Activation of Corticotropin Releasing Factor Receptors in the Rostral Ventrolateral Medulla is Required for Glucose-Induced Sympathoexcitation

Megan E. Bardgett, Amanda L. Sharpe, Glenn M. Toney.Am J Physiol Endocrinol Metab (September 30, 2014). doi:10.1152/ajpendo.00291.2014.

Glucose leads to increased energy expenditure  through activation sympathetic nerve activity (SNA). The mechanism that leads to the activation of SNA is unknown. PVN and RVLM play an important role in the control of SNA and BP.  We already know that PVN not directly projects down to the IML in order to control SNA and BP but there is a direct projection to RVLM from PVN. Also neurons in the PVN are activated by glucose. The activity of RVLM is controlled mainly by Glutamate and GABA but we also know that there are other neurotransmitters that may be involved such as corticotropin releasing factor (CRF). So it has been shown that there are CRFergic neuron in the PVN. The hypothesis for the study in this article was that glucose leads to increases in SNA through activation of CRFergic neurons in the PVN that ultimately lead to activation of RVLM neuron by activating CRF receptor that lead to sympathoexcitation.  In order to prove this they provided anatomical along with function data. The data provided showed that glucose infusion elevates both LSNA and SSNA. They also demonstrated that glucose does, in fact, activate neurons in both RVLM and PVN. Not only that but the majority of PVN neurons that expressed c-fos was also a CRFergic neuron. As for RVLM, they showed that glucose activated a significant portion of TH RVLM neurons. Blockade of CRF receptor blunted the increases in LSNA and SSNA in response to glucose infusion. They also showed that kyn blocks the increase in SNA in response to glucose. Final they demonstrated that blockade of PVN blocked the response to glucose infusion. These data demonstrate that glucose leads to activation of SNA by activating CRFergic neurons in the PVN and this leads to the release of CRF and this leads to activation or the CRF receptor and this may facilitate the release of glutamate in the RVLM and this leads to increases in SNA and thus energy expenditure. I wonder how SNA in our model might behave in response to glucose infusion…-MD

CNS neuroplasticity and salt-sensitive hypertension induced by prior treatment with subpressor doses of ANG II or aldosterone

Sarah C. Clayton, Zhongming Zhang, Terry Beltz, Baojian Xue, and Alan Kim Johnson.

Am J Physiol Regul Integr Comp Physiol 306: R908–R917, 2014.First published April 2, 2014; doi:10.1152/ajpregu.00010.2014. Hypertension is obviously a problem in today’s society.  About 25% of all hypertensive patients are salt sensitive. However the mechanism that is responsible for salt sensitivity leading to hypertension is still not clearly understood. It has been shown that pretreatment with  nonpressor doses of ANG II and aldosterone can lead to enhanced responses to ANG II. This sensitization depends on a functional brain renin angiotensin aldosterone system. Long term changes in the CNS could occur in response to sensitization. This studied investigated whether BDNF a protein that plays a role in many models neuroplasticity is involved in the changes that are occurring in the lamina terminalis (LT) in response to sensitization to ANGII.  the study showed that pretreatment to ANGII or Aldosterone in the brain can lead sensitization to salt. They also showed that the BDNF is enhanced along with p38 MARK and pCREB in response to pretreatment to ANGII and aldosterone. These data demonstrate that pretreatment with subpressor doses of ANGII and aldosterone can lead long term structural changes by increasing the the BDNF pathway in LT and this contributes to the development of salt sensitivity. I wished they would have investigated the RVLM, wouldn’t that be interesting.-MD

Medullary lateral tegmental field mediates the cardiovascular but not respiratory component of the Bezold-Jarisch reflex in the cat.

Am J Physiol Regul Integr Comp Physiol. 2005 Dec;289(6):R1693-702. Epub 2005 Aug 11.

Barman SM, Phillips SW, Gebber GL.

In this study, they tried to find out if the medullary lateral tegmental field (LTF) had a role in mediating the Bezold-Jarisch reflex in cats. They did this in two ways – they inactivated LTF neurons using microinjections of either the GABAA agonist, muscimol, or with the excitatory amino acid receptor antagonist, AP5, and got similar results with both methods. These results were that the phenylbiguanide-induced Bezold-Jarisch reflex, which normally decreases SNA, heart rate, and MAP, was either sharply attenuated or reversed. However, two aspects of the Bezold-Jarisch reflex, an increase in apnea and a decrease in phrenic nerve activity, were not affected. When they switched from AP5 to NBQX, a non-NMDA receptor antagonist, they did not see these effects.

They came away with three main conclusions from this paper. 1) that the LTF plays an important role in activating the Bezold-Jarisch reflex, 2) that there is a separation in the respiratory and cardiovascular effects of the Bezold-Jarisch reflex in the LTF, and 3) the Bezold-Jarisch reflex is mediated by NMDA receptors. They closed this paper by mentioning that people normally think of the CVLM as the major inhibitory control over the RVLM, and note that their experiments in this paper don’t contradict this since they use cats when most people use rats and rabbits, so they don’t know if species differences come into play here or not. -DH

Fractal noises and motions in time series of presympathetic and sympathetic neural activities.

J Neurophysiol. 2006 Feb;95(2):1176-84. Epub 2005 Nov 23.

Gebber GL, Orer HS, Barman SM.

So I read this paper, but I think I’m going to have to read it a few more times with a lot of different highlighters if I’m really going to understand it. I understand that they were trying to analyze rhythmic activity among LTF and RVLM neurons in cats that correlates with SNA. This is not easy since not every action potential correlates with bursts in SNA, and a neuron will often NOT fire in a lot of places where a simple rhythm would dictate that it should fire. However, using spike-triggered averaging, they analyzed the interspike intervals of neurons to see how they would correlate with cardiac SNA. They showed that even though some neurons might miss more than 10 bursts before firing a second action potential, they still were able to construct histograms that showed how the action potentials correlated with SNA, and what phase of the cardiac cycle and SNA burst the neuron was most likely to fire at. After that, the paper kind of got away from me. Although this is the exact kind of stuff we’re trying to get in to, so I’ll be reading this a little more closely and brushing up on my math skills so that I can understand all of this a little better. -DH

Role of the hypothalamic arcuate nucleus in cardiovascular regulation

Hreday N. Sapru.Autonomic Neuroscience. Volume 175, Issues 1–2, April 2013, Pages 38–50.DOI: 10.1016/j.autneu.2012.10.016.  In ths review article the role of the arcuate nucleus was discussed.The arc is located in the hypothalamus and it has some projections that go to RVLM, NTS, CVLM, IML just to name some. The PVN receives the highest amount of projections from the arc when compared to the other regions mentioned above.  The arc extends to the median eminence, which is area that lacks a BBB. This allows for the arc to be exposed to leptin, glucose and angiotensin.  What I found most interesting was that stimulation of the arc can cause both depressor and pressor responses. When blood pressor is normal activation of the arc leads to depressor responses ( release of GABA, neuropeptide y, and beta endorphin) most likely due to inactivation of neurons in the PVN that project down to the spinal cord or RVLM and lead to decreased sympathetic activity. However when blood pressure is low, activation of neurons in the arc leads to pressor responses (most likely due to the release of glutamate). The baroreceptor reflex may play also play role in how blood pressure is modulated in response to activation of the arc neurons. It has been shown that following barodenervation, stimulation of the arc leads to pressor responses. This may suggest that the baroreceptors are important for providing inhibitory input on to glutamate releasing arc neurons. overall I thought that this could be a region that we investigate in the future since it could be

 playing a role in both  activation and inhibition of the rvlm neurons. -MD

Role of the caudal pressor area in the regulation of sympathetic vasomotor tone

Full cite: Campos, R.R., Carillo, B.A., Oliveira-Sales, E.B., Silva, A.M., Silva, N.F., Futuro Neto, H.A., Bergamaschi, C.T. Role of the caudal pressor area in the regulation of sympathetic vasomotor tone  (2008) Brazilian Journal of Medical and Biological Research, 41 (7), pp. 557-562.

Role of the caudal pressor area in the regulation of sympathetic vasomotor tone

R.R. Campos¹, B.A. Carillo¹, E.B. Oliveira-Sales¹, A.M. Silva¹, N.F. Silva²,

H.A. Futuro Neto^3,4 and C.T. Bergamaschi⁵

1Departamento de Fisiologia, Disciplina de Fisiologia Cardiovascular e Respiratória, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil

2Laboratório de Neuromorfologia,

3Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Espírito Santo, Vitória, ES, Brasil

4Escola Superior de Ciências, Santa Casa de Misericórdia de Vitória (EMESCAM), Vitória, ES, Brasil

5Departamento de Biociênicas, Universidade Federal de São Paulo, Campus Baixada Santista, Santos, SP, Brasil

This review examines how the Caudal Pressor Area (CPA) controls sympathetic outflow. The CPA is a relatively undefined region that is located at the caudal end of the Caudal Ventrolateral Medulla (CVLM). This region has shown to illicit sympathoexcitatory responses when it is directly stimulated, suggesting that the CPA either contains SPNs or innervates SPNs via a direct or indirect pathway. The CPA is not widely considered to be one of the main contributors to SPN innervation, however, there is quite a bit of ambiguity as to where the SPNs receive their excitatory drive from. One of the main statements of this paper is that the CPA provides a significant source of tonic excitatory drive to the RVLM, which I find that surprising considering this is one of the very few times I have read about the CPA, so there must be some complication to the CPA that I simply do not know yet. Apparently the effect of the CPA is mediated through the RVLM, suggesting some direct projection from CPA to the RVLM. According to this review, the CPA projects to the CVLM as well, and that inhibition of the CPA neurons has the greatest effect on the slow frequency neurons in the RVLM, not the fast ones. This response may suggest that the CPA has a selected enhancement of C₁ neurons. It would be interesting to see how the CPA relates to our model, especially if it does have as significant of an effect on RVLM neurons as this review suggests. It would also be interesting to see if the Caudal Pressor Area is stimulating the RVLM via glutamate or a different excitatory agonist. Also the CPA could be selectively innervating particular neuronal beds in the RVLM which could account of the differential control of nerve activity results that we have shown previously. It is certainly possible those different brain regions are responsible for stimulating different subsets of RVLM neurons and that these brain regions are stimulated by different phenomena throughout the body. That being said, I do not necessarily know of any evidence of preferential control of RVLM in regards to different brain regions, however, I believe that it is certainly possible given the fact that the RVLM has to manage MANY inputs and I do not believe that it is sophisticated enough to act as an integration center as well as a delivery center without some help from its neuronal circuitry.  - M.T.L.

Phenotypic identification of rat rostroventrolateral medullary presympathetic vasomotor neurons inhibited by exogenous cholecystokinin.

J Comp Neurol. 2003 Oct 27;465(4):467-79.
Sartor DM, Verberne AJ.
I guess I'm having a "reading Sartor and Verberne week" in honor of the assistance and advice coming from their way. And their stuff is highly related to my stuff, so I like trying to steal tips where I can. This paper sort of addressed the idea of differential control by looking at how cholecystokinin (CCK), a peptide released by the gut, can inhibit the activity of presympathetic neurons that may control splanchnic nerve activity. They examined RVLM neurons, verified them to be spinally projecting by antidromic action potentials, verified them to be presympathetic by seeing them decrease firing while they raised blood pressure via aortic occlusion, re-verified them to be presympathetic by seeing them decrease firing during i.v. administration of phenylbiguanide (PBG) aka the bezold-jarisch reflex, and then tested them with i.v. CCK to see if they would be activated or inhibited. Neurons were either inhibited, activated, or not affected by CCK administration. Conduction velocities were slow and similar between not-affected and activated groups, so these two populations were pooled in to one. In general, neurons that had slow conduction rates also had a low firing frequency. On the other hand, they also found neurons with high firing frequencies had faster conduction rates. The neurons that were inhibited by CCK fell into this group.
So far, the experiments were pretty indirect and unable to say that any particular neuron controlled the splanchnic nerve. Directly testing this would be extremely difficult and a technical nightmare... so they did the next best thing, which was to electrically stimulate the region of the periaquaductal gray which is responsible for activating RVLM neurons to cause mesenteric vasoconstriction. They found that 79% of the neurons that were inhibited by CCK were activated by PAG stimulations. Only 50% of the neurons not inhibited by CCK were activated by PAG stimulation. The conclusion from this experiment was that stimulation of the PAG was more likely to activate neurons that get inhibited by CCK than it is to activate non-CCK sensitive neurons. They also did juxtacellular labeling but did not find any correlation between phenotype and response to PAG stimulus. However, they did find that the fast-firing, fast-conducting, CCK-sensitive cells were more likely to be non-C1 (but only at 59%) Cells not inhibited by CCK were more likely to be slow firing and conducting C1 cells (79%).
What I did find very interesting is that they said in this paper that "All medullospinal (presympathetic) C1- and non-C1 neurons are inhibited by elevation of arterial blood pressure..." which is in contrast to what was said in the other paper from them that I blogged this week, which was that some presympathetic neurons are NOT barosensitve, but are sensitive to neuroglucoprivation.  However, there's about 7 years in between these two papers, with this one being the early one - so I guess that just shows that I have to keep track of publication dates and learn the timeline of discoveries. -DH

Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats

Andrew D. Chapp , Le Gui , Michael J. Huber , Jinling Liu , Robert A. Larson , Jianhua Zhu , Jason R. Carter , Qing-Hui Chen. American Journal of Physiology - Heart and Circulatory Physiology Published 1 September 2014Vol. 307 no. 5, H701-H709DOI: 10.1152/ajpheart.00005.2014. Ingestion of alcohol leads to pressor and sympathoexicition. This purpose of this study was to elucidate the mechanism responsible for the activation of the sympathetic nervous system. By using microinjection technique they demonstrated that ethanol and acetate, a byproduct of ethanol caused pressor and increases in SSNA and LSNA when injected into the amygdala (CeA).  In order to determine whether this response is being mediated by glutamate, they injected kyn, an EAA receptor blocker, NMDAR blocker and a non NMDAR blocker mixed with ethanol. They found that Kyn and the NMDAR blocker both attenuated the pressor response and SSNA. Then in order to determine whether the RVLM was playing role in the response kyn was injected into the RVLM they found that this also attenuated the response. Finally they showed that anatomically there was an connection between RVLM and CeA and that NMDAR were present on CeA-RVLM neurons. These data show that alcohol and alcohol metabolites can lead to activation of RVLM neurons which may be the cause of increase sympathetic outflow in response to alcohol consumption.-MD

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla

 

Pasarapa Towiwat, Siripan Phattanarudee,Timothy J. Maher, Ahmmed Ally. Mol Cell Biochem DOI 10.1007/s11010-014-2218-9.  The exercise pressor reflex leads to an increase in BP in response to static exercise. This reflex mediates its control over the cardiovascular system  by modulating the activity of CVLM and RVLM neurons. As we already know, there are different neurotransmitters that modulate the activity neurons in the RVLM and CVLM. It has been demonstrated in the literature that microdialysis of L- arginine into the CVLM lead to enhanced pressor response during static exercise along with decreased GABA release.  However, when L-arginine was infused into the RVLM blocked the cardiovascular responses to static exercise.  Blockade of both nNOS and eNOS lead to enhanced GABA and decrease glu in CVLM. Blockade of both nNOS and eNOS in the RVLM lead to enhanced Glu and decrease GABA.  Since it has been shown that blockade of iNOS in SHR leads to attenuation of there hypertensive state, This study wanted to further investigate the role of iNOS in the exercise pressor response.  They found that blockade of iNOS by infusion into the RVLM,  lead to attenuated HR and blood increase in response to activation of the exercise pressor reflex. they also showed a decrease in iNOS protein expression in the RVLM following blockade of iNOS in the RVLM and no change in CVLM. However, when the antagonist for iNOS was infused into CVLM they observed the response to activation of the exercise pressor reflex was potentiated. The protein expression of iNOS was not different when compared to controls for RVLM and CVLM. These data demonstrate that iNOS may be lead playing a role in the exciting neurons in the RVLM possibly through enhancing Glu and/or decreasing GABA release.-MD

Rostroventrolateral medullary neurons modulate glucose homeostasis in the rat.

Verberne AJ, Sartor DM.

Am J Physiol Endocrinol Metab. 2010 Nov;299(5):E802-7.

I came across this paper by accident, but it is dealing with presympathetic RVLM neurons, so I read it. It’s not about the ones we normally study, but it very much could be about some of the neurons I’ve been finding in my recordings. In this paper they looked at spinally projecting unmyelinated RVLM neurons that were NOT barosensitive, but were sensitive to changes in glucose concentration. These neurons increased their activity during 2-deoxyglucose (2-DG) induced neuroglocoprivation, presumably to drive increased epinephrine release from the adrenal glands in order to compensate for hypoglycemia. When they injected bicuculline in to the region of the RVLM where these glucosensitive were found, not only did they see the expected increase in arterial pressure, but they also saw that the rat’s blood glucose levels went up. This did not occur in rats that had undergone bilateral adrenalectomy. Interestingly, the barosensitive neurons they found were either not affected by 2-DG, or were slightly inhibited – so this is another subpopulation of neurons in the same region, which may support the idea of differential control that we’ve been looking at. -DH

Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase c signaling

Jones, W. Keith, et al. "Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase C signaling." Circulation 120.11 suppl 1 (2009): S1-S9.

Ischemia reperfusion injury associated with myocardial infarction is a major contributor to cardiovascular related death.  Unfortunately, the only treatment we have for cardiac ischemia, is reperfusion of oxygen to the ischemic tissue, which in itself can also lead to cell death.  Importantly, ischemic preconditioning (IPC) has been shown to limit the damaged induced by ischemia/reperfusion and act as a cardioprotectant.  However, IPC is somewhat impractical in a clinical setting in terms of feasibility.  This study was able to show that a preconditioned non-ischemic remote trauma was also able to induce cardioprotection through neurogenic mechanisms.  Specifically they were able to show that stimulation of pain receptors located in the skin activated an antidromic spinal reflex, which in turn activated cardiac sympathetic nerves via calcitonin gene-related peptide (CGRP).  CGRP then induced NE and Bradykinin release, stimulating beta-AR and BK2R.  Activation of these receptors then caused an upregulation of PKC-epsilon (mediator of cardioprotection) and a down regulation of PKC-delta (mediator of cell-death via necrosis).  They believe that one of the cardioprotective effects of PKC-epsilon is the activation of mitochondrial Katp channels, which when inhibited attenuated the cardioprotective effects.  Conclusively, this study may provide to be extremely important clinically due to the ease and efficiency of the remote preconditioning of trauma on reducing I/R injury following an MI.

~JI 

Methods of analysis and physiological relevance of rhythms in sympathetic nerve discharge.

Barman SM, Kenney MJ. Clin Exp Pharmacol Physiol. 2007 Apr;34(4):350-5.
As we all figured out at a recent meeting, I'm desperately in need a crash course in sympathetic nerve and RVLM unit coherence analysis. So I did the only thing I know how to do; I found a review paper from Dr. Barman and started reading. This review gives some background on frequency analysis and shows how all the burst we see in our splanchnic nerve recordings can be examined and linked to respiratory or cardiovascular rhythms, or both. Figure 1 (included) is very simplified, but it shows how a fast Fourier transformations can pull apart one complicated waveform and show the contribution of different sine waves, or rhythms. Baby steps.
It gets a bit more complicated from there, immediately going in to how different nerves can be affected by the same stimuli. It also starts to hint at how coherence analysis can show that the nerves are showing the same influence and rhythmicity, even though the averaged nerve recordings look to be completely different. This is what I was really hoping to learn about, but I guess it's important to learn to walk before trying to run that marathon, right? -DH

Role of ventrolateral medulla in generating the 10-Hz rhythm in sympathetic nerve discharge.

Barman SM, Gebber GL. Am J Physiol Regul Integr Comp Physiol. 2007 Jul;293(1):R223-33 I'm kind of new to the details of the 10Hz rhythm. It's seen in sympathetic nerves and when it becomes apparent, it is linked with an increase in arterial pressure. It seems to be always somewhat in effect, but it's most noticeable after barorecepter denervation. Generation of the 10Hz rhythm seems to originate in the brainstem, since decerebration doesn't stop it, but spinal transection does. The activity of individual brainstem neurons in RVLM, CVLM, and medullary raphe is correlated with 10hz. This is somewhat strange because CVLM neurons do not have spinal projections, but they're still correlated with this 10Hz activity. They wanted to look at how these different regions interact, so they used microinection of drugs in to these regions to figure out the microcircuitry involved. What they found is that inection of GABAergic antagonists in to the VLM (rostral OR caudal) could eliminate the 10Hz signal, even with only unilateral injections. Strangely, glutamatergic antagonists could reduce the 10Hz too. They think this comes down to a network of GABAergic interneurons in the CVLM and the RVLM communicating with the neurons that we generally think of when we think of those regions. This should make Max happy, since he's pretty excited about interneurons. -DH

GABAergic mechanism in the rostral ventrolateral medulla contribute to the hypotension of moxonidine

Wang et al. cardiovascular research (2011)89 473-481; doi:10.1093/cvr/cvq289. Moxonidine is a antihypertensive drug that is thought to act centrally. This study wanted to investigate whether GABA A receptor blockade would affect the effects of mononidine on the cardiovascular system, also whether GABA release and receptor expression was affected by moxonidine. What they found was that microinjection of moxonidine leads to decreases in BP, HR and RSNA.  When GABA A receptors and GABA B receptors were unilaterally blocked and they injected moxonidine into RVLM they were able to attenuate the BP, HR and RSNA decreases. They also did bilateral blockade of GABA A receptors in RVLM and gave moxonidine systemically and showed that bilateral blockade of GABA A receptors lead to attenuated decreases in BP, HR, and RSNA.  Intravenous administration of moxonidine leads to increase in the release of GABA. In response to a week of icv treatment with moxonidine, GABA ARα1 and GABA BR1 when compared to acsf control group. These data demonstrate that GABAergic neurotransmission in the RVLM are important for mediating the effects of moxonidine. -MD

Brain angiotensin- converting enzyme type 2 shedding contributes to the development of neurogenic hypertension

Huijing Xia, Srinivas sriramula, Kavaljit H. Chhabra, Eric Lazartigues.Circ Res.2013;113:1087-1096; doi:10.1161/circresaha.113.301811.

It has been shown that overactivity of the renin angiotensin system can lead to hypertension. Ace 2 is an enzyme that can convert angII to ang 1-7. Ang1-7 has an vasodilatory affect on vasculature.  Ace 2 is down regulated in hypertension further contributing to the dysfunction of the RAS. What is really interesting is that it has been shown that ectodomain of ACE2 is cleaved and can be found in CSF. Thus hypertension leads to ace 2 shedding. The question becomes what is responsible for the removal of ace 2 from the cell membrane. So the hypothesis was adam 17 is responsible for the cleavage of ace 2 and leading to the development of hypertension.  So they used transgenic mice in order to test there hypothesis. Using mice that had overexpression of Ace2 in neurons that were DOCA- high salt diet mice improved the spontaneous Baroreflex function. Ace2 overexpression also leads to decreased vasopressin release, norepinephrine, and ang II. Along with that vagal tone was restored and sympathetic drive to the heart and vasculature was brought back down to control levels. They knockdown adam 17 and showed that this prevented the development of hypertension and lead to a decrease in ace 2 shedding in the csf. These data demonstrate that hypertension that is due to a high salt diet may be leading to increased adam 17 expression and leading to decrease ace 2 expression on neurons and ultimately leading to hypertension. –MD

Attenuated baroreflex control of sympathetic nerve activity in obese Zucker rats by central mechanisms

Full cite: Huber DA & Schreihofer AM (2010). Attenuated baroreflex control of sympathetic nerve activity in obese Zucker rats by central mechanisms. J Physiol 588, 1515–1525.

Attenuated baroreflex control of sympathetic nerve activity in obese Zucker rats by central mechanisms

Domitila A. Huber and Ann M. Schreihofer

Department of Physiology, Medical College of Georgia, Augusta, GA, USA

This study is looking at how reflex control of the vasculature is affected by obesity; the main technique they are using in this paper is electrophysiology. It is widely known that obesity is a risk factor for the development of hypertension, in addition to this; obesity has been shown to be an independent contributor to the elevation of sympathetic nerve activity (SNA). The goal of this paper is to illuminate how obesity alters sympathoregulation, in an attempt to tie the condition to other disease states. The main component of sympathetic control examined is the sympathetic baroreflex responses, with an attempt to see if it is altered due to altered sensory or central mechanisms. It has been a long while since I’ve read a Zucker rat paper, adult obese Zucker rats (OZRs) vs. lean Zucker rats (LZRs) were the rats used in this study. The OZRs weighed about 600g and the LZRs weighed about 400g on average. The OZRs had significantly higher resting sympathetic nerve activities and blood press, interestingly enough they did not have significantly different heart rate. Stimulation of the aortic depressor nerve (ADN) elicited blunted responses in the OZRs, whereas the net responses in the LZRs were significantly greater. This result suggests that OZRs are less sensitive to stimulation of the ADN. In addition, stimulation of the vagal afferent nerve brought about significantly greater responses in the LZR when compared to the OZR. These results suggest that the vagal afferents have inherently less sensitive in the OZR rats. This reduction in baroreflex sensitivity in the OZRs may contribute to the likelihood of cardiovascular diseases in overweight individuals. -MTL

Intrinsic chemosensitivity of RVLM sympathetic premotor neurons in the in situ arterially perfused preparation of rats.

Koganezawa T, Paton JF. Exp Physiol. 2014 Jul 11.

We normally think of the RVLM as something that responds to incoming signals from other areas and adjusts its activity accordingly. However, it has been shown that if you block synaptic transmission, the neurons still fire on their own despite a lack of incoming signals. Other groups have looked at their “pacemaker” properties, but in this paper, they decided to look at the intrinsic chemosensitvity of presympathetic neurons – something we haven’t really looked at. They used a modified version of the working heart brainstem preparation so that they could record the activity of nerves and neurons while they switched the perfusion solution to ones that would induce periods of eucapnia, normoxia, hypercapnia, hypoxia, etc. First, they blocked fast synaptic transmission and looked at spontaneous activity under the different conditions and found that in all 26 presympathetic neurons, activity was not significantly affected. During hypercapnic-hypoxia, they found that all presympathetic neurons increased their firing rate by an average of 130%. This indicates that these neurons are themselves chemosensitive, independent of afferents from chemoreceptors. They also tested the combined effects of cyanide and hypercapnia on presympathetic neurons during synaptic blockade and found that 7 had their activity depressed by -82%, while 3 were slightly excited by it, though the effect was not significant due to a wide variability in response. Interestingly, and in terms of differential control, they found out that if they block a particular sodium current known to be involved in C1 neuron activity, they can block the preparation from “gasping” during asphyxia, but sympathetic chain activity remains unaffected. Personally, my take-home message from this paper is that I really need to watch my blood gas measurements and make sure that I’m not putting my rat into a state that could compromise my recordings. -DH

Leptin into the rostral ventral lateral medulla (RVLM) augments renal sympathetic nerve activity and blood pressure

Barnes and McDougal.Front. Neurosci.8:232.doi:10.3389/fnins.2014.0232.

Leptin is released from fat cells in order to promote satiety and increases energy expenditure. One mechanism by which leptin affects energy expenditure is by increasing sympathetic nerve  activity. Leptin signaling is mediated through the hypothalamus and has been shown to increase blood pressure and renal sympathetic nerve activity (RSNA).   we know that PVN sends projections to the RVLM and to the spinal cord to modulate sympathetic nerve activity. This study investigated if the leptin receptor is present in brainstem regions that control RSNA./For their anatomical studies they injected PRV into the  cortex of the kidney and did immunofluorescent staining for the leptin receptor (obrb). As for the functional studies they microinjected leptin and leptin receptor antagonist into the rvlm and recorded blood pressure and RSNA.  They found obrb positive cells in the C1/A1 region, VMM, caudal raphe and A5 area. Most of the PRV positive cells were found in the rostral portion of the RVLM (similar Nick’s paper) and found no difference between ipsilateral and contralateral RVLM.  They found that 65% of the C1/A1 region was double labeled.  As for the functional studies, they found that leptin lead to an increase in RSNA and BP. Injection of the antagonist prior to the leptin injection attenuated the BP and RSNA response.  Conclusion leptin release from fat cells is leading to increased energy expenditure via increasing sympathetic nervous system activity through activation of obrb receptors in the RVLM. MD

 

Cardiovascular effect of angiotensin-(1–12) in the caudal ventrolateral medullary depressor area of the rat

Tetsuya Kawabe, Kazumi Kawabe, and Hreday N. Sapru.Am J Physiol Heart Circ Physiol 306: H438–H449, 2014. First published November 27, 2013; doi:10.1152/ajpheart.00628. 2013. This study investigated whether ang 1-12 is leading to increased neuronal activity through AT1 Receptors in the cvlm. In Wistar rats, they recorded GSNA, BP, and HR  and used microinjection technique. They found that microinjections of ang 1-12 into cvlm lead to decreases in BP, HR and GSNA. Ang 1-12 mediates it response through AT1R. The activation of AT1R leads to the release of GABA from the terminals of CVLM neurons and acts on GABA receptors in the RVLM in order to cause sympathoinhibition.  In conclusion, Ang 1-12 maybe playing a role in the activation of neurons in the CVLM. I wonder if the level of Ang 1-12   is changing in response exercise…MD

Temporal Changes in the T1 and T2 Relaxation Rates (delta R1 and delta R2) in the Rat Brain Are Consistent With the Tissue-Clearance Rates of Elemental Manganese

Chuang, Kai‐Hsiang, Alan P. Koretsky, and Christopher H. Sotak. "Temporal changes in the T1 and T2 relaxation rates (ΔR1 and ΔR2) in the rat brain are consistent with the tissue‐clearance rates of elemental manganese." Magnetic Resonance in Medicine 61.6 (2009): 1528-1532.

One of the main points of recent MeMRI study was to determine the time course of influx and efflux of manganese in the RVLM following systemic injections of manganese.  As a trace mineral, manganese can be found in multiple forms in the body that effect both its rate of efflux from a cell and its effect on proton relaxation rates.  While we discuss signal intensity in our study as manganese uptake within a cell, signal intensity may also be dictated by the relative state in which the manganese is able to change the proton relaxation rates.  To determine the extent that changes in relaxivity (how effectively manganese is able to change relaxation rates) are contributing to changes in signal intensity MeMRI studies were done in combination with NMR, directly examining manganese concentrations in neuronal tissue.  Initial results showed that T1 and T2 relaxation times, as well as delta R1 and delta R2 peak 1 day after administrations of manganese, which corresponded to greatest concentrations of manganese in neuronal tissue.  In all cases manganese uptake was seen to be greater in the olfactory bulb than in the cortex.  Both relaxation rates and relaxivity levels return to baseline levels 4 and 5 weeks post injections, conclusive with absolutely manganese concentrations also returning to baseline at 4 weeks.  Conclusively, this study was able to show that T1 and T2 relaxation times were mainly influenced by transport of manganese in and out of the cell, compared to manganese taking different forms while remaining in the cell (relaxivity).  This is important, because it further validates manganese enhancement being measured following systemic injections of manganese is an appropriate representation of in vivo neuronal activity.

~JI

Tonic glutamatergic input in the rostral ventrolateral medulla is increased in rats with chronic heart failure

Full cite: Wang WZ, Gao L, Wang HJ, Zucker IH, Wang W. 2009. Tonic glutamatergic input in the rostral ventrolateral medulla is increased in rats with chronic heart failure. Hypertension 53:370–374.

Wang WZ, Gao L, Wang HJ, Zucker IH, Wang W.

Department of Cellular and Integrative Physiology (W.-Z.W., L.G., H.-J.W., I.H.Z., W.W.), University of Nebraska Medical Center, Omaha; and the Department of Physiology (W.-Z.W.), Second Military Medical University, Shanghai, China.

The headline of this paper essentially sums up the message of this paper; chronic heart failure animals have heightened glutamatergic tone. While the finding of this paper is pretty straightforward, what I thought was particularly cool was how they used triple barrel pipettes and paired it when electrophysiology. They used kynurenic acid, an NMDA/non-NMDA receptor antagonist, non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and NMDA antagonist D-2-amino-5-phosphonopentanoate (D-AP5) to block glutamate reception and recorded the responses via single-unit electrophysiology. In addition to the triple barrel pipette they used, they had a 5-barrel micropipette containing an electrode. Using a penta barrel pipette sounds like a huge technical difficulty, that being said, if we could get some way for it to work, we could easily test the responsiveness of neurons to various direct stimuli, something we’ve never done before. With a 5-barrel micropipette, it might actually be possible to record in one piece of the pipette, have neurobiotin in another, and maybe some kind of antagonists, say bicuculline, xanthurenic acid, and kynurenic acid. I could imagine that it would be really difficult to ensure that the pipette is successfully able to inject out of each portion, but the ability to sit on a neuron and inject a slew of drugs would be incredibly powerful. It would be cool to do a similar study in runners & seds in order to get some definitive proof onto the tonic input of glutamate in seds. I believe though, we would likely see a prevalence of glutamatergic tone in the sedentary animals; it just would not be as distinct as that of the heart failure animals. -MTL

Specific respiratory neuron types have increased excitability that drive presympathetic neurones in neurogenic hypertension.

Moraes DJ, Machado BH, Paton JF.

Hypertension. 2014 Jun;63(6):1309-18.

In this paper they were trying to find out what differences between spontaneously hypertensive (SH) rats and WKY control rats are responsible for the increases in nerve activity and blood pressure seen in the SH group. To investigate this they looked at a lot of nerves, a lot of neurons, and a lot of factors that our lab often don’t consider. Their primary focus was on seeing if the increased respiratory modulation RVLM neurons seen in hypertensive rats was due to changes in the RVLM neurons themselves, or if it was due to changes in synaptic input. To make a long paper short, they found differences in nerve activity and neuronal behavior between groups (SH rats showed increases in both categories) that pretty much disappeared when they blocked synaptic input to the RVLM. This suggested that the changes in neuron and nerve activity actually came from upstream sources, altering synaptic communication in the RVLM.

They then started looking at the pre-BotC to see if the differences might be there, because respiratory modulation of RVLM neurons seems to be sourced from a place full of respiratory neurons.  What they found was that pre-inspiratory neurons in the pre-BotC were more excitable in SH rats, and it seemed to be due to reductions in the leak-K current compared to WKY rats (which should make Dr. Holt happy to learn). They also looked at post-inspiratory neurons and found these to be more excitable in SH rats as well. However, in their case, it appeared that it was due to reductions in the big calcium-activated potassium current. In fact, when they blocked the BKca channels in WKY rats, the recordings from WKY rats started to look like those from SH rats. There was no effect of the drug on SH rats, indicating that this current was pretty much non-functional, so they couldn’t drug it out.

This was a difficult paper to read and it had a lot of detail, but it really does take a good look at some of the inputs to the RVLM, and makes me remember that the RVLM isn’t an island, and it’s not the only thing that changes in hypertensive rats. -DH

Regulation of visceral sympathetic tone by A5 noradrenergic neurons in rodents.

Kanbar R, Depuy SD, West GH, Stornetta RL, Guyenet PG.

J Physiol. 2011 Feb 15;589(Pt 4):903-17

We’re pretty focused on the RVLM, but we know it’s not the only region of the brain that controls the same things we study. In this paper they looked at the A5 region, which also has spinal projections that go to IML. It has long been implicated in regulation of the autonomic system and respiration In fact, this function of the A5 has been suspected  since 1982. The cells here have been shown to have slow regular resting discharge, activated by nociceptive stimuli, inhibited by A2 agonists, activated by hypoxia and show respiratory modulation, but seem to be unaffected by changes in BP. The problem with pinpointing these effects was that there aren’t many A5 cells and they’re scattered around a small area that is surrounded by other areas that would affect nerve activity – this means microinjection studies were very hard to conduct, and A5’s effect on the control of SNA and blood pressure was difficult to establish. In this paper, they used an optogenetic approach (PRSX8-ChR2 again), combined with electrophysiology (including antidromic action potentials in the spinal cord) and juxtacellular labeling (along with immunology/TH staining), to isolate the cells of the A5 and see how they contribute to SNA.

They found 2 main types of neurons in the A5 region – the first group never showed antidromic action potential and had a huge response to increases in CO2. These cells were the most caudal and ventral ones, indicating that they were actually RTN cells living at the A5/RTN border. The other type were slow firing (<1Hz) bulbospinal ones that slightly increased activity with increasing CO2, or had no response. They were strongly activated by chemoreceptor activation (with cyanide). 4 out of 10 that they recorded were pulse modulated. Through juxtacellular labeling and immuno, these cells were shown to be TH positive, and optogenetic activation of these cells caused an increase in renal SNA (but had a very small effect on lumbar SNA). The cool thing about this paper was that they were able to combine old and new techniques to get a lot of information that would not have been possible through any of the individual techniques alone. -DH

Role of serotonergic input to the ventrolateral medulla in expression of the 10-Hz sympathetic nerve rhythm

Full cite: Orer HS, Gebber GL, Barman SM. Role of serotonergic input to the ventrolateral medulla in expression of the 10-Hz sympathetic nerve rhythm. Am J Physiol ReguI Integr Comp Physiol 2008;294:R1435-44.

Hakan S. Orer, Gerard L. Gebber, and Susan M. Barman

Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan; and

Department of Pharmacology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Submitted 7 January 2008; accepted in final form 10 March 2008

A majority of Dr. Barman’s research seems to involve the study of the 10 Hz rhythm in sympathetic neurons. In this particular study she is looking at the role of serotonin in the maintenance of this rhythm. The 10 Hz rhythm is correlated to sympathetic nerve discharge (SND) which raises blood pressure and is one of the main properties examined in sympathetic tone. Dr. Barman demonstrated in a previous study that the 10 Hz rhythm was GABA-mediated, as such; the rhythm was abolished with injection of a GABA antagonist into the ventrolateral medulla. Interestingly enough, it seemed that both the rostral and caudal portions of the VLM were contributing to the 10 Hz rhythm, which seemed weird to me because the rostral and caudal VLMs have entirely different functional roles – based on what I have gathered from prior reading. The caudal medullary raphe seems to play a modulatory role in the 10 Hz rhythm presumably by serotonergic neurons to the VLM. By injecting serotonin antagonists in to VLM, they found that the 10 Hz rhythm was significantly reduced, while the lower frequency bursts were maintained. It was also demonstrated that the decrease in 10 Hz was paired with a significant fall in MAP. By stimulating the caudal raphe, they were able to demonstrate an increase in the 10 Hz rhythm and blood pressure. It would be interesting to look at the input of serotonin within our model (presuming we haven’t done it before). In addition it would be interesting to look at 5-HT2 receptors and see if they are changing following physical (in)activity. It is strange—in my opinion— that blocking serotonin in both RVLM and CVLM decreases blood pressure. Of course, this suggests that the relationship between RVLM and CVLM are EVEN more complicated that what we had originally thought, much less the implications on physical activity dependent changes. The RVLM has such complex relationships with its inputs that it seems nearly impossible to illuminate the web of communication between RVLM and other brain regions based off of the current methods at our disposal, adding any physiological state such as heart failure, hypertension, high-salt, obesity, (in)activity, makes it seemingly impossible for us to come up with any conclusive and overarching understandings about any of the more intricate functions of RVLM. - MTL

Voltage-dependent calcium currents in bulbospinal neurons of neonatal rat rostral ventrolateral medulla: modulation by alpha-2-adrenergic receptors

Li, Yu-Wen, Patrice G. Guyenet, and Douglas A. Bayliss. "Voltage-dependent calcium currents in bulbospinal neurons of neonatal rat rostral ventrolateral medulla: modulation by α2-adrenergic receptors." Journal of neurophysiology79.2 (1998): 583-594.

Previous research has shown that both high voltage activated (HVA) and low voltage activated (LVA) calcium channels are present in the brain and may play different physiological roles.  The HVA calcium channels consist mostly of N type, P/Q-type, and to a lesser extent L-type components which also contribute to different physiological processes such as neurotransmission, and calcium mediated gene expression.  Until this study, little was known about calcium channel properties in the RVLM.  However, it was known that C1 neurons in the RVLM expressed alpha 2A-adrenergic receptors that had been shown to inhibit calcium channels in other neurons via activation of an inwardly rectifying potassium conductance.  Through the combination of using 2A-adrenergic receptor agonists and antagonists with electrophysiology, this study was able to characterize calcium channels and their regulation via norepinephrine for the first time.  Results showed that the HVA current consisted mainly of N-type calcium channels, with a significant but lesser contribution from P/Q-type. They also found small, but present L-type calcium channel activity.  As for LVA current, they found it was present in the RVLM and was activated with a much lower (~-50mV compared to ~0mv) as expected.  Conclusively, the study revealed that norepinephrine mediated activation of alpha 2-adrenergic receptors is able to inhibit HVA currents, specifically N-type and P/Q-type, but not LVA currents.  This study was relevant to MeMRI studies because manganese enters neurons through active L-type calcium channels.  If little L-type calcium channel current is present, it would need to be addressed for future studies using MeMRI to examine neuroplasticity in the RVLM.

~JI

Rhythmic activity of neurons in the rostral ventrolateral medulla of conscious cats: effect of removal of vestibular inputs.

Barman SM, Sugiyama Y, Suzuki T, Cotter LA, DeStefino VJ, Reighard DA, Cass SP, Yates BJ. Am J Physiol Regul Integr Comp Physiol. 2011 Oct;301(4):R937-46 In most electrophysiological studies of RVLM neurons, animals are either anesthetized (like we do) or decerebrated (like in the half-rat prep). In this paper, they went ahead and examined neurons in conscious/behaving cats, and correlated their activity with carotid blood flow. They found that around 8% of RVLM neurons showed their spiking to be most likely to occur during periods of decreased blood flow (in between heart beats). Their previous work showed that the vestibular system normally suppresses RVLM responses to body rotation in intact/awake rats (think like the Dampney paper where they inhibited the colliculi in order to get sympathetic activation following normally non-stimulating cues). So in this paper, they looked at what removal of vestibular inputs would do the cardiac-linked RVLM activity and found that vestibular removal caused them to have an increased likelihood of finding neurons that showed cardiac related activity. What I find most interesting though, is that their data came in bursts - some days they didn't get any neurons with flow-related activity, while other days up to 60% of the neurons they recorded had it. Maybe that's the problem with my recordings - I just keep picking the wrong days to do experiments... -DH

Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats

Barman, Susan M., and Hakan S. Orer. "Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 299.5 (2010): R1269-R1278. Previous to this study, work by Dr. Barman and her laboratory had shown that the medullary lateral tegmental field (LTF) neurons set the pattern for resting sympathetic nerve activity (SNA), not the NTS, CVLM, or RVLM. She was very open in stating that sympathetic tone to most of the vasculature was not driven by the RVLM, and that the RVLM, CVLM, and NTS were not the only neuronal pathways that contributed to SNA modulation resulting from the baroreflex, chemoreflex, or vagal afferents. However, these hypotheses were based upon various microinjecitons into directly into the LTF while recording SNA, so this study looked to further characterize the role the LTF plays in the sympatho-sympathetic reflex in response to afferent stimulation. After the separate microinjection of 1) non-NMDA receptor antagonist (NBQX) 2) NMDA receptor antagonist (D-AP5) 3) muscimol into the LTF there were no differences seen in sympathoexcitatory response recorded from the right inferior cardiac nerve, following stimulation of either the left inferior cardiac afferent nerve (CN) or sympathetic afferent nerve (SN). This same protocol was then repeated in the RVLM and injections of NBQX reduced the sympatho-sympathetic excitatory response to ~25% from baseline levels, as well as increased the onset latency following the electrical stimulation to the CN and SN. Like in the LTF, microinjections of D-AP5 produced no significant differences in the sympatho-sympathetic excitatory response. Muscimol, however, like NBQX significantly reduced the responses to CN and SN stimulation. In most cases the response seen was negligible, with little to no excitation. Conclusively, there were no changes in the excitatory responses following identical microinjections into the NTS and CVLM. Results from this study disproved the original hypothesis and instead showed that the RVLM is a critical region involved in sympatho-sympathetic cardiovascular responses. Dr. Barman also goes on to hypothesize that non-NMDA receptors play a primary role in this reflex compared to NMDA receptors. ~JI

Exaggerated Cardiovascular Stress Responses and Impaired β-Adrenergic–Mediated Pressor Recovery in Obese Zucker Rats

Gerard D’Angelo, James D. Mintz, John E. Tidwell, Ann M. Schreihofer,David M. Pollock, David W. Stepp.Hypertension. 2006;48:1109-1115.Obesity is a major risk factor for development of metabolic disorders. Besides the increased risk for metabolic disorders there increase in the pressor response that results from mental, physical and acute stress. Since α and β adrenergic receptors are important for determining vascular tone, changes in either receptor expression or sensitivity to norepinephrine or epinephrine. In a previous study this laboratory demonstrated that pressor responses to ganglionic blockade was not different between obese and lean zucker rats. They also demonstrated that isolated mesenteric resistance arteries responses to exogenous norepinephrine is attenuated when compared to lean zucker rats. They concluded from this data that there is elevated peripheral vascular resistance. Furthermore, it may be due suppressed β-adrenergic-mediated vasodilation. In this study they hypothesized that pressor responses to environmental stressors would be greater in the obese versus the lean zucker rats. The elevated pressor response in the obese zucker will be most likely due to attenuated vasodilation as a result of impaired β- adrenergic mechanisms. Obese and lean zucker rats ( and LZR, respectively)were implanted with telemetry probes in order to record blood pressure under conscience conditions. Following recovery animals were brought to a soundproof room and then the animals were put into restrainers and a baseline blood pressure and heart rate were monitored for fifteen minutes prior to the air jet stress. In a separate group of obese and lean zucker rats, following the fifteen minute control period propranolol was administered and then the air jet stress was initiated. They also instrument animals so they could determine blow flow to the mesenteric arteries and aorta.They also looked at cardiac output along with blood glucose, plasma levels of cholesterol and triglycerides. They also looked at thyroid hormones T3/T4.They found that OZR had higher baseline BP and HR variability. In response to stress the OZR the area under the curve (AUC) for blood pressure was significantly higher in obese untreated when compared to the LZR. the AUC 20 minutes post stress was significatly greater in the OZR when compared to LZR. There was also a significant increase in AUC following treatment with propranolol in the LZR. They further analyzed the role of β-adrenergic receptors following ganglionic blockade. prior to ganglionic blockade, β-adrenergic receptor blockade resulted concentration dependent decreases in MAP in both groups, however the OZR had attentuated response when compared to LZR. Following ganglionic blockade resulted in a similar trend. They also looked at hindlimb and mesenteric conductance. There were no differences in hindlimb conductance between OZR and LZR ganglionic blockade in response to β- adrenergic blockade. As for mesenteric conductance both group exhibit increases in conductance, however only at 0.05,0.1, 0.5 μmoles of isoproterenol is there an attenuation in the OZR response when compared to LZR. As for cardiac output, there is an increase in response to isoproterenol in both group however the OZR had attenuated changes in CO at 0.05 and 0.5 μmoles. These data suggest that Obesity may lead to attenuated β- adrenergic mediated vasodilation. This β- adrenergic mediated vasodilation is playing an important role in the recovery period for stress.-MD

Increased reactive oxygen species in rostral ventrolateral medulla contribute to neural mechanisms of hypertension in stroke-prone spontaneously hypertensive rats.

Kishi T, Hirooka Y, Kimura Y, Ito K, Shimokawa H, Takeshita A. Circulation. 2004 May 18;109(19):2357-62. Epub 2004 Apr 26. In this paper, the authors wanted to look at how baroreflex sensitivity (BRS) is impaired in cardiovascular disease (CVD) models, and how it can be linked to changes in reactive oxygen species (ROS). Their previous work had shown that by causing increased expression of manganese superoxide dismutase (MnSOD), they could reduce ROS and cause sympathoinhibition in hypertensive (HT) rats. However, that was kind of an acute study, so this time they wanted to look at BRS in HT rats, using a chronic redution in ROS via MnSOD overexpression. To do this, they injected the RVLM of spontaneously hypertensive rats (SHRs) with a construct causing expression of MnSOD. They found strong reductions in MAP and HR 5-9 days after injection, and a strong reduction in urinary norepinephrine at day 7. For the BRS, they found that the SHRs started with lower sensitivity than WKY-MnSOD controls, but became more sensitive after transfection (days 5-8). WKY-MnSODs did not demonstrate this increase in sensitivity, nor did LacZ-SHR controls. I have a bit of confusion about this paper... I'm honestly not sure exactly how they measured BRS, partially because I didn't look up the two papers describing how BRS is calculated. I admit, not knowing this is kind of a big hole in my interpretation of the paper. In my defense, the two papers they referenced on the technique seem to be calculating BRS in humans, and they never referenced any papers showing how it was done in rats, so I can't yet be really confident about the technique. I dug up a couple of this group's other papers and didn't see it in those ones either, so I might have to go look in to this one more, or consult our resident expert. I'm also a little confused about how all of the effects peaked at one week before returning to baseline levels, as soon as day 9. I know that viral expression isn't always permament, but is 7 days "chronic?" Is there a combined effect of the transfection failing AND some kind of compensation happening to maintain the MAP/HR/BRS? The levels of MnSOD were only shown at days 0,7, and 21... I'd like to know if their expression graphs match the effects - that might answer part of this question. -DH

Blood pressure is maintained during dehydration by hypothalamic paraventricular nucleus-driven tonic sympathetic nerve activity.

Holbein WW, Bardgett ME, Toney GM. J Physiol. 2014 Jun 27. In this paper, they wanted to look at how what happens during dehydration that allows the blood pressure to be maintained. To do this they looked at the PVN , because previous papers suggested that it might be the region controlling th effect. Their first experiment was to look at the differences in SSNA between dehydrated (DH) and euhydrated (EH) rats. They found that MAP and phrenic nerve activity (PNA) that wasn't different between groups, but DH rats had a much higher baseline SSNA. When they gave injections of muscimol in to the PVN to block its activity, they saw almost no effect in the EH rats, but in the DH rats they a strong and rapid reduction MAP and SSNA. For PNA, both groups showed only a slight decrease in burst frequency, but not amplitude, after the injection of GABA. They also noted that, even though comparison of SSNA between animals is touchy, the post-inhibition SSNA was not different between EH and DH rats, suggesting that the entire process may be mediated by the PVN. This also suggests that the splanchnic nerve is critical to maintaining blood pressure during dehydration, though they have yet to examine nerves leading to other vascular beds. -DH

Efficacy of an L- and N-type calcium channel blocker in hypertensive patients with neurovascular compression of the rostral ventrolateral medulla

Aota, Yasuko, et al. "Efficacy of an L-and N-type calcium channel blocker in hypertensive patients with neurovascular compression of the rostral ventrolateral medulla." Hypertension Research 32.8 (2009): 700-705. It has been previously shown in clinical studies that essential hypertension may be linked to neurovascular compression (NVC), and that NVC maybe be mediating hypertension through increased levels of sympathetic nerve activity. Neurovascular decompression studies have been shown to decrease blood pressure in patient with essential hypertension, but requires a rather intricate invasive surgery. The purpose of this study was to examine the effects of non-invasive, sympatholytic, L- and N-type calcium channel antagonist (Cilnidipine) for the use of a treatment for essential hypertension mediated by NVC. In this trial, 46 patient with essential hypertension (22 –NVC, 24 +NVC) were treated with Cilnidipine for 16 weeks, with clinical follow ups at 0, 8, and 16 weeks. At 8 and 16 weeks, systolic and diastolic blood pressure in both groups was significantly reduced from baseline levels. Correspondingly, systolic and diastolic pressures were significantly reduced in the +NVC group, compared to the –NVC group. To examine SNA, norepinephrine levels were taken at each time point, revealing that baseline levels of norephinephrine were significantly higher in the +NVC group than the –NVCs. That being said, at 16 there was a significant decrease in NE levels in the +NVC, but not –NVC group. With no difference in NE levels found between the groups. Finally, after measuring left ventricular mass index (LVMI) at each time point, it was observed that LVMI decreased in the +NVC, but not the –NVCs. Conclusively, this study was able to show the use of Cilnidipine by patients with EH mediated by NVC may be a non-invasive antihypertensive treatment. However, because Cilnidipine is not only an N-type calcium channel antagonist, but also an L-type vascular calcium channel antagonist more studies are being done to distinguish if the results seen were due to sympatholic or vascular effects. ~JI

The sympathetic control of blood pressure

Patrice G. Guyenet Nat Rev Neurosci. 2006 May; 7(5):335-46 I’ve been writing my introduction and figured this paper would be a great resource, so I read it *again*…………… The overall purpose of this review is to examine sympathoregulation and how altered sympathoexcitation can result in hypertension and other cardiovascular diseases. It is also important to mention that while SNA has been correlated to CVD, there are likely other mechanisms involved in the development of CVD, including hypertension. Apparently renal SNA is selectively regulated by volume receptors whereas the other sympathetic nerves are not. Also the rostral ventromedial medulla (RVMM) and medullary raphe are involved in cutaneous circulation, whereas the rostral ventrolateral medulla (RVLM) is not. I also did not know that the C1 neurons were only one of three clusters of adrenaline-synthesizing cells in the CNS, which is particularly interesting because we still don’t particularly know the role that adrenaline synthesis is even having in the RVLM, aside from being a good marker. Here are C1 cells that are non-barosensitive that control adrenaline-releasing chromaffin cells. Guyenet mentions that the baroreflex can be set (towards a higher level) in the case of different activities that require heightened sympathetic tone, exercise for example. I do not know the mechanism by which the baroreflex can be altered; I know that reflexes like the muscle metaboreflex exist and different types of hypoxia can raise blood pressure above baroreflex regulation. Something interesting, which is slightly unrelated (however mentioned in the review), is that obstructive sleep apnea (OSA) increases SNA throughout the waking period of the day, which I thought was surprising because I figured it would only have an effect during the night. Something that I found particularly interesting (as well as something that I don’t necessarily agree with) is the remark that “sympathetic efferents that innervate the kidneys are commonly presented as the only ones that are capable of influencing 24-h average BP.” I don’t think that renal controlling neurons are the only neurons involved in tonic cardiovascular regulation, I think that looking at only the neurons controlling the kidneys is totally short sighted. It would be interesting to look at a review with a similar approach as this, but considering all of the papers presented after 2006 as well. -MTL

Olfactory exposure to males, including men, causes stress and related analgesia in rodents

Sorge, R.E., Martin, L.J., Isbester, K.A., Sotocinal, S.G., Rosen, S., Tuttle, A.H., Wieskopf, J.S., (...), Mogil, J.S. Olfactory exposure to males, including men, causes stress and related analgesia in rodents (2014) Nature Methods, 11 (6), pp. 629-632. doi: 10.1038/nmeth.2935 Dr. Berkowitz had mentioned this paper…………………… The goal of this paper is to look at the responses of male and female mice to different stimuli which are intended to gauge the stress or withdrawal response to the presence or perceived presence of a male or female researcher or animal. The first experiment consisted of an injection of zymosan, an inflammatory agent. The researchers studied the facial grimacing of mice in the presence or absence of an experimenter (male or female, seated at a distance of ~0.5m). All of the four male observers elicited significant changes in grimaces, while all four of the women did not. They conducted the same study, but instead of using the researcher, they placed a shirt worn the previous night by the male or female experimenters ~0.5 away from the mice. They found that they were able to see the same trend of responses. Interesting enough, the rats did not show the response if both the male and female shirts were placed together. Next, they placed the mice in different bedding materials from either unfamiliar male mice, nonpredator male guinea pigs, rats, cats, and dogs, and once again saw the same decreases in facial grimacing. Castrated male mice, cats, and dogs did not produce the same effect in mice, suggesting that this response is likely in large part due to androgens. A longitudinal study was performed to examine their previous experiments and look at the baseline and peak responses of mice, and found that mice tested by male experimenters displayed lower baseline pain sensitivity. This stress induced analgesia in the mice is shown only when in the presence of a male. The stress caused by male experimenters is short lasting, however, it is something to consider when conducting animal research, particularly when interested in different stress responses. -MTL

Developmental changes in GABAergic neurotransmission to presympathetic and cardiac parasympathetic neurons in the brainstem

Olga Dergacheva , Carie R. Boychuk , David MendelowitzJournal of Neurophysiology Published 1 August 2013Vol. 110no. 672-679DOI: 10.1152/jn.01054.2012. This article was interesting because it investigated how postnatal development altered gabaergic neurotransmission specifically, the effects on parasympathetic cardiac and presympathetic neurons in the brainstem. This group was interested in how hypoxia and hypercapnia (H/H) may play role in this. They used retrograde labelling with CTB injected in the T2-T4 region of the spinal cord. Then at certain days postnatal p5, p20, p30 and they looked at GABAergic neurotransmission and how it was altered by hypoxia and hypercapnia. In the parasympathetic cardiac vagal neurons there was IPSCs in response to strychnine in P5, P20, and P30. However, the P20 had increased frequency and amplitude of IPSCs than the other groups. As for the cardiac parasympathetic neurons on the P20 frequency was altered in under control conditions when compared to the P5 and P30. In response to H/H in the P5 and P30 the IPSCs were reversed however in the P20 frequency and amplitude IPSC not altered for presympathetic neurons. H/H decreased IPSC frequency in P5,P20, and P30. In only decreased the amplitude in the P20. These data demonstrate P20 is an important developmental stage for parasympathetic and sympathetic development. The reason why this study is so important is because this age in rats is equivalent to the age when children tend to develop SIDS. This increased sensitivity to hypoxia/ hypercapnia may be a possible reason why SIDS. -MD

Exercise Training Lowers the Enhanced Tonically Active Glutamatergic Input to the Rostral Ventrolateral Medulla in Hypertensive Rats

Yan-Ping Zha, Yang-Kai Wang, Yu Deng, Ru-Wen Zhang, Xing Tan, Wen-Jun Yuan, Xiao-Ming Deng & Wei-Zhong Wang. doi: 10.1111/cns.12065 The study investigated how exercise can modulate glutamate neurotransmission. Using WKYs and SHRs that were sedentary (sed) or exercise trained (ext) they looked basal blood pressure (BP), which was significantly reduced in the SHRs-ext when compared to the SHRs-sed. HR was also significantly reduced in the SHR-ext when compared to the SHR-sed. Similarly, they saw that exercise also reduced the decrease in BP and HR that is caused by giving a glutamate receptor blocker. Kyn had little affect ion bp and HR responses in the WKYs. In order to determine whether the concentration of glutamate is altered they used HPLC and found that SHR-sed had a higher concentration of glutamate when compared to WKYs(both sed and ext) and also SHR-ext. Western blot was done in order to examine the amount of vglut2 protein being expressed in the RVLM . Results showed that the SHR-SED had more vglut2 compared to the other groups. Finally they wanted to determine where this glutamatergic input is originating from. Western blot was done on punches from the NTS, PVN and PRF for glutaminase2. They found a significant increase in NTS and PVN in SHR-seds for glutaminase2 when compared to SHR-ext. Overall message, exercise reduces glutamatergic input via reduction in concentration of glutamate in rvlm and also a reduction in glutamatergic inputs from NTS and PVN. Maybe we should start looking at NTS also because it could be providing some glutamatergic input to rvlm.-MD

Discharge of RVLM vasomotor neurons is not increased in anesthetized angiotensin II-salt hypertensive rats.

Pedrino GR, Calderon AS, Andrade MA, Cravo SL, Toney GM. Am J Physiol Heart Circ Physiol. 2013 Dec;305(12):H1781-9. I might get in trouble for saying this... but I kind of dislike this paper for personal reasons. These people are also interested in differences in RVLM neuron firing rates between normotensive and hypertensive rats. They looked at the difference in RVLM unit activity between normotensive (NT) rats and rats that had become hypertensive (HT) due to three weeks of AngII infusion and consumption of a high-salt diet. Neurons were confirmed to be presympathetic and spinally projecting via baroinhibition and antidromic action potentials. They noted that the neurons fired one spike per heart beat, which was likely to fire during mid-late systole, which was not different between groups. The discharge rate at resting MAP was similar between groups, so they looked at barosensitivity and found that HT rats required a higher increase in MAP before they would stop firing. They also used nitroprusside to unload baroreceptors and found that both groups had similar maximum discharge rates and requisite change in MAP needed to achieve these max rates, but within a certain range of MAPs (120-150mmHg) neurons in HT rats were more likely to be firing at a higher rate. This says that just like we would expect to see in our exercise vs inactivity model, unhealthy rats are reluctant to decrease a high sympathetic tone even when there is no physiologic need for it. They also did juxtacellular of some cells found that in NT rats, 3 were C1 (PNMT positive) and 4 were non-C1. In HT rats, 6 were C1 and 6 were non-C1. -DH

Disinhibition of the midbrain colliculi unmasks coordinated autonomic, respiratory and somatomotor responses to auditory and visual stimuli.

Müller-Ribeiro FC, Dampney RA, McMullan S, Fontes MA, Goodchild AK Am J Physiol Regul Integr Comp Physiol. 2014 Aug 6. Previous work has shown that if you cause blockade of GABA-A receptors in the colliculi of awake rats, the rats will show defensive/escape responses to stimuli that normally wouldn't bother them. This suggested that the colliculi are involved in activation of the sympathetic nervous system, but that the neurons are usually under tonic GABAergic inhibition. Based on that idea, this paper studies the effect of GABA-A blockade in the superior and inferior colliculi and its effect on splanchnic, sciatic, and phrenic nerve activity in anesthetized rats. Normal, anesthetized rats should have no increases in nerve activity or MAP after the rat is exposed to clapping, flashes of light, and paw-pinches. That's exactly what they saw in their preparation. They then blocked GABA-A receptors with picrotoxin (which rarely caused changes in baseline nerve activity), and gave the rats the same stimuli. After blockade, they saw that all three of those stimuli could cause synchronized increases in nerve activity among all three of the nerves. This is strong proof that the colliculi are relay points for autonomic activity that are normally kept under GABAergic inhibition. They followed this up by trying to figure out where this GABA might be coming from - to do this they removed the forebrain and saw the exact same effects. Because they saw the complete response in decerebrate rats, they can now say that GABA is coming from either the midbrain or the brainstem, tonicically inhibiting the colliculi's activation of nerve activity, and also that whatever signal is driving that GABAergic tone is ALSO located in the midbrain or brainstem. -DH

Role of voltage-gated L-type calcium channel isoforms for brain function

Striessnig, J., et al. "Role of voltage-gated L-type Ca2+ channel isoforms for brain function." Biochemical Society Transactions 34.Pt 5 (2006): 903-909. This is a more detailed study examining the role of both L-type calcium channel (LTCC) isoforms 1.2 and 1.3 within the brain. Like discussed previously in my last blog, because the LTCC agonist and antagonist do not show specific binding to either 1.2 or 1.3, it has been difficult to differentiate the roles each plays. However, more recently discovered, two groups of genetically modified mice have enabled scientist to conduct much needed studies better characterizing these two calcium channels. The first mouse group Cav1.3-/- has a selective knockout for all Cav1.3 channels. Whereas the second modified mouse group Cav1.2-/- has a modified α1 subunit that inhibits the binding of agonists and antagonists from the channel. With the creation of these mice, creative studies can now selectively examine either isoform 1.2 or 1.3 depending on which pharmacological substances are utilized. Recent studies have now found that homologous knockouts of isoform 1.3 causes complete deafness, as well as sinoatrial node dysfunction. These results lend to the hypothesis that 1.3 specific calcium channels play a vital role in cochlear hair and sinoatrial node cell signaling transduction. Similarly, experiments utilizing Cav1.2-/- have revealed the importance in Ca2+ influx through Cav1.2 in regulating smooth muscle and cardiac contractility. This study also reiterates the importance of both isoforms in synaptic plasticity pertaining to memory, learning, and the storage of fear induced memories. Interestingly, when sensitivity to direct Ca2+ channel activators was inhibited in Cav1.2-/-, the efflux of glutamate from neurons in the ventral striatum was completely abolished, providing evidence for the importance of Cav1.2 in neurotransmission of glutamate. Again, I think this paper provides plausible evidence that L-type calcium channels may play an important role in synaptic plasticity within the RVLM. ~JI

Role of rostral ventrolateral medulla centrally mediated pressor responses

Full cite: Kiely JM, Gordon FJ. Role of rostral ventrolateral medulla centrally mediated pressor responses. Am J Physiol Heart Circ Physiol 267:H1549–H1556, 1994. Role of rostral ventrolateral medulla centrally mediated pressor responses James M. Kiely and Frank J. Gordon Department of Pharmacology, Emory University of Medicine, Atlanta, Georgia 30322 This paper is one of the first to look at how the RVLM mediates different central pressor responses, in particular how EAA receptors play a role in the proliferation of these responses. In this particular experiment they used female Sprague-Dawley rats, recorded BP, and instrumented for sciatic nerve stimulation (SPR). Electrodes were positioned into the periventricular nucleus (*which I thought was pretty cool*). Then a head surgery was performed to expose the brainstem, similar to how we prepare for our microinjection experiments. Kyn was injected bilaterally and there was no BP response, similar to in our experiments. When Kyn was injected in the RVLM, the hypothalamus’s pressor responses were intact. Injection of kainic acid increased BP for a short period of time (= 10s), after that point BP fell around 35 mmHg (~5 min). SPR and PFH responses were attenuated, PVN and AH responses were intact. PFH and PVN responses were reduced following injection of muscimol; lidocaine into RVLM abolished SPR, and greatly reduced the PFH and PVN responses. This paper is likely one of the first to demonstrate that the PVN and PFH have spinal projections of their own and are able to function at least somewhat independently from RVLM. Also it seems that SPR are almost exclusively mediated through the RVLM. It would be interesting to see how the activity of PVN and PFH might be altered during an RVLM knockout or blockade. I also do not know whether or not PVN or PFH are barosensitive. I think that at some point we are either going to have to integrate PVN into our experiments or knock it out in some way, because it may very well be playing a role in our responses. MTL

The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain functions

Berger, Stefan M., and Dusan Bartsch. "The role of L-type voltage-gated calcium channels Cav1. 2 and Cav1. 3 in normal and pathological brain function." Cell and tissue research 357.2 (2014): 463-476. There are multiple types of voltage-gated calcium channels (VGCCs) including L-type, T-type, P/Q-type, R-type, and N-type that are defined by their pharmacological responses. Each channel is made up of five subunits, with the main pore forming subunit being alpha1. For this discussion I will mainly be focusing on the L-type calcium channels(1.1-1.4) which are believed to be the channels that allow manganese entry into a depolarized cell when using manganese enhanced MRI. More specifically, I will be focusing on 1.2 and 1.3 which are expressed by in large in the body compared to the more restricted 1.1 and 1.4. Cav1.2 and Cav1.3 can be found on multiple organs in the periphery including the hear, smooth muscle, pancreases, and adrenal glands, but most importantly for my studies, both of these channels are also expressed on neurons in the brain. With some cells expressing both 1.2 and 1.3, leading to the hypothesis that each channel may have its own specific function and importance. Some studies using radioreceptor assays have suggested that 89% of all Cav isoforms in the brain are 1.2, where as only 11% are 1.3. However, other studies utilizing western blotting have stated that within neurons in the hippocampus, cerebral cortex, and cerebellum 1.2 and 1.3 isoforms are equally abundant. As for location within a cell, it appears 1.2 are mostly found on the post synaptic dendrites, compared to the 1.3 which is most dense around the cell body. Electrophysiologically, 1.3 isoform channels are activated more rapidly and in more hyperpolarized membranes. They are also inactivated with a slower current than 1.2 isoforms. In both channels calmodulin acts as an imperative calcium sensor that 1) initiates the inactivation of the calcium channel preventing intracellular calcium toxicity 2) causes phosphorylation of the channel which increases the probability of an open state during repeated or prolonged activation and 3) enables the expression of calcium dependent genes within the cell. Unfortunately, there still is not a well established antibody for the isoform 1.3 so these studies may be less reliable then more recent studies being undertaken using Cre recombinase knockout mice. Using genetically modified mice, pharmacological experiments have showed that LTCCs play a role in synaptic plasticity involving learning and memory. Interestingly, injections of LTCC antagonists into the hippocampus have shown increases in acquisition and retention of spatial reference and working memory. Correspondingly, chronic injections into older animals has shown prevention of age-related hippocampal-dependent memory loss. It is believed this change is linked to the loss of NMDA-receptor-independent form of late long-term potentiation. Related, it has also been shown using LTCC antagonist injections into the amygdala that blocking LTCC also blocks the formation of fear memories. Finally, other studies have shown a link between LTCC's and the modulation of the mesoccumbal dopamine signaling pathway, which plays a major role in the reward system and addiction. Compared to animal studies, few human studies have been done analyzing LTCCs. However, in the past few years the hypothesis that LTCC's play an important role in psychiatric diseases is becoming more and more accepted. Stemming from patients with Timothy Syndrome, upregulated LTCC activation leads to the upregulation of tyrosine hydroxylase expression, causing increased concentrations of norepinephrine and dopamine. They have also shown that stimulation of TS-mutated calcium channels 1.2 cells led to dendritic retraction. Since the GWA the CACNA1C gene, associated with the alpha1 subunit on Cav1.2, was identified as a common risk factor allele for bipolar disorder, schizophrenia, and major depression. As for physiological defects in the isoform 1.3, recent studies are showing Cav1.3-mediated vulnerability of the dopaminergic neurons affected by Parkinson's disease. Specifically, increases in calcium entry increases alpha-synuclein aggregates present in Parkinson's disease. Conclusively, very few studies have been done examining the role of VLCC's in normal human cognition up to date. I think studies examining the role of L-type calcium channel activity within the RVLM would be interesting, and may shed light on differences in neuroplasticity between sedentary and physically active animals. ~JI

Role of presympathetic C1 neurons in the sympatholytic and hypotensive effects of clonidine in rats

Ann M. Schreihofer and Patrice G. Guyenet . American Journal of Physiology - Regulatory, Integrative and Comparative Physiology Published 1 November 2000Vol. 279no. R1753-R1762. The rostral ventrolateral medulla (rvlm) is important to control of blood pressure (BP) and sympathetic nerve activity (SNA). Clonidine is an antihypertensive medication that acts as an agonist for α1 adrenergic receptor on axons and terminals of rvlm neurons. The purpose of this paper was to determine whether clonidine acted on spinally projecting c1 or no c1 in the rvlm in order to lower SNA and BP. What they found was that after injecting DβH- saporin the response to clonidine. This finding along with previous data from other labs that showed that injections of clonidine into the nucleus tractus solitarius (NTS) and in the intermediolateral cell column (IML) demonstrates that clonidine may act on several areas in the brain in order to have the antihypertensive affect that is known for.-MD

Stress-induced elevations of y-aminobutyric acid type A receptor-active steroids in the rat brain

ROBERT H. PURDY, A. LESLIE MORROW, PERRY H. MOORE, JR., AND STEVEN M. PAUL. Proc. Nati. Acad. Sci. USA. Vol. 88, pp. 4553-4557, May 1991 Neurobiology. They investigated the effects of stress on the production of pregestrone metabolites in the brain. They used adrenectomized and non-adrenalectomized rats in order to determine how csf levels of progesterone, allopregnanolone and pregnanolone in rats were altered in response to swim stress. They used a couple of techniques such as chromatography, and RIA or Radioimmuno assay. What they found was that in the adrenalectomized, adrenalectomized stressed and controls had undetectable amounts of allotetrahydroDOC in plasma and cortex, however the stressed rats had significant enhanced levels of allotetrahydroDOC in both cortex and plasma. As for progesterone was not altered in response to stress when compared to the control group however the adrenalectomized rats had significantly lower progesterone when compared to the control rats. As for the allopregnanolone, in response to swim stress the level was significantly higher when compared to control animals in the cortex and plasma. Another interesting finding is that the in the adrenalectomized rats there was reduced allopregnanolone. The most important point I got from this article is that allopregnanolane and allotetrahydroDOC have the ability to bind to the benzodiazepine site on the GABA receptor, thus potentiating the effects of GABA. Since it seems that doing a adrenalectomy, abolishes the increase in these steroid metabolites, adrenal gland may be important to modulating GABAergic mechanisms in the brain. -MD