Neurons of the rostral ventrolateral medulla mediate somatic pressor reflex

Stornetta RL, Morrison SF, Ruggiero DA, Reis DJ.

Department of Neurology, Cornell University Medical College, New York, New York 10021

This article was investigating whether rostral ventral lateral is mediating the somatic pressor reflex. The somatic pressor reflex is activated when the sciatic nerve for example is stimulated, this will cause an increase in arterial pressure along with SNA. The SPR had been identified in cats and dogs however not in rats, so they investigated this response in rats. Where the information from the stimulation travel up afferent nerve was been integrated into a response that would cause changes in Arterial Pressure (AP) was unknown. Since they knew that RVLM neurons played role in tonically and in reflex control of blood pressure along with the fact that the neurons in RVLM had an excitatory effect on sympathetic preganglionic neuron in the IML, this lead them to believe that the SPR may be mediated by the RVLM. So what the concluded from the study was the following:

1.       Stimulation of the sciatic, sural and saphenous caused increases in AP and HR  which is similar dog and cat

2.       Stimulation of muscle nerves such as tibial or femoral nerves resulted in decreases in AP and HR.

3.       After transection of the anterior pons the SPR was still functioning suggesting that the SPR is controlled by a brain region of somewhere below the pons

4.       Lesions of the LRN  reduced the SPR but when kainic(before lesion) was injected into the LRN the SPR was still functional meaning that when the LRN was not the brain region mediating this responses but when the area was lesion there may have been nerve fibers destroyed or because LRN is so close to RVLM some of the neurons in this area could have been damaged

5.       Finally they found that if there is unilateral lesion of an RVLM and the contralateral sciatic is stimulated then the response will be blocked.  

Spontaneously Hypertensive Rats Are Highly Vulnerable to AMPA-Induced Brain Lesions

Clotilde Lecrux et al.,
University of Caen, France.
Stroke. 2007
http://stroke.ahajournals.org/content/38/11/3007.full.pdf+html


The fact that chronic arterial hypertension is a major risk factor for cerebral ischemia is well known. However the mechanisms by which arterial hypertension induces brain damage is unknown. In this study the authors were interested in two important steps that could possibly result in understanding the mechanisms. First they compared the evolution of cerebral blood flow during transient ischemia, using a Doppler flowmetry probe between SHRs and WKYs. Secondly they examined the amount of brain damage induced by the administration of NMDA or AMPA (because following ischemia there is overactivation of these ionotropic glutamate receptors) into the striatum using 20 um section stained by thionin. Thirdly since in the SHRs the hypertension develops over a period of time as a control, hypertension was induced in a group of 5 weeks old WKYs rats by renal arterial stenosis and tested over a period of time. Finally they tested whether the increased sensitivity of SHRs to AMPA receptors is linked to the differential expression of its subunits in the striatum using western blot and RT-PCR. The present study provides evidence that SHRs show more specificity to AMPA receptors activation. Even though the levels of AMPA receptors subunits GluR1 and GluR2 were similar between both the strains, the phosphorylated form, pGluR1 is significantly higher in SHRs compared to WKYs. Further SHRs also expressed higher levels of CamKIIalpha protein in the striatum compared to WKYs. In addition, inhibition of this kinase significantly reduced the deleterious effects of AMPA receptors overactivation in the SHRs. This article suggests  that phosphorylation of AMPA receptors, at least in part may explain the propensity of this strain (SHRs) to stroke.

- Madhan

Monosynaptic connection from caudal to rostral ventrolateral medulla in the baroreceptor reflex pathway

S.K. Agarwal and F.R Calaresu

Department of Physiology, University of Western Ontario, London, Ont (Canada)

We already know that CVLM projects to RVLM; this article is one reason why that conclusion has been made. So the authors hypothesized that the CVLM neurons are activated by activation arterial baroreceptors, activation of NTS and there is a monosynaptic between the CVLM and RVLM.  In urethane anesthetized rats they recorded from several neurons within that CVLM. The tested the activity of these neurons by giving a bolus of PE to increase blood pressure and in order to activate the CVLM neurons. By using electrophysiology, they were able to record the activity of the neurons directly. They found that 20 were actually activated. Only 6 were activated by stimulation of the NTS. The same 6 neurons were activated by antidromatic stimulation of the RVLM.   The conclusion that they came to from the experiment were the following 1.) NTS has excitatory inputs to CVLM  2.) CVLM has neurons that are barosensitive and send inhibitory inputs to RVLM 3.) There is a monosynaptic connection between the CVLM and RVLM.

Mandatory Electrocardiographic Screening of Athletes to Reduce Their Risk for Sudden Death. Proven Fact or Wishful Thinking?

A little off-topic, but interesting nonetheless.
Because of their tragic nature, the media-at-large often emphasizes reports of young athletes dying of sudden cardiac arrest (the heart stops) during a practice or a game.  To address this obvious problem, many community groups and even some medical organizations have called for mandatory screening of young athletes for the kinds of electrical heart problems that may lead to a sudden arrest. A few of these programs sprung up in the early and mid-80s in Italy, Israel, and Minnesota USA. Well, the pendulum is beginning to swing the other way, and doctors are questioning the cost/benefit of screening all young athletes. That is the focus of this article.
A recent analysis of Italy's (The world's Ultimate Nannystate) ECG screening program, which ran for 25 years, showed a significant reduction in sudden cardiac death (SCD) during the program period. These authors questioned that result since, by comparison, SCD rates in Italy were high to start out with. A similar program was enacted in Israel in 1986, and the authors retrospectively analyzed the yearly standardized rates of SCD in competitive athletes from 1986-2006 to see if the screening program had an impact.
It did not.
In addition, a similar analysis of the Minnesota program showed the same result. Moreover, the authors concluded that even if one were to take the results of the Italian study as fact, it would cost around $1,500,000 in screenings and additional tests to save one life.Finally, because of the extrememly low incidence of SCD and the less-than-perfect specificity of the ECG, many many more healthy athletes would be excluded from participating because of palse positives than lives would be saved.
I'm not saying that morally one can put a price on human life, but since governments are often amoral they can, and they do.
Draw your own conclusion on how much a random young life is worth to the taxpayer.
-Nick

GABA(A) receptor activation at medullary sympathetic neurons contributes to postexercise hypotension.

Am J Physiol Heart Circ Physiol. 2002 May;282(5):H1615-24.

Kajekar R, Chen CY, Mutoh T, Bonham AC.

Department of Internal Medicine, University of California at Davis, Davis, California 95616, USA.

A single bout of mild or moderate exercise in a hypertensive individual leads to post exercise hypotension (PEH). Two important features of PEH are a reduction in sympathetic nerve activity (SNA) and an intact baroreflex system. In this study the authors investigated the possible mechanisms underlying PEH. The experiments were performed in male spontaneously hypertensive rats (SHR) seperated in two groups, PEH group were subjected to single bout of exercise on a motor driven treadmill at 15m/min, 10 degree for 40 min or to a sham exercise group (sham-PEH) placed in treadmill with no exercise for 40 min. This study provides some interesting findings, during PEH the spontaneous firing activity of the cardiovascular sympathetic neurons in the RVLM are significantly decreased along with a reduced lumbar SNA. The reduced neuronal activity were mediated at least in part by a GABAa receptor mechanism, which was tested by injection of muscimol and bicuculine on RVLM. Muscimol and bicuculine produced a current-related decrease and increase in unit activity respectively supporting the above findings. Finally they also found that during PEH the baroreceptor control of RVLM activity is reduced. These findings suggest the possibility that increased signaling of GABA at RVLM neurons may contribute to PEH by decreasing sympathetic outflow.

- Madhan

http://ajpheart.physiology.org/content/282/5/H1615.full.pdf+html

Depressor neurons in rabbit caudal medulla act via GABA receptors in rostral medulla

W.W. Blessing

Department of Medicine, Centre for Neuroscience, flinders University of South Australia, Bedford Park, 5042 South Australia, Australia

This article is an older article by Blessing. This laboratory investigated whether cardiovascular responses can be affected by CVLM and if RVLM is necessary for the transmission of the signal. They used microinjection technique, in order to investigate their question. Using male rabbits, they recorded from the renal nerve along with the arterial pressure and heart rate. They used horseradish peroxidase for histological identification of the areas that they injected into. Microinjections of bicuculline (bic), a GABA_A  receptor antagonist at different doses into the RVLM was done. Then after every bic microinjection they waited 5 minutes and then glutamate was injected in the CVLM.  For another group of rabbits they used a mixed agonist antagonist (Muscimol and bic) that was microinjected into the RVLM and then they either microinjected strychnine (glycine blocker) or Phenotolamine (α-adrenergic receptor blocker) and then injected glutamate in the CVLM and recorded the responses. What they found by doing the following experiments was that GABA is playing a role in modulating RVLM effects on sympathetic output by acting on GABA receptors in the RVLM. Also they found that CVLM does release GABA that acts on the GABA receptors in the RVLM to decrease sympathetic output. Finally they demonstrated that CVLM has an effect on cardiovascular responses by acting on GABA receptors in the RVLM and not by acting on glycine and α-adrenergic receptors.

Differential drives from rostral ventrolateral medullary neurons to three identified sympathetic outflows.

McAllen RM, May CN.


This article dates back to more than a decade ago. Microinjection of glutamate in the RVLM of cats have shown to drive the sympathetic outflow differentially based on the area stimulated. Muscle vasoconstrictor activity were driven when the injections were lateral whereas skin vasocontrictor responses were driven when the injections are medial with in the RVLM. In this study the authors were interested in investigating whether seperate RVLM neurons controlling different vasomotor outflows holds true for tissues other than skin and kidney. In addition they further studied the relative strenghts of such specific drives and the location of their cells of origin. Several microinjections of sodium glutamate were given in a grid like pattern covering the RVLM. All the recording were made in cats anesthetized using chloralose. Muscle vasoconstrictor (MVC), visceral vasoconstrictor (VVC), and renal sympathetic nerves (RSN) were recorded. Findings from this study suggest that each of these sympathetic outflow are driven by a seperate population of neurons with in the RVLM.

American Journal of Physiology, 1994
http://ajpregu.physiology.org/content/267/4/R935.full.pdf+html

-Madhan

Dendritic morphology of neurons in medial prefrontal cortex, hippocampus, and nucleus accumbens in adult SH rats.

The brain is very susceptible to hypertension-induced damage. In addition to the well-known increased risk of stroke in hypertensive patients, hypertension alone (rat model) can induce changes in neuron structure in a cognitive brain center. The aim of this study was to characterize the differences in neuron morphology in three cognitive centers, the medial Prefrontal Cortex (mPFC), Hippocampus (Hcx), and Nucleus Accumbens (NAcc). They utilized Golgi-Cox staining to analyze 1260 (!!) neurons in 21 spontaneously hypertensive rats (SHR) and 21 Wistar-Kyoto rats (WKY). They also measured blood pressure via a tail cuff in both groups to confirm the presence of hypertension. Finally, they assesed activity in a novel environment (i.e. curiosity). Dendritic spines, a morphologic structure, usually indicate the presence of a synapse. In general, spine density decreased in both groups over time while overall dendritic length remained the same. Spine density decreased more in SHR versus WHY. The activity level (or curiosity) of SHRs was greater than WKYs. The authors hypothesize that alterations in dopaminergic regulation in these centers is related to the behavioral differences documented between the two strains. They go on to suggest that alterations in these and other structures during hypertension are an important component of the hypertensive syndrome.

-Nick  

Altered regulation of the rostral ventrolateral medulla in hypertensive obese Zucker rats

Huber DA, Schreihofer AM.

Dept. of Integrative Physiology, Univ. of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.

Am J Physiol Heart Circ Physiol. 2011 Jul;301(1):H230-40. Epub 2011 May 2.

http://ajpheart.physiology.org/content/301/1/H230.full.pdf+html

Numerous elegant studies by Professor Alan Sved and other investigators have shown that elevated sympathetic nerve activity (in spontaneously hypertensive rats, Dahl salt-sensitive rats and other hypertenive rat models) is associated with increased tonic activation of RVLM by glutamate and angiotensin II and decreased tonic GABAergic inhibition.  Obese Zucker rats (OZR) have elevated renal and splanchnic sympathetic nerve activity and mean arterial pressure, however the mechanisms are unknown. In order to understand the mechanisms the authors did mutliple experiments and made some significant findings. When RVLM was inhibited it produced a greater decrease in MAP in the OZR compared to their lean counterpart. When AT1 receptors were blocked it produced a modest decrease in splanchnic SNA and MAP in the OZR but not in lean zucker rats (LZR). Ionotropic glutamate receptor antagonists produced comparable differences in the SNA, MAP and HR in OZR and LZR. GABA-A receptor antagonist produced smaller increase in SNA, MAP and HR in OZR compared to LZR. Finally inhibition of CVLM or the NTS produced a smaller increase in SNA and HR in OZR compared to LZR but the pressor responses were normal.  The authors suggest some possible explantion for this unexpected finding. One possibility is that the pressor response is a summation of changes in splanchnic SNA with other other sympathetic responses and another possibility could be related to a threshold effect because of changes in vascular reactivity. Overall the results suggest that elevated MAP and SNA in the OZRs are a measure of increased angiotensinergic activation and reduced GABAergic inhibition of the RVLM.

(In)activity dependent alterations in resting and reflex control of splanchnic sympathetic nerve activity

Mischel, NA and Mueller, PJ.  Am J Physiol Regul Integr Comp Physiol (resubmitted)

I wanted to be the first to post a blog on a hot new paper that is destined to be accepted in AJP: Reg in the next couple of weeks.  It's the first first author paper from a student in an up and coming laboratory examining neuroplasticity in cardiovascular regulation that occurs following sedentary versus physically active conditions.  In the study the author observed increased resting and stimulated sympathetic nerve activity.  Neuroplasticity in the brainstem is a possible mechanism since one of the methods used to increase sympathetic nerve activity was with direct microinjections of the excitatory amino acid, glutamate.  Interestingly, the authors also observed enhanced sympathoexcitation with unloading of arterial baroreceptors with the vasodilator nitroprusside.  Although baroreceptor unloading is typically associated with withdrawel of GABAergic inhibition of the RVLM (i.e. disinhibition), there are a few studies that have implicated glutamate in the the baroreflex (see Maryov and Head, 2003).  Lastly, the authors test vascular sensitivity by injections of the alpha one agonist phenylephrine.  In a classic move, they test the responses in the presence of ganglionic blockade to remove any compensatory actions of the baroreflex from influencing their results.  All an all an interesting paper that is likely to be cited by several since it implicates the splanchnic circulation as a target for physical (in)activty dependent neuroplasticity.  As in their review from 2010, they make use of a "physical (in)activity" nomenclature to promote the idea that these changes could be the result of remaining sedentary, being physically active or both.  They suggest future studies are warrant to distinguish between these possibilities.

-Posted by Pat (upon resubmission of Nick's 1st paper, of course!

Distribution and projection of the medullary cardiovascular control neurons containing glutamate, glutamic acid decarboxylase, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rats

Takashi Suzuki, Kiyoshige Takayama, Mitsuhiko Miura

The aim of this study is to determine the distribution and projection of the medullary cardiovascular neurons that contain the neurotransmitters or the enzymes involved in cardiovascular regulation. Six male Wistar rats were used, and a mixture of WGA-HRP was injected into the depressor caudal ventrolateral medulla (D-CVLM) and the pressor rostral ventrolateral medulla(P-RVLM). Immunohistochemistry was performed to identify HRP-labelled neurons which were stained with antiserum to glutamate (GLU),glutamic acid decarboxylase(GAD),tyrosine hydroxylase(TH) or phenylethanolamine N-methyltransferase (PNMT). The investigators in this study counted all the single HRP-labeled and the double labeled neurons in the NTS, D-CVLM and P-RVLM either ipsilaterally or contralaterally to HRP injection. They found that: (1)the HRP/GLU-labeled neurons in the NTS project to both the D-CVLM and P-RVLM which indicates divergent excitatory projection, (2)the P-RVLM receives innervation from HRP/GLU- labeled neurons in the D-CVLM, while the D-CVLM receives innervation from HRP-GLU-labeled neurons in the P-RVLM indicating the mutual innervation,(3)the HRP/GAD- labeled neurons in both the CVLM and the NTS project to the P-RVLM indicating the convergent  inhibitory  projection,(4) there are projection of both  HRP/TH- and HRP/PNMT- labeled neurons  to the P-RVLM but they are much less than the HRP/GAD-labeled neurons projections to the P-RVLM .  From these results, they concluded that catecholaminergic  neurons paly a minor role in inhibition of the sympathetic activity of the RVLM neurons, the glutamatergic NTS neurons excite both the P-RVLM and D-CVLM neurons, and the GABA ergic NTS and CVLM neurons inhibit the P-RVLM neurons. 

Hot off the press!! Splanchnic sympathetic nerves in the development of mild DOCA-salt hypertension.

Hi everyone, sorry for the gap since my last post, but I hope I can make up for it with this exciting new paper from our friends down the road at Michigan State.

Recent work from Greg Fink's lab (last author) and others has highlighted the importance of examining splanchnic sympathetic activity in the development and/or maintanance of hypertension. For example, experimental hypertension induced in rats using exogenous angiotensin II and a high salt diet is characterized by differential sympathetic responses depending on which "sympathetic regions", including splanchnic, are examined. In that model, renal nerve activity is decreased and lumbar nerve activity remains unchanged as measured by chronic nerve recordings in conscious rats. If the splanchnic sympathetic bed is denervated by removing the celiac ganglia (CGx) in the same model, blood pressure falls dramatically, suggesting that sympatehtic activity to the splanchnic bed is increased.
In this new study, they examine the role of the splanchnic bed in another clinically relevant model of hypertension, the DOCA-salt model. Similar to the ANGII-salt model, it appears that the combination of high salt with overexposure to a blood pressure regulating hormone, in this case the aldosterone-like molecule deoxycorticosterone acetate (DOCA), produces significant hypertension.
The lead author implanted a long-term release DOCA pellet in rats at the same time as implanting a radiotelemeter to measure arterial pressure chronically. Some rats were subjected to  CGx and some underwent the same surgery except not denervated. Over a period of two weeks to a month, blood pressure significantly increased in both groups, but the increase was significantly attenuated in CGx rats. At the end of the experiment, tissue supplied via the spalnchnic circulation was harvested and analyzed for norepinephrine (NE) content. NE was markedly diminished in the sampled tissues, indicating that CGx was effective. Additionally, the lead author infused radiolabeled NE into the rats until it reached steady state concentrations, then simultaneously sampled arterial and venous splanchnic blood for radiolabeled and endogenous NE levels. A calculation of the differences between arterial and venous values of radiolabeled and endogenous NE indicates how much endogenous NE is has been used by sympathetic nerve terminals as a neurotransmitter and then "spilled over" into venous blood. This procedure is a way to measure NE handling in specific tissues, with increased NE spillover indicating increased sympathetic outflow to the tissue. They found that whole-body NE spillover and NE plasma levels were decreased in CGx during the control period, but not during DOCA-salt. Similar findings were observed with splanchnic NE spillover.
The data indicate that the splanchnic bed certainly contributes to hypertension in this model but is not wholly responsible for it. In addition, splanchnic SNA does not appear to be increased in this model when assessed by the NE spillover method. Thus, it is possible that, in this model, increased reactivity of the splanchnic vasculature decreased arterial and venous compliance and leads to increased central arterial pressure.

-Nick

Angiotensin-II-induced reactive oxygen species along the SFO-PVN-RVLM pathway: implications in neurogenic hypertension

V.A. Braga, I.A. Medeiros, T.P. Ribeiro, M.S. França-Silva, M.S. Botelho-Ono and D.D. Guimarães
Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, João Pessoa, PB, Brasil.

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2011007500088&lng=en&nrm=iso&tlng=en

This is an interesting review about how angiotensin-II (Ang-II)-induced reactive oxygen species (ROS) produces some forms of neurogenic hypertension. The most interesting aspect is that it discusses about the three important areas, subfornical organ (SFO), paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) and their contributions individually and in combination with one another for the pathogensis of neurogenic hypertension. Direct microinjection studies in the brain using Ang-II have shown that oxidative stress is an important mechanism by which Ang-II increases blood pressure. However the question of how circulating Ang-II acts on these brain centers without crossing blood brain barrier, resulting in increased sympathetic nerve activity and causing hypertension remains to be answered. Numerous hypothesis have been postulated, one widely accepted hypothesis is that Ang-II acts on the neurons in the circumventricualr organ (CVO) which lacks blood brain barrier, which inturn alter the other brian regions by increasing the production of local Ang-II. This review mainly address the mechanisms by which Ang-II acts on the neurons in the SFO (one of the CVOs), and how SFO communicates with PVN and RVLM in ROS production, regulating sympathetic activation and altering blood pressure. This review points out at numerous studies that directly and indirectly have shown how ROS production in all the three regions SFO, PVN and RVLM could contribute to neurogenic hypertension. The figure in the last page summarizes how Ang-II could induce the production of ROS and inturn neuronal firing.  The possible pathway interconnecting all these regions to the pathogenesis of neurogenic hypertension could have been more elaborate.

- Madhan

Chronic AT1 receptor blockade normalizes NMDA-mediated changes in renal sympathetic nerve activity and NR1 expression within the PVN in rats with heart failure

Allison C. Kleiber, Hong Zheng, Neeru M. Sharma, and Kaushik P. Patel

Am J Physiol Heart Circ Physiol. 2010 May;298(5):H1546-55. Epub 2010 Feb 19.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.

In my previous post, I summarized the article which discusses about how normalization of glutamatergic mechanisms in the paraventricular nucleus could be a possible mechanism by which exercise training (ExT) normalizes sympathetic outflow in heart failure (HF). As a follow up, I'm summarizing this article in which the authors investigated whether Ang II type 1 (AT1) receptors are involved in the normalization of PVN glutamatergic mechanisms. Studies by the same group have previously observed that ExT reduced the increased plasma Ang II levels associated with HF. Studies have shown that intravenous infusion of Ang II elevated the Fos immunoreactivity within the PVN, suggesting the possibility that PVN gene expression can be regulated by plasma Ang II levels. This let to the hypothesize that chronic AT1 receptor blockade in rats with HF would normalize PVN NMDA mediated renal sympathetic nerve activity (RSNA) responses and increased NR1 expression within the PVN. Chronic AT1 receptor blocker losartan was given at a dose of 50 mg/kg/day for a period of 3 weeks in drinking water. Three weeks of treatment with losartan normalized the NMDA induced (microinjected into the PVN) increase in RSNA.  In addition losartan treatment also normalized the elevated mRNA and protein expression of NMDA receptor subunit NR1. To address the question whether PVN received direct inputs from plasma ANG II since it cannot cross the blood brain barrier the authors suggested the possibility that the circumventricular organs such as subfornical organ (SFO) has direct projections to the PVN and it could have received signals from the SFO. The results from the present study suggest that normalization of plasma Ang II levels is one possible mechanism by which  exercise training normalizes enhanced gluatamatergic mechanisms associated with HF.

-Madhan

Chronic intermittent hypoxia augments sympatho-excitatory response to ATP but not to l-glutamate in the RVLM of rats

Daniel B. Zoccal, J. Pablo Huidobro-Toro b, Benedito H. Machado a, 

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil,

Nucleotide Research Lab, Department of Physiology, Faculty of Biological Sciences, P. Catholic University, Santiago, Chile 

Chemoreceptors located in the carotid bodies sense oxygen levels in the blood and are able to make respiratory and cardiovascular adjustments by activating neuronal mechanisms. When there is an increase in the activation of these chemoreceptors it can lead to the development of cardiovascular and respiratory pathological conditions. In this article, they were interested in obstructive sleep apnea (OSA), many of us already know that OSA can lead to the development of hypertension. Individuals that suffer from OSA experience chronic intermittent hypoxia (CIH is basically alteration between low and ambient air conditions). As we already know, glutamate is major neurotransmitter it has been shown that Glu release in the VLM cause excitation of pre-sympathetic and respiratory neurons. ATP may also play a role in this too. So the authors wanted to see whether CIH modulated the expression of Glutamatergic (Glu) and purinergic (ATP) receptors. They used microinjection and western blot techniques, in order to investigate their question. The investigators in this article were specifically interested in Bötzinger complex (Bötc) area. Bötc is important for respiratory control. When they were trying to functional identify this area using glutamate, they were looking for increase in sympathetic excitation along with a decrease in phrenic nerve activity.

For the microjection results, they found that there was no difference between the control and the CIH group in the response to glutamate. Then by western blot, they showed that there was no difference in the expression of Glutamatergic receptor protein between the groups. The glutamate receptors that they looked at using western blot were NMDAR1 and GluR2/3. For the purinergic receptors, they looked at using western blot were P2X1, P2X3, P2X4 and P2Y2. In the microinjection experiments, they found that there was a greater response to ATP in the CIH compared to the control suggesting that there is a possible up regulation of purinergic receptors due to CIH. The western blot results showed that there was increase in P2X3 and P2X4 protein in the CIH compared to control, which further supports their data. In CIH, there is increased SNA in these individuals. This article shows a possible factor that may contribute to the increase in SNA may be mediated by increased P2X3 and P2X4 in the VLM.

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

Xin Ni1, Ding-Feng Su2, WeiWang4, Ming-Juan Xu5*, and Wei-ZhongWang1,6*

1Department of Physiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China; 2Department of Pharmacology, School of Pharmacy, Second Military MedicalUniversity, Shanghai 200433, China; 3Department of Neurobiology and Physiology, Ningxia Medical University, Yinchuan 750004, China; 4Department of Physiology, Nebraska MedicalCenter, Omaha 68198, USA; 5Department of Obstetrics and Gynecology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; and 6Key Laboratory of Molecula rNeurobiology, Ministry of Education, Second Military Medical University, Shanghai 200433, China Received 18 March 2010; revised 15 August 2010; accepted 1 September 2010; online publish-ahead-of-print 9 September 2010

Cardiovascular Research (2011) 89, 473–481 doi:10.1093/cvr/cvq289

This article investigated the effects of moxonidine, an antihypertensive drug on GABA _A  and GABA _B receptors along with GABA transmission in the RVLM. Mox activates  I₁R  that may mediate the release of GABA. So the investigators used several techniques, a couple that we currently use in the lab. They used microinjection, western blot and microdialysis. The major findings were that the effects of mox are mediated through the GABA receptors. They also demonstrated that there is an upregulation of GABA _A  and GABA _B  receptors and this was shown by western blot. By using microdialysis, they showed that there is an increase in the amount of GABA released in the RVLM after the use of mox.  These findings suggest that mox leads to an increase in GABAergic transmission in the RVLM that causes hypotension and an increase in sympathoinhibition.

GABAa α1 and α2 receptor subunit expression in rostral ventrolateral medulla in nonpregnant and pregnant rats

As most of you already know the RVLM contains the neurons responsible for excitatory input to preganglionic sympathetic neurons that produce vasoconstriction and increases in heart rate and contractility--- and you all definitely know that the neurotransmitter GABA inhibits sympathetic transmission in the RVLM. GABA_A receptors are heteroligomeric proteins that form ligand gated Cl^- channels. Expression of the different subunits can be regulated by numerous physiologic and pathophysiologic conditions. In this study the effects of pregnancy hormones (specifically estrogen and progesterone) on the expression of GABA_A subunits α_1-3. Foley et al. hypothesized that GABA_A receptors in the RVLM of late term pregnant rats may have higher expression of α₁GABA_A receptor subunit than the expression of α₂ or α₃ subunits.

Using PCR, quantitative PCR and immunoblots as their methods they set out to identify if there was indeed an effect by the progesterone metabolite 3α -OH-DHP (3α -hydroxy-dihydroprogesterone) on the α₁ receptor. The results showed that in the RVLM, the levels of GABA_A α₁ and α₂ receptor subunit mRNA and protein were similar between nonpregnant and late term pregnant rats, and the ration of GABA_A α₁ and α₂ receptor subunit mRNA expression was not different between groups. It seems that further studies have to be conducted on different GABA subunits that can be affected by neurosteroids.

The ventrolateral medulla and sympathetic regulation of arterial pressure.

ANN M. SCHREIHOFER AND ALAN F. SVED.  The ventrolateral medulla and sympathetic regulation of arterial pressure. In: Central regulation of autonomic functions.  2^nd Edition. Eds: I.J. Llewellyn-Smith and A.J. Verberne Oxford University Press, Inc. New York, 2011.

So in honor of our guest from Australia I thought it was timely to provide a posting on one of the chapters from Ida's recent book that will likely be considered the bible on neural control for at least the next 5-10 years.  The particular chapter I chose is of course most relevant to our studies on the RVLM, although other chapters in this book are also important in terms of our understanding of brainstem control of arterial pressure.  In this chapter Ann Schreihofer and Alan Sved provide a nice overview of the history and development of what the current knowledge is on brainstem control of sympathetic outflow.  They do a great job in discussing the number of techniques used to examine the ventrolateral medulla in term of it's anatomy and physiology.  Several nice figures combine immunohistochemistry, histology, BP and SNA responses etc. to illustrate the relationship between the CVLM and the RVLM.  There is also some clarification on the confusion caused by the original nomenclature of the C1 cells and that while serving as a useful marker for barosensitive, bulbospinal neurons controlling SNA, use glutamate not epinephrine serves as the primary neurotransmitters of RVLM neurons.  A thorough discussion of the roles of the RVLM and CVLM is provided.   There is a section of the caudal pressor area which while interesting, seems to pale in comparison to the importance of the CVLM and RVLM.  Finally, the last two sections are devoted to differential control and the role of the VLM in hypertension.  Both nice summaries but also highlight the number of unaswered questions that remain to be answered, some of which are being addressed in our laboratory.  Overall this is a chapter that every student, postdoc and faculty working in the field needs to be well-versed in so if you haven't done so recently or already, I would suggest giving it a look soon.

Posted by Pat

Hot off the press! Patterning of somatosympathetic reflexes reveals non-uniform organization of presympathetic drive from C1 and non-C1 RVLM neurons

It is well known that activation of muscle afferents by contracting muscle causes an increase in blood pressure. This is known as the somatopressor reflex. The reflex is driven by the SNS, and neurons in the RVLM mediate it. Some differential patterning of responses between different sympathetic nerves has been observed before, but not thoroughly investigated. This study investigates differential sympathetic nerve responses to graded activation of the afferent arm of the sciatic nerve, a.k.a. the somatosympathetic reflex. Furthermore, the relative contributions of C1 to non-C1 neurons in this response were examined.
Briefly, anesthetized rats were instrumeted to record arterial pressure and sympathetic activity from four nerves, cervical, splanchnic, lumbar, and renal. Stimulating electrodes were implanted on the left or right sciatic nerve. They performed sciatic nerve stimulation (ScNS) at varying intensities and amplitudes in normal rats. An additional set of experiments was performed in C1 neuron-depleted rats in which only splanchnic SNA was recorded.
Under normal conditions, ScNS produced double-peak responses in all but the cervical nerve. The peaks in the lumbar and renal nerves had slightly longer latency than splanchnic and cervical. In addition, the first peak of the splanchnic nerve was of larger amplitude than the second, but the opposite was seen in renal and lumbar nerves. In rats which had about 60% depletion of C1 neurons, the second peak in splanchnic SNA was abolished, but the first peak was only slightly attenuated.
These results suggest that, indeed, different sympathetic nerves have specific roles in the SNS response to muscle contraction. Moreover, C1 and non-C1 neurons are both involved in the response, each producing distinct peaks in splanchnic SNA, possibly according to the conduction velocity of their axons (C1 fast, non-C1 slow). This study provides even more evidence that SNA to specific vascular beds may be differentially controlled. It also highlights the importance of recording from multiple versus a single sympathetic output(s) in a single animal when performing in vivo experiments.
-Nick

Exercise training normalizes enhanced glutamate-mediated sympathetic activation from the PVN in heart failure

Kleiber AC, Zheng H, Schultz HD, Peuler JD, Patel KP.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.

Am J Physiol Regul Integr Comp Physiol. 2008 Jun;294(6):R1863-72. Epub 2008 Apr 2.

 

Heart failure patients have high sympathetic outflow. Studies have shown that exercise training (ExT) normalizes muscle sympathetic nerve activity (MSNA), however the mechanisms are unknown. In this study the authors hypothesized the mechanisms by which ExT reduces MSNA is by normalizing the increased glutamatergic mechanisms within the PVN. Previous studies from the lab have shown that when PVN was injected with N-methyl-D-aspartic acid (NMDA) (a glutamate agonist that acts at the NMDA receptor), there was augmented renal sympathetic nerve activity (RSNA) in the heart failure rats compared to their normalized control. In this study the authors determined whether ExT reduces the RSNA response to NMDA in heart failure. Further they also determined whether ExT normalizes the expression of NMDA receptor subunit NR1 in heart failure rats. Heart failure was induced by left coronary artery ligation. Three weeks after inducing heart failure animals were given ExT using treadmills for 3 weeks. The animals ran for a period of 1hr/day at a speed of 20-25 m/min. RSNA and microinjection procedures were performed 6-8 wk after heart failure surgery or sham surgery. Microinjection of NMDA to hearf failure rats that are on ExT produced no changes in RSNA or MAP compared to control. This shows that ExT normalizes the augmented RSNA in response to NMDA microinjection in the PVN of heart failure rats. ExT also reduced the gene expression and protein levels of NMDA receptor subunit NR1 in the heart failure rats compared to heart failure rats that were not on ExT suggesting a possible mechanism by which RSNA responses to NMDA injected in to the PVN. These results suggest normalization of glutamatergic mechanisms is one possible way by which ExT normalizes sympathetic outflow in heart failure.

 

- Madhan

Local anaesthetics for acute reversible blockade of the sympathetic

Melissa M.J. Farnham, Paul M. Pilowsky

∗Australian School of Advanced Medicine, Level 1, Dow Corning Building, 3 Innovation Rd., Macquarie University, Macquarie, NSW 2109, Australia

Sinoaortic denervation (SAD) is a technique used to determine the role that the baroreceptors play in the control of blood pressure in different pathological states. In this article they discussed an alternative method to the current way of doing a SAD. They should how the use of lignocaine (10 – 30 min) and the bupivacaine (30 – 60 min) can yield result very similar to a complete (fig 2 and 3). The reversible SAD allows for more protocols can be done and also experiments can be done before and after the SAD. This new method allows for both intact and barodenervation to be investigated in the same animal this allows for greater statistical power and will reduce the number animals to be used for experiments. There are some disadvantages to this technique as well. Local anesthetics can have toxic effects on the cardiovascular system. This method will allow for more experiments in animals, along with increasing statistical power but another important reason to try this method that it reduces the amount of animals necessary for experiments thereby making this method more ethical.

Systemic Cholecystokinin Differentially Affects Baro-activated GABAergic

Susan C. Mobley, Daniel A. Mandel, and Ann M. Schreihofer
Department of Physiology, Medical College of Georgia, Augusta, Georgia

J Neurophysiol

First published August 16, 2006; doi:10.1152/jn.00526.2006


  In this article, the actions of Cholecystokinin (CCK) on CVLM and SNA were investigated. CCK will cause a decrease in splanchnic nerve activity (sSNA). CCK is released after the ingestion of a meal. First they looked how CCK will affect sSNA and arterial pressure (AP). They recorded the neurons activity in the CVLM during all procedures. what they found was that in the majority of animals depressor responses within the first 15 seconds. They also found that the response was not a smooth but that it decrease some at first and then came back up and was followed by a greater depressor response in AP and also in sSNA (see fig 1A). In a few animals there was no AP depressor response however, there was a decrease in the sSNA. Then the neurons were labeled with biotinamide and the GABAergic phenotype was confirmed.Next they investigated how CCK would infleunce the activity of baro-inhibited neurons. By using aortic snare AP was raised and those neurons that had decrease in activity were considered baro-inhibited CVLM neurons. Then CCK was injected and the activity of these neuron was decreased and several neurons were silenced. In order to determine if CVLM was essential to the effects of CCK, CVLM was inhibited and CCK was injected. Before the muscimol (mus), a GABA A receptor agonist, they gave phenylbiguanide (PBG) to show the maximal activity of CVLM, next CCK was given they saw a decrease in sSNA, AP and HR. Then they gave mus bilaterally and then injected CCK what they saw was an increase in SNA and a decrease in HR. As for the PBG after mus it was reversed also instead they saw increase in all responses. Because inhibiting the CVLM causes an increase in SNA it could be possible that CCK could be potentiating the affects of Acetylocholine at the nicotinic receptor. So they gave hydralazine a vasodilator to cause an increase in sSNA and then gave CCK which inhibited the sSNA. Therefore the increase seen in the muscimol protocol was not due to the elevation of SNA.

 The interesting thing is that PBG maximally activates CVLM neurons but CCK only activates about 30% of the CVLM neuron population suggesting that certain neurons are resposible for the control of specific neurons in RVLM that control specific nerves. for instance injecting CCK causes increase in lumbar SNA. CCK also has cardiovascular responses it mediate, it can cause vasodilation at the blood vessels. It can also cause bradycardia by acting at CCK A receptors on the heart, this is most likely why after inhibiting the CVLM bilaterally, there was still a decrease in HR. CCK therefore does not depend on the CVLM pathway to control HR. PBG however does which is why after the muscimol there was increases in HR, AP and sSNA.

 

Lateralisation of projections from the rostral ventrolateral medulla to sympathetic preganglionic neurons in the rat

Elizabeth A. Moon, Ann K. Goodchild, Paul M. Pilowsky
Brain Res. 2002 Mar 8;929(2):181-90.

It is well known that presymapthetic neurons in the RVLM is important in tonic and reflex control of blood pressure in the arteries and sympathetic nerve activity. In this article the authors were interested in identifying how the sympathoexcitatory neurons form connections with sympathetic preganglionic neurons (SPN). They tested this by three different approaches. 1) Retrograde tracers were used to label the SPN that project to the adrenal gland or the superior cervical ganglion (SCG). Antrograde tracers were used from the pressor sites in the RVLM in order to identify whether lateral projections from the bulbospinal neurons project to the SPN that innervates the SCG and adrenal gland. 2) To identify the degree of lateralization retrograde tracers were injected unilaterally in to the spinal cord. 3) Finally they used a electrophysiological approach to stimulate RVLM with glutamate and recorded the activity from adrenal and cervical sympathetic nerves in order to examine the functional aspect of lateralization.
The significant findings of the present study are anterograde and retrograde experiments showed that the presympathetic neurons from the RVLM that form connections with SPN that innervate the SCG are bilateral. Interestingly projections to the adernal gland are ipsilateral. The second study showed that projections from RVLM to both the upper and lower level of the thoracic spinal cord (intermediolateral cell column) are predominantly ipsilateral. Inspite of this differential anatomical lateralization, the final study with glutamate microinjection showed no difference in nerve response even when different sides of the RVLM were injected.

- Madhan

Effect of renal sympathetic denervation on glucose metabolism in patients with resistant hypertension: a pilot study.

As most of you probably know, a study came out a couple of years showing that renal sympathetic nerve denervation can dramatically lower blood pressure in a hypertensive patient.  While the reno-centric scientists out there said "See! I TOLD you it was the kidney!", the evidence as a whole suggested that a more global phenomenon was occurring, since whole body norepinephrine spillover and sympathetic activity to the muscle were reduced in these patients.  In addition to affecting blood pressure, symptahetic overactivity can negatively affect glucose metabolism.  When beta adrenergic receptors in the liver are stimulated by cuirculating epinephrine, liver cells increase glucose production and subsequently plasma glucose and inculin levels increase. In turn, insulin stimulates sympathetic nerve activity, forming a cycle of activation. The authors guessed that glucose metabolism would benefit from renal denervation as well.
They performed the procedure in 37 patients with resistant hypertension and assigned 13 to the control group. A catheter was inserted into the renal artery and radiofrequency pulses were used ot heat the tip of the catheter and ablate the artery at various spots. This procedure kills any nerves running in and around the artery and causes a scar to form that prevents reinnervation. Blood pressure and markers of glucose metabolism were measured before and 1 and 3 months after the study.
The authors found that renal denervation had a marked effect on glucose metabolism. It is important to note that the patients' glucose and insulin levels were only slightly above normal before the study. fasting gluicose decreased, baseline insulin levels decreased, and blood pressure decreased as expected. Interestingly, some members in the trestment group were 'cured' of diabetes or pre-diabetes, depending on how you look at it. Now, does this prove that denervation blunts central sympathetic outflow to the adrenal gland and thus epinephrine secretion to cause these effects? No. Is it enough evidence to be suggestive? To me, yes. But then again, the appearance of the moon may suggest that it is made of cheese.  In any case, this study reminds one to remember the BIG PICTURE. Sympathetic overactivity means much more than just blood pressure.

Link to article

-Nick

RNA integrity and the effect on the real-time qRT-PCR performance Simone Fleige, Michael W. Pfaffl

Real time polymerase chain reaction is a very sensitive and powerful technique that is used to amplify a small amount of cDNA, reverse transcribed from mRNA, into large quantities to study low abundance gene expression. For successful and reliable gene expression data, it is important to start with intact RNA, since working with low quality RNA may strongly compromise the results. It is known that RNA is very sensitive to degradation due to many factors either from the extraction process or from inefficient lab management that may introduce exogenous contaminants to tissue samples. The RNA quality can be measured using different techniques, in this study Agilent Bioanalyzer was used, and RNA integrity number (RIN) that is higher than five was considered as good quality and higher than eight as perfect RNA quality.

In this review, the researchers compared the RNA quality of different bovine tissues and cell cultures to identify the influence of degraded RNA on the performance of qRT-PCR . In a study from Fleige and Pfaffl (2006) the purity and integrity of RNA samples derived from different bovine tissues and cell lines was measured using Bioanalyzer (2100). One distinct bovine tissue was degraded by enzymatic digest or with ultraviolet light to examine the effect of RNA integrity. The effect of RNA quality on RT-PCR performance was investigated by correlating RIN values with the cycle number(CP) of the PCR runs of four genes (18S,28S,B-Actin,IL-1B).  They found that a high quality RNA determined a lower CP than by less quality RNA, and there is significant correlation between RIN and CP that indicates with increasing RNA integrity the variability of RT-PCR results was decreased; however, after normalization of the CP by a reference gene (B-Actin) to decrease the RIN dependency, and by correlating RIN values with dCP values. They found that the normalized results showed minor influence of RNA quality on the expression results and the significant effect of RNA quality decreased to a minimum.

To get accurate RT-PCR results, it is important to start with high quality RNA; however, this review illustrates that moderate degraded samples may still give a reasonable data especially with the normalization; only the non-normalized values show a correlation between RNA quality and CP.

Posted by Wafa

New Approaches to Quantifying Sympathetic Nerve Activity

Burke SL, Lambert E, Head GA.
Curr Hypertens Rep. 2011 Jun;13(3):249-57.

http://www.springerlink.com/content/515x5218g67h2l58/


        This review focus on some of the advances in quantifying symapthetic nerve activity (SNA). Sympathetic nerve activity (SNA) is given as a value that relates to both the burst frequency and amplitude. More often than not it is rectified, integrated and averaged over time.
        Some of the indirect methods by which global SNA can be assessed are ganglionic blockade and spectral analysis. In order to measure regional SNA, norepinephrine isotope dilution method is widely used.The main advantage of this method is that it can be applied to various vascular beds to measure the regional SNA. Microneurography is being used for direct recording of multifiber SNA in humans.
        An important discussion in this review is whether single unit recording is a much better approach to quantify SNA. In this method the firing properties of individual muscle vasoconstrictor neurons are assessed. The properties of the firing sympathetic neurons are analyzed based on mean firing frequency, firing probability and the number of spikes a unit generates per cardiac interval. The reviewers believe that this method will be highly useful to determine the underlying pathology since many healthy individuals have high multiunit SNA but with low firing probabilities, rates and low incidence of multiple firing. 
        A number of methods have been used to compare integrated multifiber SNA between different groups, one such method is to measure all SNA as a percentage of baseline SNA. Another way is to show SNA as a percentage of maximum response to unloading the baroreceptors. In addition to the above methods, a combination of methods are also used, after measuring SNA as a percentage of baseline, it was compared in raw microvolts. In another study SNA was expressed as percentage changes from baseline and the frequency of bursts in baseline SNA was compared. 
        This review also discusses about the usage of telemetry for chronic recording of SNA. One of the pressing problems that remains to be solved in this method is the accounting for signal decay over time and comparing nerve activity between different animals, this makes calibration of nerve activity difficult.

Posted by Madhan

"Rewiring" the nervous system after a spinal cord injury: How WSU-SOM secretly produces really good scientists

I came across this article last week while browsing on Pubmed. Two things jumped out at me. One, it's a Nature  paper and, two, the first author used to be Harry Goshgarian's PhD. student. I gave it a look and here's what I learned.

Spinal cord injuries are terrible. In particular though, injuries which occur above the motor neurons innervating the diaphragm (phrenic motor neurons, PMNs in C3-C6) are imminently life-threatening and the prognosis is very poor. The first obvious problem is that PMNs no longer recieve input from higher centers. In addition, inflammation due to degeneration of afferent fibers leads to upregulation of extracellular matrix molecules which potentially inhibit re-innervation. The authors hypothesized that enzymatic digestion of these molecules would improve recovery from injury. They injected the enzyme chondroitinase ABC into the phrenic motor nucleus at the same time as recieving a hemisection at C2. With the enzyme alone, the treated animals showed improved recovery of breathing, but the authors took it one step further. In another group of animals, they grafted a section of the tibial nerve from the site of the lesion (C2) to C4. Remarkably, animals in this group showed near-normal breathing activity 12 weeks after the lesion. Immunohistochemistry showed that fiber regeneration was robust and extensive at the lesion site and at the distal graft site. If this procedure works with fiber tracts to other skeletal muscles, then WOW.

Posted by Nick

RVLM GLYCINE RECEPTORS MEDIATE GABAA AND GABABINDEPENDENT SYMPATHOINHIBITION FROM CVLM IN RATS. Cheryl M. Heesch,1 Jennifer D. Laiprasert,2 and Lyudmyla Kvochina1 Brain Res. 2006 December 13; 1125(1): 46–59

As we know, the neural regulation of blood pressure is carried through baroreflex which is always on and detects any blood pressure changes through the baroreceptors, and the rostral ventrolateral medulla (RVLM) is the most important brain region in the regulation of cardiovascular function through the baroreflex. In addition, the caudal ventrolateral medulla (CVLM) has been shown to have an important role in the modulation of the presympathetic RVLM neurons. Many studies have been done to prove that there is a tonically active GABAergic pathway from CVLM to RVLM, and the aim of this study is to evaluate the role of the RVLM GABAergic receptor subtypes and glycine receptors in mediating CVLM sympathoinhibition.

In this study, experiments were performed in 26 virgin female Sprague Dawley rats. This group of rats was divided into three groups each for different protocol. In protocol 1 and 2, the right nucleus tractus solitarius (NTS) was electrically lesioned to eliminate the effects of afferent baroreceptors input from the right side. The left CVLM and RVLM were functionally mapped based on the response to microinjection of GABA. In protocol 1, the changes in mean arterial blood pressure(MAP), heart rate(HR), and renal sympathetic nerve activity(RSNA) after microinjection of GABA in the left CVLM were measured before and after blockade of GABA A receptors in the left RVLM with bicuculline. The microinjection of GABA in the left CVLM before GABA receptors blockade resulted in significant increase in MAP and RSNA. After 10 minutes, GABA A receptor antagonist was microinjected in the left RVLM which produced an expected increase in both MAP and RSNA .Within 2 minutes, microinjection of GABA in the CVLM produced further increase in MAP and RSNA which indicated that there is a source of inhibition from the CVLM is not mediated by GABA A receptors in the RVLM. Based on that, the investigators of this study hypothesized that RVLM GABA B receptors might contribute to both tonic baroreflex independent sympahtoinhibition and the remaining CVLM sympathoinhibitory influence after RVLM GABA A receptors blockade. In protocol 2, they tested this hypothesis by measuring MAP, HR and RSNA responses to microinjection of GABA in the left CVLM before and after blockade of GABA A and GABA B receptors in the left RVLM. Following the combined GABA antagonists microinjection, the pressor response to inhibition of the CVLM was decreased while MAP and RSNA responses persisted, suggesting that GABA B receptors do not make a major contribution to GABAergic inhibition from CVLM to RVLM.

Although in protocol 1 and 2  NTS lesion on the right side eliminated the effect of right baroreflex, they considered that the combination of minor effects of baroreceptors from the left NTS to CVLM pathway and baroreflex independent inhibition from the left CVLM to the right RVLM could be accounted for residual responses  to inhibition of left  CVLM.  Therefore, in protocol 3, bilateral blockade of GABA A and GABA B receptors in the RVLM was performed and changes in MAP, HR and RSNA after microinjection of GABA in the CVLM were measured before and after bilateral microinjection of the combined GABA antagonists.  The inhibition of CVLM after bilateral blockade of RVLM GABA receptors resulted in increased pressor response. In addition, glycine receptors blockade was evaluated in the presence of bilateral GABA A and GABA B receptors blockade to determine if RVLM glycine receptors contribute to the remaining inhibitory influence of the CVLM. They found that inhibition of glycine receptors resulted in elimination of the increase of MAP and RSNA that had been observed after the inhibition of CVLM in the presence of RVLM GABA receptors blockade.

To sum up, following ipsilateral blockade of RVLM GABA A(protocol 1) , ipsilateral GABA A+GABA B receptors (protocol 2) in rats with contralateral NTS lesion and following bilateral blockade of the RVLM GABA A+GABA B receptors the pressor and sympathoexcitatory responses persisted. These results indicate that there is no significant contribution of GABA B receptors to tonic baroreflex independent GABAergic inhibition from the CVLM to the RVLM and no evidence for influences form contralateral CVLM. Moreover, glycine receptors mediate GABA A and GABA B independent inhibition from the CVLM to the RVLM.    

A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.

Card JP, Kobiler O, Ludmir EB, Desai V, Sved AF, Enquist LW.  A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.  PLoS One  2011;6(6):e21141. Epub 2011 Jun 16.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116869/pdf/pone.0021141.pdf 

This is a second article from the group at Pittsburgh that is using viral tract tracing in order to understand more about the pathways of sympathetic innervation of various organs involved in blood pressure regulation.  In this latest work, they continue the use of viral tracers that can cross synapses and retrogradely label sympathetic post-ganglionic; pre-ganglionic; and pre-motor neurons including those in the RVLM.  What's unique about this study is they inject two different viruses, one into the left and one into the right kidney. Although both viruses express the mtomato label which expresses a red color, each virus has its own characteristic labeling pattern.  One fills the cytoplasm and dendrites whereas the other provides only punctate (i.e. dotted) staining of the neurons.  Also by use of the Brainbow cassette and Cre recombinase, when a cell is infected by both viruses (i.e. a cell presumably innervates both kidneys) the ability of the cell to express the red label is excised and the cell now expresses blue or yellow.  Furthermore once the first cell in this pathway is dually-infected, not only does it turn yellow or blue, one of the virus loses it's ability to replicate in any subsequent cell that is transynaptically labelled.  That means that a cell with punctate staining in yellow or blue is a neuron that was infected transynaptically by the cell that had the original dual labelling by both viruses.  In this way the authors can examine what are called 1st and 2nd order dually infected neurons in succession. 
Posted by Pat

Identification of an efferent projection from the paraventricular nucleus of the hypothalamus terminating close to spinally projecting rostral ventrolateral medullary neurons.

Neuroscience. 1999;88(3):949-57
Pyner S, Coote JH.

http://www.sciencedirect.com/science/article/pii/S0306452298002553
As we know both RVLM and PVN are important brain areas which are known to be involved in the regulation of cardiovascular activity. The purpose of this study was to determine whether paraventricular axons project to the rostral ventrolateral medulla and whether they are closely apposed to reticulospinal neurons in this region. In this study they have used anterograde tracing to see whether neurons in the PVN send efferent fibers that terminate on or near the retrogradely labelled RVLM spinally projecting neurons.Biotin dextran amine (BDA, Molecular Probes 10,000 mol.wt 10% in 10 mM phosphate buffer pH 7.25) was iontophoretically deposited in and around the PVN over a period of 20 min. The injection sites in the PVN covered a wide range of areas around the PVN. BDA labelled axons were found in the RVLM as a result of each of these injections.After 10–14 days animals were re-anaesthetized and 1 μl of 4% wheatgerm agglutinin–horseradish peroxidase (WGA–HRP) pressure injected into spinal segment T2 after stabilizing the vertebral column with a clamp on T2 spine. BDA filled axons located in the RVLM and originating from the PVN injections sites were thin in appearance and purple in colour compared to the black/brown WGA–HRP labelled RVLM neurons.The present study using anterograde and retrograde tracing methods has identified axons originating from the PVN that intermingle with RVLM reticulospinal neurons. Many of these PVN axons have terminal varicosities that are either apposed to or closely associated with RVLM reticulospinal neuronal cell bodies and dendrites. This article concludes that axons originating in the PVN terminate close to spinally projecting RVLM neurons which are assumed to terminate on sympathetic preganglionic neurons(SPN). So the PVN could involve in the cardiovascular activity through its descending projection to the SPN.



Madhan

Quantifying sympathetic nerve activity: problems, pitfalls and the need for standardization

Telemetry Research Ltd, Auckland, New Zealand

Guild SJ, Barrett CJ, McBryde FD, Van Vliet BN, Head GA, Burke SL, Malpas SC.

Exp Physiol. 2010 Jan;95(1):41-50. Epub 2009 Aug 21.

http://ep.physoc.org/content/95/1/41.long

In this article Guild et al, are proposing that there needs to be a more consistent way to interpret SNA so that the results can be compared across different groups. In the article they discuss how to define a good recording versus a bad one. They discuss alternative ways of figuring out noise in the recording. They propose using the quiet period in between bursts as the zero level for SNA. They also discuss the use of long term SNA recording. The problem with the long term SNA recordings is that there could be growth of tissue around the electrode that affects the signal that is pick from the nerve and another issue is how  one figures out the noise level in these long term recordings. The noise level could change every day making it difficult to figure out the amount noise that is present throughout the long term recording. Also they discuss reporting not only the % change in comparison to baseline but also the frequency and the amplitude along with the absolute SNA. 

Unique levels of expression of N-methyl-D-aspartate receptor subunits and neuronal nitric oxide synthase in the rostral ventrolateral medulla of the spontaneously hypertensive rat

Mark A. Edwards, Rhonda A. Loxley, Kellysan Powers-Martin, Janusz Lipski, Douglas J. McKitrick, Leonard F. Arnolda, Jacqueline K. Phillips
Molecular Brain Research 129 (2004) 33-43

NMDA receptors are made up of one NR1 subunit and at least one NR2A-D subunit.  NR1 (of which there are several splice variants) is required for a functioning NMDA receptor, but none of the NR2 subunits are.  Different splice variants and NR2 subunits can be combined to produce NMDA receptors with different pharmacological properties.  SHR rats are known to be more sensitive to glutamate in the RVLM, and the authors of this paper are interested in seeing if the SHRs have different numbers or types of NDMA receptors than the WKYs do.  Also of interest is the action of NO, which is involved in regulating sympathetic output from the RVLM.  NO is produced by three different enzymes: neuronal NO synthase (nNOS), inducible NO synthase (iNOS), and endothelial NO synthase (eNOS), which have different stimuli (and thus different amounts of activity) and can produce different effects on sympathetic activity.  In addition, NMDA receptors can trigger the production of NO by nNOS when activated, and NO can cause modification of the NMDA receptor.  Conventional and RT-PCR were used to study the potential differences in gene expression related to these processes between the SHRs and WKYs.

Tissue punches were performed in the RVLM and RNA was isolated.  Reverse transcription was performed on the samples to obtain cDNA.  Conventional PCR was used to detect NR1 splice variants and these products were sequenced to confirm the presence of specific splice variants.  RT-PCR was done with Taqman probes to detect expression of NR1, NR2A-D, nNOS, and iNOS, which were normalized to neuronal specific enolase (NSE), the reference gene they chose.  Within each sample, NR2 subunits were compared to NR2A to determine relative abundance, because NR2A did not vary between SHRs and WKYs.  Finally, immunohistochemistry was used to detect nNOS in the RVLM.

There was no difference in the presence of NR1 splice variants between the two strains (but these were not quantified).  NR1 was found in highest concentration in both rat strains, and along with NR2A and NR2B, did not change significantly in concentration between the them.  NR2C had the highest expression among the NR2 subunits, and along with NR2D, was found in lower concentration in the SHRs than in the WKYs (NR2C was expressed only 0.37 times as much, and NR2D was expressed 0.26 times as much).  nNOS levels were higher in the WKYs than in the SHRs.  Because the composition of subunits affects the receptor's activity, it can be hypothesized that the difference in composition of NDMA receptors in the RVLMs of the two strains may contribute to the difference in sympathetic nerve activity that is seen (which would be a topic for another paper; this one did not address cause and effect).