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.  

Hemodynamic and Neurohumoral responses to acute hypovolemia in conscious mammals

By: James C. Schadt and John Ludbrook (Dalton Research Center and Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211; and Cardiovascular Research Laboratory, Department of Surgery, University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia)

During the initial studies on hemorrhage, two phases of hemodynamic responses were observed. The first being peripheral resistance and heart rate(HR) increases to compensate for the fall in cardiac output (CO). The second phase involves vasodilation, as opposed to vasoconstriction in phase 1, which is combined with bradycardia, which will cause blood pressure (BP) to drop. Phase 1 has also shown to have an increase in sympathetic nerve activity (SNA). Important contributors to this entire process are the baroreceptors and the receptors in cardiopulmonary regions. They are important for sensing the hemodynamic changes that occur on a beat to beat basis. This information is then relayed to the brain, which will then cause the vasoconstriction in phase 1, followed by vasodilation in phase 2.

Phase 1 features sympathoexcitation, which is the cause of the constricting blood vessels. This process lasts until blood loss exceeds 25-35% of blood volume. The process compensates for the drop in blood volume so that the arterial pressure doesn't fall with it. Norepinephrine (NE) is seen as the major contributor to the constricting blood vessels. This was proven by giving an alpha 1 adrenoceptor blocker, which prevented the blood vessels from constricting. HR is the second factor in phase 1, but does not contribute as much. The rise in CO during tachycardia is not enough to maintain the blood volume. Plasma renin activity also has been shown to rise during phase 1. This also helps in counteracting the fall in BP by increasing peripheral resistance.

When studying the baroreceptor, sinoaortic barodenervation (SAD) was used to prove what type of control the baroreceptor had on hemodynamic responses. During chronic SAD, the responses that occur in phase 1 were abolished, which prevented the ability to compensate for the blood loss. This study was important to show that the afferents are vital to sense the changes that are occurring, which will then signal the body to react.

Phase 2 involves a drop in blood volume and BP passed a critical point that the body is not able to compensate. HR decreases with a decrease in BP. CO is unrelated to the onset of hypotension in this phase. The fall in BP is due to a drop in peripheral resistance. SNA is decreased followed by an increase in vasodilation.

Opioids have also seen to affect acute hemorrhagic hypotension. Naloxone increases BP during a hemorrhage. During normal conditions, it does not have the same affect. This could mean that mechanisms that are antagonized by naloxone are inactive in normal conditions.

I probably should've read this review before I read the experimental studies, but this paper does a great job summarizing the different mechanisms and effects that take place during a hemorrhage. I will use this information during my experiments to visualize what is mentioned on paper, in a real life setting.

-Tsetse Fly

Defense reaction alters the response to blood loss in the conscious rabbit

By: James C. Schadt and Eileen M. Hasser (Am J Physiol Regulatory Integrative Comp Physiol
280: R985–R993, 2001.)

When blood loss occurs, mammals respond to this in two different phases. The first phase is a sympathoexcitatory phase where sympathetic nerve activity (SNA) and blood pressure (BP) increase to compensate. This occurs through the baroreflex, which senses the changes. The second phase of blood loss is the sympathoinhibitory phase, where BP and SNA fall below critical levels and not able to compensate for the blood loss. The purpose of the study is examine the response to blood loss when the defense reaction (fight or flight, i.e. increase BP, HR, and skeletal muscle blood flow) is activated. The study hypothesized that exposure to air jet would lower the rabbits ability to maintain BP during blood loss.

31 rabbits are used in this study. To start the sensory stimulation, air or no stimulation occurred (sham). To induce blood loss, blood was removed at a rate of 8-9 ml/min. The end point of hemorrhage was 5ml after BP reached 40 mmHg. Blood was reinfused at the end of the experiment.

Following the experiments, results showed that air increased BP and HR in both groups. Pressor responses caused an increase in hindquarters blood flow and decrease in mesenteric blood flow. Also, there was a decrease, followed by an increase of renal flow, combined with an increase in cardiac output. In the presence of air, rabbits were better able to defend BP. A greater blood loss was needed to cause a decrease in mean arterial pressure and HR.

The results of the study showed that air jet stimulation extended defense of arterial BP. This was the opposite of what was hypothesized. When a hemorrhage occurs, it usually coincides with stress responses. The data showed that as stressors become greater, it tends to defend the BP more during hemorrhage. Of course, there is still a critical point where the inhibitory phase kicks in and the body cannot compensate any longer. The mechanism by which stressors defend BP is not known. My guess would be an increase in NE and EPI due to the activation of the sympathetic nervous system causes vasoconstriction to occur at a longer rate. Could be more neurotransmitter release or slower breakdown.

This study shows how vital the sympathetic nervous system is to sustaining BP during hemorrhage. The contribution is already high, but when stressors are introduced, it gets even greater. In our lab, we study the SNA that the RVLM contributes too. We can also use the process of blood loss to see how the RVLM compensates to keep BP and HR from falling.

-Tsetse Fly

Oxidative Stress in the Rostral Ventrolateral Medulla Contributes to Cardiovascular Regulation in Preeclampsia

Jiu-Qiong Yan, Fang Huang, Fan Hao, Xiao-ling Su, Qi Meng, Ming-Juan Xu (October 2017)

Pre-eclampsia (PE) is a disorder that occurs during pregnancy and is characterized by high blood pressure and a significant amount of proteins released in the urine. Although there is a substantial amount of research on PE, the exact pathogenesis is not known. Reactive Oxygen species (ROS) are a compound produced during metabolism that play a role in cell signaling, but can have many harmful effects in high concentrations. Oxidative stress is caused by too much ROS production and not enough ROS clearing mechanisms. This study attempts to show how ROS in the RVLM are related to the cardiovascular problems associated with PE.
         

Ninety Sprague Dawley rats were used in this study. Desoxycorticosterone acetate (DOCA) is a hormone that causes signs of PE such as high blood pressure and high levels of proteins in urine. Pregnant rats were treated with DOCA to produce an animal model of PE. This study involved four groups of rats: a non-pregnant control group, a non-pregnant group treated with DOCA and saline (NPS), a pregnant group without any DOCA (NP), and a pregnant group treated with DOCA and saline (PDS). Vaginal smearing was used to confirm that the rats were pregnant. After about 18 days of pregnancy, 24 hour urine samples were collected. High-performance liquid chromatography and an enzyme linked immunosorbent assay were used to measure the levels of proteins in the urine samples. To detect ROS, fluorescence microtopography was used, specifically Dihydroethidium (DHE). A microinjection of Tempol, which is a catalyst that can limit ROS, was performed on the RVLM to see the effects on blood pressure, heart rate, and nerve activity. Sympathetic nerve activity was measured at the renal nerve.

The urine samples collected after 18 days of pregnancy showed that the protein concentration was significantly increased in the PDS group, compared to the control. While there was a small protein concentration level increase in the NPS and NP groups, it was not significantly different from the control. The PDS group also exhibited significant increases in blood pressure and levels of norepinephrine in the urine. The NP and PDS groups both exhibited a significant increase in heart rate when compared to the control group. Examining the results of the fluorescence microtopography showed that ROS levels in the RVLM were significantly higher in the PDS group, compared to the control. ROS levels were shown to be about seven times higher than the control. The enzyme NOX4 was also shown to be upregulated in PDS group, which is responsible for the production of ROS. The enzyme SOD1, which can limit the damaging effects of ROS, did not significantly change in any of the groups when compared to the control. The microinjection of Tempol into the RVLM demonstrated a significant decrease in blood pressure, heart rate, and renal sympathetic nerve activity in the PDS group. The NPS and NP groups did not show a significant change compared to the control.

In conclusion, DOCA was shown to increase protein levels in urine, including norepinephrine. DOCA also caused an increase in heart rate, blood pressure, and ROS. NOX4, which helps produce ROS, was shown to increase when DOCA was injected, while the ROS limiting enzyme SOD1 stayed constant. Overall, DOCA was able to produce a mouse model that exhibits PE symptoms. When treated with Tempol, this mouse model exhibited improved symptoms. Tempol is demonstrated to be a possible therapy for PE or other problems involving oxidative stress in the RVLM. In relation to what we are working on lab, I would like to further understand the role of ROS in the RVLM when comparing different lifestyles. Does a sedentary lifestyle always lead to an increase of ROS in the RVLM? If so, I’m curious what factors are causing ROS to upregulated when compared to a more active individual.

-Paul

SEX DIFFERENCES IN THE SUBCELLULAR DISTRIBUTION OF ANGIOTENSIN TYPE 1 RECEPTORS AND NADPH OXIDASE SUBUNITS IN THE DENDRITES OF C1 NEURONS IN THE RAT ROSTRAL VENTROLATERAL MEDULLA

By J. P. PIERCE, J. KIEVITS, B. GRAUSTEIN, R. C. SPETH, C. IADECOLA, and T. A. MILNER 
Neuroscience, 2009

Division of Neurobiology, Department of Neurology and Neuroscience, Weill Cornell Medical College, 407 East 61st Street, New York, NY 
Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 
University of Mississippi School of Pharmacy, Department of Pharmacology, University of Mississippi, University, MS 

There are many studies that document the sex-differences of cardiovascular disorders in humans. Women have a lower risk of developing heart disease compared to men until the onset of menopause—at which point it flips. The sympathetic nervous system has been argued to contribute to the development of hypertension. The C1 neurons in the rostral ventrolateral medulla (RVLM), which express tyrosine hydroxylase (TH), are the main type of neuron that controls the sympathetic nervous system activity (SNA). They are tonically active, working to maintain the sympathetic vasomotor tone of individuals. When the activity of these neurons is increased, the vasoconstriction of the blood vessels increases and causes a similar increase in blood pressure. Aside from expressing TH, the C1 neurons also express angiotensin 1 (AT1) receptors. When angiotensin acts on these receptors, both the SNA and the blood pressure increases. In order to produce its effects, angiotensin binds to the AT1 receptor which stimulates NADPH oxidase after the phosphorylation of p47, an NADPH oxidase subunit. P47 then translocates to the cell membrane. NADPH oxidase then produces reactive oxygen species (ROS) to increase the blood pressure. Aside from the NADPH oxidase subunits that translocate to the cell membrane, there are also transmembrane units that act as an internalized pool, including p22. Thus, both pools in the RVLM C1 neurons need to be measured to understand the influence of NADPH oxidase on the development of hypertension. Additionally, the AT1-NADPH oxidase interaction may be where the sex-differences in hypertension originate.

Male, proestrus (high estrogen) female, and diestrus (low estrogen) female Sprague-Dawley rats were used. Vaginal smear cytology determined the stage of the estrus cycle for the female rats. The expression of p47, p22, and AT1 were measured by tagging them with antibodies (ImG antibody). After being tagged, the brains underwent electron microscopy to image the different densities of receptors and subunits. Furthermore, C1 neurons were labeled with ImP using the “avidin-biotin-peroxidase complex method” previously used in other studies.

The dendrites of the C1 neurons I both male and female rats expressed a high volume of AT1, p47, or p22 ImG labeling. They were expressed less in the presynaptic processes, suggesting that the action of ANG II occurs only at the C1 neurons to increase the sympathetic nerve activity. There were also two ImG populations found also the C1 membranes: 1. Membrane-associated and; 2. Internal particles. The internal pools were suggested to be p47, since it translocates to the membrane to form NADPH oxidase after the AT1 receptor is activated. The membrane-bound pool is theorized to be the p22 and AT1 since both of the receptors are transmembrane proteins. These pools were found to be distributed in a nonrandom fashion, suggesting the ImG antibody was successful in labeling the p47, p22, and AT1 proteins.

To investigate whether or not the AT1 receptor may contribute to the sex differences in hypertension, the levels of the AT1 receptor were measured. Female rats, in general, had 2.1 times more AT1 ImG labeling on the dendrites of their RVLM C1 neurons compared to males. It is important to note that previous studies have found that juvenile rats (23 days-old) and ovariectomized female rats have similar increases in the AT1 expression compared to males. These results suggest that sex differences persist throughout the life of rats. Therefore, although the increase in AT1 receptors may make female rats more sensitive to ANG II signaling, another component may be contributing to the age-related changes seen in menopausal females. However, it would be interesting to see if older rats have a reduced AT1 expression. This future study could help determine what happens at the onset of menopause to increase women’s risk of hypertension.

Females were found to have lower p47 ImG labeling compared their male counterparts. This difference was true for both the protein on the cell membranes and in the cytoplasm. p22 expression was not different between the sexes, which could mean that this pool of the subunit is not as critical in the control of blood pressure in rats. These results are supported by the findings of other studies and suggest that there the lower amount of NADPH oxidase subunit reduces the capacity of female rats to produce ROS in the C1 RVLM neurons. A reduced capacity leads to a lowered capability to increase SNA upon AT1 activation. Therefore, the female rats are unable to tonically increase their blood pressure as much as males. More studies should be done to further investigate the relationship since other pathways may be involved in the control of the expression of both the p47 protein and AT1 receptor.

Interestingly, AT1 and the NADPH oxidase subunits changed depending on the stage of the estrus cycle each rat was in. During proestrus (high estrogen), AT1 was much higher on the cell membrane compared to diestrus (low estrogen). The cytoplasm levels did not differ during the stages, nor did the expression of p22 or p47. This may make females more sensitive to ANG II during proestrus. However, the lower amounts of NADPH oxidase subunit prevent the increase in the blood pressure by reducing the capacity of the females to produce ROS. Without the increase in ROS, the SNA cannot increase with the ANG II signaling. The researchers describe this relationship as "counterbalancing" and may explain why females have an attenuated blood pressure response. 

In summary, the current study suggests that there are sex-differences in the C1 neurons and the ANG II signaling proteins involved in the maintenance of blood pressure. There may also be a direct impact from estrogen on the expression of the receptors.

-LivInLaVida