial.

Exercise successfully reduced social aversion in rats within the social exploration protocol. Additionally, exercise prevented the increase in corticosterone levels post-social exploration. Therefore, the study concludes that the rats were protected against the stress induced by tail shocks prior to interacting with a novel rat. The rats felt more at-ease to interact with the novel rat and did not experience the stress that coincides with the protocol. 

Interestingly, stress from the tail shock protocol reduced cage crossings during the social exploration protocol regardless of prior exercise. These results suggest that all rats, including the exercise rats, were anxious within their new environment and did not “feel” comfortable enough to explore. This anxiety-like behavior was enhanced the longer the rat stopped wheel running so exercise could be protective in that regard. Therefore, Greenwood et. al conclude that exercise protected the rats from developing the behaviors associated with stress despite having an increase in anxiety (associated with the reduced number in cage crossings). The protective measures disappeared sometime between 15 and 25 days after exercise cessation, suggesting that exercise can protect against the behavioral developments for quite some time.

Only the sedentary rats showed stress potentiated, shock-elicited freezing. The running rats were “protected” from the behaviors associated with stress. However, the cessation of exercise itself did lead to the shock-elicited freezing. That is, the rats who were a part of the “No Stress” group still froze when presented with the cues associated with foot shocks. The freezing occurred as a function of time, affecting the rats who stopped running for 15 and 25 days more so than the 0- and 5-day rats. The anxiety-like behavior of exaggerated fear still emerged, which suggest that the exercise cessation itself is stressful to the rats.

Previous wheel running experience was associated with a removal of the shuttle box escape deficit that is seen in sedentary, stressed animals. Prior exercise exposure did not affect the first fixed ratio latency, which makes sense because no rat would have has learned to associate the keys with the foot shocks yet. Interestingly, the exercised rats were more likely to remember the cues during the second fixed ratio and escaped the foot shocks while the sedentary animals were slower to move to the other side. The sedentary animals are suggested to have impaired fear learning without the protective factors of exercise. These effects were present at 15-days exercise cessation and no longer present at 25 days. Therefore, the protective factors lasted well past the cessation of exercise.

The current study suggests that 6-weeks of running wheel leads to resistance to stress and the associated behavioral sequelae. These protective measures are shown to persist between 15 to 25 days after ending exercise. While the rats still showed signs of anxiety-like behaviors, there were still reductions in the consequences of stress and reductions in the corticosterone response. Therefore, the rats still develop symptoms similar to anxiety, but it does not lead to the negative behaviors that normally arise. Future studies could look into the neuronal plasticity that lead to these behavioral changes in rats and what changes in the brain to allow these behaviors to persist over time. Additionally, researchers should investigate the stress that could arise from the cessation of exercise as this adds a confounding factor in other studies.

-LivInLaVida

Estrogen Increases Locomotor Activity in Mice through Estrogen Receptor alpha: Specificity for the Type of Activity

SONOKO OGAWA, JOHNNY CHAN, JAN-ÅKE GUSTAFSSON, KENNETH S. KORACH, AND DONALD W. PFAFF

Endocrinology 2002

                Past literature has demonstrated that estrogen can regulate running wheel activity in both female and male rats and from a previously blogged paper it was shown that this running activity is highest in estrous stages where plasma estrogen is elevated. Studies have also shown that the medial preoptic area is one of the brain sites responsible for the effects of estrogen. There are two different nuclear types of estrogen receptors: alpha and beta. These are both shown to be localized to the medial preoptic area. The purpose of this study was the assess the effect that estrogen would have on mice that were lacking either the alpha or beta receptor.

                172 mice were used, both male and female. Mice were either lacking the gene for estrogen receptor alpha or beta or were wile type mice. They were kept on 12:12 light dark cycle and group housed. At 9-11 weeks mice were gonadectomized and separated into groups. Groups consisted of estrogen receptor beta and alpha knock outs and their respective wild types in both males and females. Each of these groups either received placebo or 16 ng/d or 160 ng/d replacement of estrogen benzoate. Mice were given one week to recover from surgery and implanted with the treatment or placebo and then given another week before they were given access to a running wheel. At this point the mice were housed individually and the revolutions of the wheel per day was recorded.

                Results of the study show that in both wild type female groups there was significantly increased running activity in the estrogen treated groups as compared to the placebo. Significant difference was only seen in the high dose in the alpha wild type group but was seen in both doses in the beta wild type group. In the alpha knock out group this effect of estrogen was not seen and there was no difference between treatment and placebo. In the beta knock out group both doses of the treatment increased running compared to placebo. The effect of estrogen treatment was lower in the beta knock out than in the beta wild type. In males, treatment increased running activity in both wild types and this was abolished in alpha knockout. Estrogen increased running activity in the beta knock out similar to the beta wild type.

                These results indicate that it is indeed primarily the estrogen receptor alpha that is responsible for modulating the running wheel activity. This is consistent with past research with this group that showed that activation of estrogen receptor beta did not increase running. The results from this study also indicate that it may be that ER beta also plays a role as the effect of estrogen was less in the beta knock out than the beta wild type. This was an interesting paper that tried to identify the mechanism that estrogen has in affecting the running wheel activity of rodents. It is interesting that they used knock out mice that were obtained from a supplier calling into question the effect that knocking these genes out early in the rats life had on its overall development.