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).

Analyzing real-time PCR data by the comparative Ct method

Thomas D Schmittgen and Kenneth J Livak
Nature Protocols Vol. 3 No. 6 1101, 2008

Note: the procedures involved in preparing for and running the PCR are covered in the paper but will not be detailed in this post.

Real-time RT PCR is a method of analyzing gene expression.  The RT stands for reverse transcription; RNA is converted to DNA and then amplified by PCR.  Specific fluorescent probes allow for detection of the amount of the gene of interest present after each cycle.  The number of the cycle during which it reaches the threshold (the red line in the graph below; set at a value below which the noise is too great to give an accurate impression of the actual fluorescence due to DNA quantity), or Ct, depends on the amount of RNA for the target gene that was in the original sample.  The cycler will give out the exact Ct for each sample, along with a graph showing the fluorescence of the sample vs. the cycle number, to show how the fluorescence changes over time.  An example graph is shown below, with one sample being probed for two different genes.  Each gene is assayed in triplicate, so the graph shows six different curves (in two close groups of three).
 
A lower Ct means more RNA, which means more gene expression.  By comparing the Ct of the target gene (VGLUT2, in the above example) to the Ct of a reference gene (a gene that is always expressed in the same amount; HPRT, in the above example), we can come up with a ratio of expression of the target gene to expression of the reference green.  If this is done in many different samples, than we can compare those ratios to determine if the samples express the target gene in the same amounts.  This depends largely on two things: picking a good reference gene that is known to be equal in all samples and using a reliable mathematical approach to compare the samples.

Schmittgen and Livak spend a good deal of time on this second concern in their paper.  The most effective approach for comparing the treatment and control groups depends on what one is attempting to study, but the initial steps are the same.  First, average the triplicates; each sample is probed for each gene in triplicate to reduce the effect of pipetting error on the results.  If the triplicates are too different (if the standard deviation between the Ct values is more than 0.3), one or all three must be thrown out.  If they are within the acceptable range, then the average Ct will be used from here on out.

Secondly, calculate the deltaCt.  This is done by subtracting the Ct of the reference gene from the housekeeping gene (see equation below).  Do this for every sample.  After this, the next step depends on whether the goal is to compare individual samples to each other or to compare groups of samples to each other.  Now it becomes important to know the origin or importance of the samples, so I will refer to them as belonging to the treatment group or to the control group.

deltaCt = Ct(target gene) - Ct(reference gene)

If the goal is to compare each individual sample from the treatment group to a matched control, subtract the deltaCt of the control sample from the deltaCt of the treatment sample to get a deltadeltaCt (see first equation below).  Next raise 2 to the power of -deltadeltaCt to obtain a fold change (see second equation below).  For example, if deltadeltaCt is 2, the fold-change in gene expression will be 2^(-2), or 0.25.  This means that the target gene is expressed one-fourth as much in the treatment sample than in the control sample.  When this is calculated for each pair of treatment and control samples, the fold-change values can be averaged to obtain an average fold-change.  Note that when the fold change is less than one, it can be presented as 1/fold change; for example, a 0.25-fold change could be stated as a 4-fold reduction.

deltadeltaCt = deltaCt(treatment group) - deltaCt(control group)

Fold change = 2^(-deltadeltaCt)

If the goal is to compare a treatment group to a control group, average the deltaCt values within the groups and calculate the SDs.  After this, there will be one treatment deltaCt and SD, and one control deltaCt and SD.  The control group deltaCt can be subtracted from the treatment group deltaCt to obtain a deltadeltaCt.  This is used as above to obtain a fold-change.

The reason that the second situation requires averaging the Ct values and the first situation does not is that the samples in the second situation are not paired with controls.  If you have a clinical study in which you have matched your treatment subjects with controls based on age, weight, or other criteria, this is the first situation.  This is quite useful with small studies that have differences between their treatment individuals that could make lumping them together suppress the effects of the treatment (which would happen when averaging blood pressures, for example, when some patients are children and some are adults; matching each child to a control child and each adult to a control adult will control for difference within the group so that differences between the groups will be apparent).  However, many studies (especially larger ones) randomize their subjects and end up with two groups but no specific pairs.  In these cases, you could pair the subjects after the fact, but it would not be as necessary or as beneficial as it is in the first situation.  Therefore, averaging the deltaCts before calculating the deltadeltaCt is the better option.

Alternately, 2^-deltaCt can be calculated for each sample.  Those can be averaged for each group, and the fold-change would then be calculated by dividing the mean from the treatment group by the mean from the untreated group (see equation below).

Fold change = [2^(-deltaCt(treatment))]/[2^(-deltaCt(control))]

Now, all of this is only valid if the target and reference genes have similar amplification efficiencies, which are influenced by the primers and the PCR conditions.  Amplification efficiency is determined by running a PCR plate with different concentrations of the same sample and then plotting the Ct vs. log(concentration).  The data points should form a line, with the efficiency equal to 10^(-1/slope).  If the efficiencies are not within 10% of each other (and 10% of 2; an amplification efficiency of 2 shows that the DNA doubles each cycle), then the PCR conditions should be altered or new primers should be ordered.

CARDIOVASCULAR EFFECTS PRODUCED BY ACTIVATION OF GABA

In this article by Menezes and Fontes, is looking at the role of GABA receptors A and B, in the RVLM in conscious rats.  They basically found that GABA A receptors are responsible for the depressor response that is observed and that this response is greater in conscious rats.  They used muscimol which is a GABA agonist and they microinjected bilaterally into the RVLM to activate the GABA A receptors. They also looked at GABA B receptors by using Balcofen a GABA B receptor agonist that they injected bilaterally into the RVLM. They found that this caused a pressor response however this difference was similar to the vehicle. It would have been interesting to see SNA in these rats however they did not do any nerve recordings. The article was interesting to me because they talked how anesthesia can potentiate GABA causing a greater depressor response. The problem I have with this article is that it is done in conscious animals; the animals could have been stressed prior to the injections because of the surgeries and the fact that they had cannula in their head. I would argue that MAPs could have been higher so there was a greater decrease observed in the conscious animals versus the anesthetized animals however Table 1 shows that they are similar. Also they only did the muscimol 200pmol in the anesthetized group. The final thing that I can think of is that they did not do each drug in the same animal instead they did a single drug in each animal.

Neurons of the Rostral Ventrolateral Medulla Contribute to Obesity-Induced Hypertension in Rats

Hypertension. 2007 Mar;49(3):640-6. Epub 2006 Dec 26
Sean D. Stocker; Rachel Meador; Julye M. Adams 

There are several possible mechanisms that contribute to the pathogenesis of obestity induced hypertension. However one of the well studied mechanism is how sympathetic nervous system plays a role in obesity-induced hypertension. In the present study the authors are interested in evaluating the role of sympathetic neurons in the rostral ventro lateral medulla contribution to obesity induced-hypertension. Rats were fed with high fat diet and were segregated into obesity prone and obesity resistant rats based on their weight gain. Mean arterial pressure was significantly elevated in the obesity prone rats than obesity resistant rats. GABA A receptor agonist muscimol decreased mean arterial blood pressure when injected bilaterally into the RVLM. The decrease in MAP was more pronounced in the obesity prone group of rats than other groups. vascular reactivity remained unaltered across the groups in response to phenylephrine and norepinephrine. The authors conclude that the tonic activity of rostral ventrolateral medulla sympathetic neurons contribute to the pathogenesis of obesity- induced hypertension.

Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus

J. Patrick Card, Oren Kobiler, Joshua McCambridge, Sommer Ebdlahad, Zhiying Shan, Mohan K. Raizada, Alan F. Sved, and Lynn W. Enquist. Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus. Proc Natl Acad Sci U S A. 108 (8): 3377-82, 2011.

My first blog, yeah!! 

     This paper that is based on a collaboration between Alan Sved, Mohan Raizada, and Lynn Enquist.  We've had previous blogs on papers by Alan Sved, a leader in our field for about 20 years.  This paper in particular highlights Alan Sved's interest in tracing the neural networks involved in blood pressure control.  In this paper they use a virus to trace in a retrograde fashion the sympathetic pathways the control the kidney.  This is accomplished by injecting the virus into the kidney, which then travels back up the sympathetic postganglionic nerves (hence retrograde tracing) to the sympathetic ganglion.  Here in the ganglion the virus crosses the synapse (a.k.a. "goes transynaptic") to infect and replicate in the terminals of the preganglionic nerves that innvervate the post-ganglionic nerves that innervate the kidney.  The virus then continues to travel up the preganglionics into the spinal and where it goes transynaptic in the intermediolateral cell column (IML) to infect the sympathetic premotor neurons that are coming from the RVLM (and other brain regions).  The interesting part about using viruses is that the longer you wait following the injection (i.e. the longer the animal's recovery time) the further back the virus traces.  There is a limit, however, as the virus will eventually make the animals sick--it's a virus right? But by comparing shorter recovery times (i.e. shorter transport times) to longer recovery times (longer transport times), it's possible to see infection 1st in the post-ganglionics, then the pre-ganglionics, and then into the CNS.  Viruses have actually been used for a number of years to trace the sympathetic pathways from various organs (heart, kidney, adrenal, skeletal muscle, etc.; see Loewy AD and colleagues).  Another limitation of viruses, however, is that the interconnnectivity and complexity of most pathways can make it difficult to truly define specific connections of a subpopulation of neurons.  Until now.....

     The truly innovative aspects of this work is the use of a peudorabies virus (PRV) that contains a specific sequence (or cassette) that causes the cells to express a red fluorescent tag.  When the virus encounters a cell that also contains Cre recombinase (Cre) the red reporter gene gets taken out and the cell will now will express a yellow or light blue tag.  The sequence (or cassette) that causes the cells to express different colors is appropriately named the Brainbow cassette (yes like rainbow, see pic below).  What these authors were able to do was get the Cre recombinase to be expressed specifically in catecholaminergic neurons (yes, our favorite C1 cells).  So by injecting the PRV into the kidney, and waiting long enough, they could label neurons (in red) that went back up to the RVLM.  When the PRV infected a C1 cell in the RVLM the color changed to yellow or light blue.  This unique approach allowed them to label specific neurons in the RVLM that projected to the kidney (red) and a subpopulation of those cells that were the C1 cells (yellow or blue).
     This paper contains some really cool diagrams and pictures of the labeling so please check it out if interested in seeing it for yourself.  I would have posted them but I believe it would violate copyright laws despite the fact that the article is available for free online.  Finally, related to the lab, we have some high hopes for a compound known as wheat germ agglutin (WGA), which our colleague Dr. Goshgarian in Anatomy has used for transynaptic labeling instead of a virus.  We recently discoved that a company makes the WGA conjugated directly to an Alexa 488 fluorophore so there is no need to perform immunohistochemistry to see the WGA and make the process much simpler.  It's like combining the Fluorogold with a virus, hopefully the best of  both worlds.  Cool huh?

Bioactive Compounds in Berries Can Reduce High Blood Pressure

Blog Post July 15, 2011

Bioactive Compounds in Berries Can Reduce High Blood Pressure

ScienceDaily (Jan. 15, 2011) — Eating blueberries can guard against high blood pressure, according to new research by the University of East Anglia (UEA) and Harvard University.

http://www.sciencedaily.com/releases/2011/01/110114155241.htm

Science Daily is a good website for stimulating potential “out of the box” topics which high school students can use in their research process. Articles can also be used as an ice-breaker for lecture and lab. 

According to Science Daily, this study is also published in the American Journal of Clinical Nutrition (could not locate). The article summarizes research completed at the University of East Anglia (UEA) and Harvard University.  The posting got me thinking about connecting the article’s findings with microinjections.

According to the article, 181,000 people were placed in cohorts for a 14 year study. At the start of the study, none of the participants had hypertension. The study found that the participants who consumed flavonoids  were less likely (8%) to be diagnosed with hypertension. Those who ate blueberries once a week were 10% less likely to become hypertensive. So what are flavonoids and how are they related to what we do in our lab?

After a quick search in PubMed (and asking Jessica and Nick), I learned that  GABA lowers blood pressure when injected into the RVLM of rats. I probably heard this response to GABA during the Thursday lab meetings, but it finally clicked for me today--light bulb moment! A PubMed search for  “GABA + Flavonoid”, provided me with information that there is a flavone-binding site in the GABA(A)-receptor. This receptor is a ligand-gated ion channel and it responds to the neurotransmitter gamma-aminobutyric acid. Since microinjected GABA lowers blood pressure when injected into the RVLM and if consumption of blue berries lowers the risk of developing hypertension by 10%. Could we connect the two into an experiment in our lab? 

The receptor for GABA is a flavone-binding site, so what would happen if we injected a flavonoid into the RVLM? Would it decrease the blood pressure? And, what is the specific molecular conversation here? Would the flavonoid enhance the function of the receptor, serving like a cofactor or coenzyme?  Is there a conformational change in the receptor in the presence of flavonoid? 

Chronic estradiol-17β exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: reversal by resveratrol

This very well-written study investigates the effect of chronic exposure to estradiol on blood pressure regulation in female rats. It is known that hormone replacement therapy (HRT) in post-menopausal women causes a slight, but significant, increase in blood pressure and cardiovascular disease risk. In addition, a large number of young women in industrialized countries take forms of estrogen orally for contraceptive purposes. Since the rostral ventrolateral medulla plays such an important role in blood presure control, they focused on changes occurring in the RVLM associated with changed in blood pressure. Specifically, they were interested in superoxide production since many studies have shown increased oxidative stress in the RVLM of animal models of cardiovascular disease.
Female rats were implanted with slow release estradiol pellets, or not, for three months. During the third month they were implanted with radiotelemetry transmitters and their BP was monitored for about two weeks. At the end of the experimental period, animals were sacrificed. Brains and trunk blood were removed and frozen for later study. In a second experiement, estradiol implanted and sham animals were divided into two groups, one recieving the antioxidant resveratrol in their food and the other not. Animals were sacrificed at the end of the 2nd experiment as in the 1st.
The author's original hypothesis was right. They showed that animals recieving chronic estradiol developed higher blood pressures than those who did not. Also, treated animals were shown to have increased superoxide levels in the RVLM. Most interesting was that treatment with resveratrol reversed these deleterious effects. These data suggest that increases in blood pressure seen in women recieving HRT and young women on contraceptive therapy may be due to oxidative stress in the RVLM. This effect can be mitigated by simply adding resveratrol in the diet. "A glass of red wine for me keeps my RVLM happy"
Althought these data are interesting and certainly compelling, the study could have been greatly strengthened by adding an additional group of ovarectomized animals. This would make the study much more applicable to post-menopausal women recieving HRT. For shame authors, for shame. :-)

Dietary salt enhances angiotensin-II-induced superoxide formation in the rostral ventrolateral medulla

Autonomic neuroscience, basic and clinical 2010 Jun 24;155(1-2):14-8. Epub 2010 Jan 6.
Valdir A. Braga

In this article the authors investigated the association of dietary salt and angiotensin-II infusion on hypertension and superoxide formation in the RVLM. Ang-II or saline was subcutaneously infused to male Wistar rats for 14 days. Two different doses of salt was given in the drinking water. As expected on the 15th day rats that received Ang-II and low salt in water exhibited higher levels of baseline arterial blood pressure than rats that received saline and low salt. Rats that received Ang-II and high salt had a significantly greater hypertension compared to Ang-II and low salt. On the other hand, rats treated with saline and high salt or saline and low salt did not become hypertensive. In addition the above treatment rats that are administrated with hexamethonium, a ganglionic blocker evoked larger decreases in mean arterial pressure in rats treated with Ang-II and high salt and rats treated with Ang-II and low salt. Dihydroethidium technique was used to measure superoxide formation in the RVLM which was greater in rats treated with Ang-II and high salt, than withAng-II and low salt and saline and high salt. These findings show that dietary salt provokes Ang-II-derived superoxide formation in the RVLM, resulting in a more severe hypertension. The authors believe that this effect could be mediated by an increase in inputs within the forebrain–PVN–RVLM axis.

Influence of GABA in the nucleus of the solitary tract on blood pressure in baroreceptor-denervated rats

In this article by Ito and Sved, they investigated the affects of 10 pmol Bicuculline (bic) , a GABA antagonist and glutamate (glu)  in the NTS of baroreceptor intact, chronic SAD and acute SAD animals. What they found is that the with the microinjections of bic the chronic animals had a greater decrease in MAP and HR compared to the control animals. They also compared the effect of bic of not only the control and chronic SAD, they also showed the change in MAP for partial, near complete and acute SAD animals. the chronic SAD had the greatest change in MAP followed by near complete SAD, partial SAD, acute SAD and then the control. Then they did a dose response with glu using 10 pmol, 50 pmol, and 200 pmol concentrations. The different groups responded similarly as they did to bic for each dose with the exception of 200 pmol. At 200 pmol the chronic SAD still has the greatest change in MAP but instead of the near complete SAD coming in second the acute SAD is next followed by the near complete SAD and the previous pattern continues. So these findings show that in the intact animals GABA is not playing an important role in tonic control of blood pressure. In the chronic SADs the Gaba A rectors is up regulated and plays a role in the control of bp.

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

Kleiber AC, Zheng H, Sharma NM, Patel KP.

Previously it is known that glutamatergic mechanisms are normalized in the PVN on exercise treatment. Also plasma Ang II levels are normalized in the rats with heart failure. In this study the authors tested whether Ang II receptor AT1 was involved in this normalization effect. In order to test this the authors used AT1 receptor blocker losartan in drinking water for 3 wk. Left ventricular end-diastolic pressure was measured. It was increased in both the heart failure groups compared to the sham animals. Losartan had a similar effect like that of exercise training. When N-methyl-d-aspartate (NMDA) is injected in the PVN, it increased the RSNA in HF animals treated with losartan compared to other groups. NMDA receptor subunit NR(1) mRNA and protein expression levels were increased in the PVN in HF animals with losartan treatment. Invitro studies also confirmed the above finding. These data suggests that AT1 receptor are involved in the normalization of glutamatergic mechanisms in the PVN in heart failure rats.

Blog Summary for Tues July 12th Journal Club Presentation

Blog Summary for Tues July 12^th Journal Club Presentation

Strong Inference

John R. Platt

Science, New Series, Vol. 146, No. 3642. (Oct. 16, 1964), pp. 347-353.

http://ecoplexity.org/files/Platt.pdf

Strong inference (SI) identifies, explains and supports a systematic use of inductive thinking. The paper describes SI as a four step method that if adopted will enhance scientific progress in both speed and accuracy of findings. SI steps include 1) devising alternative hypotheses   2) devising crucial experiment(s) to test among the alternatives for the purpose of eliminating the alternatives which are inconsistent with the data 3) running a “clean” experiment that supports the conclusions 4) Recycling the procedure by repeating steps/observations with the intended purpose of revising, rejecting or accepting the accepted hypothesis.  The implementation of multiple hypotheses, as noted in the paper, is intended to protect against bias to try to confirm our first “pet” idea. And, it forces the researcher to think outside of the box and about “all” possibilities.

In other words, imagine a world where we can look up the answers to everything! Hum! Yes, like having access to Google or Wiki or Dr. Mueller’s blog! So pretend that in this everything-at-your-finger-tips world, a young lad has car trouble. He looks up the trouble he is having by typing it in the famous search engine (no pun intended). He then determines what is wrong with his car and happily provides his personal diagnosis to the mechanic who has been hired to repair the car. In the world of Google, it is possible that the car mechanic would do whatever the customer desired. However, in a world of SI, the mechanic would follow a method for determining the car trouble. She would

1)      listen to the car owner, use her background knowledge in automechanics to determine possible problems

2)      run a series of tests based on the car symptoms, the owner’s complaint, and his automehcnaic knowledge

3)      eliminate from the list of possible problems based on the tests

4)      refine the tests as needed until a firm diagnosis was reached

5)      repair the car ($$$)

The strong-inference protocol: not just for grant proposals

Sara M. Hiebert

ADV PHYSIOL EDUC 31:93-96, 2007

Department of Biology, Swarthmore College, Swarthmore, Pennsylvania http://advan.physiology.org/content/31/1/93.full

In this paper, we focus only on the Appendix—Specific content for the chicken embryo metabolism experiment. The author of this paper contends that students in the Animal Physiology course are able “to apply the general form of the strong-inference (SI) protocol to specific experiments, with the proviso that they benefit…in the early stages from seeing a specific example on which to model their own protocols” (p94).  The students are armed with an invaluable tool—their personally designed SI protocols— to respond to the overarching question and support thinking throughout the experiment. Prior to the student- designed experiment, students are required to complete the introduction and background, methods, and outcomes (alternative hypotheses) sections of their lab reports. In this way, students know ahead of time and throughout the lab why and how the experiment is to be done. And the outcomes section provides a list of differential diagnoses for the upcoming experimental results. The paper asserts that the SI approach to conducting experiments

1)      supports the laboratory’s direction/purpose by keeping students focused on their protocol

2)      benefits student thinking by developing big idea understanding

3)      frontloads the actual writing of the lab report thereby taking the pressure off of completing an arduous task at the close of the experiment

4)      maintains the focus on the larger questions of why something is occurring and what are the implications of our experiment, as opposed to being lost in the method of step by step cookbook instructions

Tyrosine hydroxylase immunoreactivity as indicator of sympathetic activity: simultaneous evaluation in different tissues of hypertensive rats

Burgi K, Cavalleri MT, Alves AS, Britto LR, Antunes VR, Michelini LC

Dept. of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 São Paulo, SP, Brazil.

Don’t judge too harshly—this is my first time!

This journal article was brought to my attention by Nick -- since it included my favorite enzyme Tyrosine Hydroxylase! In this article the researchers are trying to quantify the amount of sympathetic activity in different tissues simultaneously. In this study they examine the kidney, heart and skeletal muscle. Jut to reiterate, tyrosine hydroxylase, is one of the enzymes in the chain that converts the amino acid tyrosine to norepinephrine— which is one of the main neurotransmitters that causes vasoconstriction as part of the sympathetic response. In this study both WKY and SHR rats were used.

Burgi et al., propose that simultaneous measurement of THir (tyrosine hydroxylase immunoreactivity—which is what Nick does in our lab) in different vascular beds can be used to determine SNA activity in SHR rats as an indication of hypertension. They then compared their findings to normotensive rats for control.

Interestingly out of the three vascular tissue beds examined (kidney, heart and skeletal muscle), they found higher THir in the kidney and heart tissues of the SHR, however in the skeletal muscle THir was not higher between the SHR and WKY groups. There are many reasons outlined in the discussion as to why this might be the case. The authors also showed in their results that differential sympathetic activity was shown by taking nerve recordings from the renal (RSNA) and lumbar (LSNA). They showed that there was increased RSNA in the SHR and unchanged LSNA activity which coincided with the results of increased THir in the kidney and heart, and lower THir in the skeletal muscles as compared to WKY.

The main point of this paper set out to prove that THir can be used as an indicator for sympathetic activity in different tissues simultaneously.

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

Domitila A. Huber and Ann M. Schreihofer
American Journal of Physiology - Heart and Circulatory Physiology 301: H230-H240, 2011

Fat rats!

Obese Zucker rats (OZR) are a genetic model of obesity and differ from lean Zucker rats (LZR) in that they lack leptin receptors.  Because leptin is a key signaler of satiety, these rats eat much more and become obese.  They also have increased sympathetic nerve activity (SNA) and increased mean arterial pressure (MAP).  It has been seen that, in human subjects, eliminating SNA causes greater decreases in MAP in obese subjects than in lean subjects, suggesting that the increased SNA contributes to the increased MAP.  In rats, this increased sympathetic tone has been traced back to the RVLM.  In some models, this is at least partially explained by increases in tonic glutamate and angiotensin II activation and decreased tonic GABA inhibition of the RVLM.

Huber and Schreihofer did recordings from the left greater splanchnic nerve, brain stem microinjections, and histological analysis to further study the source of the differences between the OZRs and LZRs.  When muscimol (a GABAA agonist) was injected into the RVLM, the SNA dropped almost to zero and the MAP and heart rate (HR) both decreased significantly, with the MAP decreasing more in the OZR than in the LZR.  In the NTS, muscimol injections caused increases in SNA, HR, and MAP, but the OZR had smaller increases than the LZR.  Injection of losartan (an AT1 antagonist) into the RVLM produced a decrease in SNA, MAP, and HR in the OZR but only a decrease in HR in the LZR.  Kynurenate (an ionotropic glutamate receptor antagonist) injection eliminated the reflex decreases in all three variables in response to stimulation of the sciatic nerve.  In the CVLM, kynurenate injections evoked smaller increases in SNA and HR in the OZR than in the LZR.  Gabazine (a GABAA antagonist) injected into the RVLM caused a reduction of the reflex decreases in SNA, HR, and MAP in response to stimulation of vagal afferents, but less so in the OZR than in the LZR.  Finally, injecting GABA into the RVLM caused similar decreases in SNA, HR, and MAP in both OZR and LZR.

To summarize the results, drug injections into the RVLM showed that the RVLM in the OZR is probably receiving less GABA than in the LZR (because the GABAA agonist caused a greater response and the GABAA antagonist caused a lesser response) and that the NTS is exerting less control in the OZR than in the LZR (because inhibiting is caused a lesser response).  The AT1 antagonist caused a greater response in the OZR, indicating that the OZR RVLM is being activated more by angiotensin II than the LZR RVLM is.  Knocking out glutamate signalling in the CVLM of the OZR had less of an effect than in the LZR, implying that the CVLM is less active in the OZR (which agrees with the NTS data).  In short, the OZR RVLM is receiving less tonic GABA input from the CVLM, receiving more tonic angiotensin II input, and receiving similar tonic glutamate input.  The OZR CVLM receives less tonic glutamate (which explains the lower tonic GABA release in the RVLM), and the NTS is also less active (because inhibiting it has a lesser effect).

Huber and Schreihofer suggest several reasons for these differences, but the experiments in this paper assessed only the differences and not their causes.  One potential problem with comparing a leptin-receptor deficient rat to a normal rat is that the leptin-receptor deficient rat has higher levels of leptin, which is known to cause increased SNA and MAP.  However, it has been shown that the OZR still have higher SNA and MAP and attenuated baroreflexes even when leptin's effects are absent.  Also, the MAP and baroreflexes are normal in juvenile OZR, even though they lack the leptin receptor.  Experiments in other animal models of hypertension show differences similar to those seen in these rats, as well.