Voluntary Exercise Induces a BDNF-Mediated Mechanism That Promotes Neuroplasticity

FERNANDO GO´MEZ-PINILLA,1,2 ZHE YING,1 ROLAND R. ROY,3 RAFFAELLA MOLTENI,1 AND V. REGGIE EDGERTON1,3 1Department of Physiological Science, 2Division of Neurosurgery, UCLA Brain Injury Research Center and 3Brain Research Institute, Los Angeles, California 90095 Received 4 March 2002; accepted in final form 15 July 2002 “BDNF is a powerful modifier of neuronal excitability and synaptic transmission”. The authors were interested to determine whether exercise induces an integrated response of BDNF and its receptors that may lead to synaptic modification at the level of neuromuscular system. To test this, they allowed 3 months old male SD rats to exercise for a period of 3 days and 7 days in running wheel with increasing load over the time period. The authors selected the lumbar region of the spinal cord and soleus muscle to examine the changes since the motor pools innervating the hindlimb muscles are located in the lumbar region of the spinal cord and running involves recruitment of soleus muscle. Further to examine whether muscle activation via locomotion is the main stimulus for the induction of neurotrophins the authors paralyzed the soleus muscle with botulinum toxin in separate groups of animals. Voluntary exercise increased the mRNA expression of synapsin I, which mediates the action of BDNF. Synapsin I mRNA levels were increased in proportion to growth associated protein (GAP-43) and signal transduction receptor (trkB). These suggest that exercise can impact synaptic growth and function. Paralysis of soleus muscle resulted in reduced BDNF and synpasin I mRNA levels suggesting that basal level of neuromuscular activity is necessary to maintain normal levels of BDNF. Overall BDNF was shown to play an important role in exercise mediated neuronal plasticity and function in the neuromuscular system. -Madhan

Running enhances neurogenesis, learning, and long-term potentiation in mice

Henriette van Praag*†, Brian R. Christie†‡, Terrence J. Sejnowski‡§, and Fred H. Gage*¶ *Laboratory of Genetics and ‡Laboratory of Computational Neurobiology, Salk Institute for Biological Studies, La Jolla, CA 92037; and §Howard Hughes Medical Institute and Department of Biology, University of California at San Diego, La Jolla, CA 92093 Edited by Charles F. Stevens, Salk Institute for Biological Studies, La Jolla, CA, and approved September 2, 1999 (received for review July 20, 1999) Neurogenesis is the formation of new neurons in the adult brain. New neurons are predominantly being formed in two regions of the brain 1) Subventricular zone lining the lateral ventricles and 2) Subgranular zone, part of the dendate gyrus of the hippocampus. Dendate gyrus is important for the memory function. The authors in this study were interested to know whether voluntary physical activity enhances neurogenesis, synaptic plasticity and learning. To test this, the authors subjected female mice to several tests, 1) The animals were injected with 5-bromodeoxyuridine intraperitoneally to label the dividing cells 2) Mice were trained on Morris water maze to measure spatial learning 3) To determine whether running affects synaptic plasticity, long term potentiation (LTP) was studied in the hippocampal slices. Running enhanced neurogenesis, showed by increased number of Brdu-labelled cells. Running increased learning showed by improved acquisition on the water maze task and finally running augmented dendrite gyrus LTP demonstrated by increased excitatory postsynaptic potential after administration of high-frequency stimuli. It will be more interesting to know the mechanism behind these correlated changes. -Madhan

Physical (In)Activity-Dependent Structural Plasticity in Bulbospinal Catecholaminergic Neurons of Rat Rostral Ventrolateral Medulla

Authors: Mishcel..Mueller Background: It is known that physical inactivity is a leading risk factor in cardiovascular disease, which is the number one cause of death in the United States. One potential consequence of physical inactivity is the development of hypertension that can also lead or contribute to cardiovascular disease. There are multiple forms of hypertension, however the one this paper focuses on is hypertension induced by increased sympathetic outflow (specifically splanchnic) from the brain region known as the rostral ventrolateral medulla (RVLM). Previous studies have shown that the RVLM is comprised of C1 and non-C1 spinally projecting neurons that decend down the intermediate medial column of the spinal cord and eventually target multiple areas in the body such renal, lumbar, and splanchnic nerves used to control blood pressure. Here the authors hypothesized through changes in the function of RVLM neurons that regulate splanchnic sympathetic outflow contribute to the development of hypertension. Methods: • Retrograde tracer CTB spinal cord injections • Immunohistochemistry • Light microscopy and digital reconstruction Results: • In both physically active and sedentary rats C1 neuron distribution varied depending on rostrocaudal level. It was seen rostral to the caudal pole of the facial nucleus most c1 neurons were arranged in clusters just ventral and medial to the motor neurons of the FN. However, in more caudal regions located caudal to the caudal pole of the FN C1 neurons were more evenly distributed and did not appear in tight clusters. • Morphological analysis following neuron reconstructions revealed C1 neuron dendritic structural differences in sedentary rats compared to physically active, with no differences in neuron somata. Overall dendritic surface area, branch points, and secondary dendritic length were increased in sedentary rats compared to physically active rats. • Finally, not only were structural differences observed between sedentary and active rats, but these morphological changes were seen on a rostrocaudal level, with increased branch points in more rostral regions of the RVLM. This rostrocaudal pattern in structural changes was not observed in the physically active cohort of rats. ~JI

The importance of stupidity in scientific research

Martin A. Schwartz Journal of cell science 121, 1771 doi:10:1242/jcs.033340 This article talked about how the author had a friend who dropped out of the phD program because she was tried of feeling stupid everyday.. The author hwas very surprised by this because he thought she was very bright. the he goes on to talk how how he ran into problems during his project and the faculty member didn't know how to help. This eventually lead him to realize that nobody knew the answers to the questions we asked and that is why we do research. in order to determine the question that may have a huge impact on healthcare people in the world.Then goes on talk about how there two major flaws in Phd programs. One is that students don't really understand how hard it is to do good research. I really agree with this. Because every scince course I have every taken everything always works out perfectly, which is not the case in a real laboratory setting. It may take months to get a technique to work. These are not things people tell you. Secondly he thinks that students are taught how to be productively stupid.meaning that we have to realize that we don't know everything. This lack in knowledge is what drives us to continue our research. He also talks about how hard it is to get funding. -MD

Evidence for descending tonic inhibition specifically affectingsympathetic pathways to kidney

Kristen Hayes, Christopher P. Yardley and Lynne Weaver Journal of physiology 1991 434 295-306 This is an early study investing whether the RVLM provides tonic inhibitory control over different sympathetic nerves. Specifically splenic , renal, splanchnic and the nerves comming from the T13 region. We know that the RVLM is important for the tonic control of blood pressure and that when stimulated with glu there is an in increase in nerve activity and bp. This study aimed to to test whether blocade of excitory neurons in the RVLM leads to an imbalance and profound infuence of inhibitory stimuli that could be balanced could counter acted by spinal cord transection. They used bilateral injection of glycine and then the did a spinal cord transection at the first cervical section. During the experiment they recorded renal, splenic splanchnic and T13 white ramus. They basically showed that renal and the T13 rami had greater response to blockade of RVLM compared to spinal transection; as for the other nerves (splenic and splanchnic)there was no difference.These data are suggesting that the majoirity os sympathoinbition is being driven at the level of the RVLM. -MD

Anesthesia differentially modulates spontaneous network dynamics by cortical area and layer

Sellers KK, Bennett DV, Hutt A, Fröhlich F. J Neurophysiol. 2013 Dec;110(12):2739-51 PMID: 24047911 Anesthetics are generally thought to depress neuronal activity, but that's not always the case. Current thinking seems to be that they work by modulating network communication, even though that may mean increasing activity in some cases. In this paper, they looked at how 0.5, 0.75, and 1.0% isofluorane modulates activity in the visual cortex and the association cortex at the field and multiunit levels in ferrets. What they found was that the different regions responded differently to anesthesia, and even different cortical layers within the same area responded differently. For instance, the overall activity of the visual cortex did not change, there was an increase in layer IV neuron activity and a decrease in other layers. So while they were able to see the slow oscillations that have previously been seen in anesthesia, they were able to measure differences between areas and layers. I wanted to read this paper because we are starting to examine neuronal activity, and we've had questions of what sedation can do to our animals. I didn't really get any answers to my questions, but I have more questions now and I realize that things could get a lot more complicated. I guess we should be careful about how we do our upcoming MeMRI studies. I guess this also makes me question some of the early studies showing greater Mn uptake as sedation wore off, but that's a whole different issue right there. -DH

Exercise counteracts the effects of short-term overfeeding and reduced physical activity independent of energy imbalance in healthy young men

Walhin JP, Richardson JD, Betts JA, Thompson D. J Physiol. 2013 Dec 15;591(Pt 24):6231-43 PMID 24167223 So, given what occurred at Thursday's departmental party and today's lab lunch, this study seems amazingly appropriate. The human participants were broken into sedentary and active groups (less than 4,000 steps/day vs 45 mins of treadmill time, respectively). Sedentary subjects increased their caloric intake by 50% while active groups increased by 75% so that both groups would have a caloric excess. They were then monitored for changes in glucose tolerance, cholesterol, and adipose tissue gene expression at the beginning and end of a 7-day protocol. The results showed that even though both groups saw increases in fat mass and systolic blood pressure, only the sedentary group increased their insulinemic response to a glucose challenge (2hr insulin area-under-the-curve). The seds also showed greater increases in total and LDL cholesterol, white blood cells, adiponectin, beta-cell function, along with gene expression of Fatty Acid Synthase, glucose transporter 4, and a number of other markers for poor health. The take-home message is that, among making life better in a billion other ways, exercise helps your body get better at dealing with short-term binge eating... just like we all tend to do over the holidays. Fine, I'll TRY to get on my exercise bike... but I can't promise that I'll pedal it much. -DH

Enhanced MRI and Acoustic Startle Reflex Testing

Avril Genene Holt, David Bissig, Najab Mirza, Gary Rajah, Bruce Berkowitz Introduction Between the military weapons and loud rock concerts emerging over the past couple decades a major growing health care concern is tinnitus, which is the perception of being able to hear a stimulus such as ringing, buzzing, or hissing that is not actually present. In the late, it has been found that tinnitus can be linked to an increase in spontaneous neuronal activity, but little is known about exact brain regions that tinnitus effects making drug targeting only partially successful. This was the first in vivo study examining specific auditory pathways and the correlated neuronal activity in subjects experiencing tinnitus. Methods • Auditory Brainstem response Testing • Salicylate and Noise Exposer induced tinnitus • Acoustic Startle Reflex Testing • MeMRI Results Implementing tinnitus inducing protocols using salicylate and noise exposer, tinnitus was induced in both groups of rats. However, it was seen that tinnitus in salicylate treated rats only experienced acute tinnitus that dissipated with the removal of salicylate. On the other hand, the noise induced group was seen to have permanent tinnitus perception showing limited ability to inhibit ASR 2 days post exposer. In MeMRI studies, the targeted brain regions were normalized to anterior pituitary uptake of manganese as well as local muscle uptake. It was seen that the DCN showed greater spontaneous activity compared to VCN in both normal hearing animals and tinnitus induced animals (no VCN activity was seen in tinnitus induced animals). Salicylate induced tinnitus animals displayed bilateral increases in manganese uptake following a systemic injection that was predicted. However, the noise induced animals also showed a bilateral increase in manganese uptake that was not predicted due to the fact that tinnitus was only induced in one ear with an ear plug protecting the opposing ear. This result was thought to be seen as a result of enough hearing preservation to successfully perform ASR testing, but enough damage to alter neural tissue activity in the IC. Finally, the DCN showed increased spontaneous neuronal activity via manganese uptake in only the salicylate group. With no significant changes in the DCN in the noise induced group, the authors’ hypothesize that the imaging time point may not have been correct at 2 days post exposer, because other studies have shown spontaneous neuronal activity 5 days post noise exposer. However in the IC both salicylate and noise-induced tinnitus groups a significant increase in manganese uptake was observed. The next study the authors’ would like to undertake is studying the auditory brain regions involved in tinnitus over a longer time frame, ie. A longitudinal study. ~JI

CCK-induced inhibition of presympathetic vasomotor neurons: dependence on subdiaphragmatic vagal afferents and central NMDA receptors in the rat

Anthony J. M. Verberne , Daniela M. Sartor Am J Physiol Regul Integr Comp Physiol. 2004 Oct;287(4):R809-16. Epub 2004 May 20 “Systemic administration of cholecystokinin (CCK) inhibits a subpopulation of rostral ventrolateral medulla (RVLM) presympathetic vasomotor neurons”. The authors investigated whether the above effects are mediated by 1)Subdiaphragmatic vagal afferents. To test this, Lidocaine, a topical anesthetic was applied to the subdiaphragmatic branches of the vagus before and after injections of CCK, phenylbiguanide and phenylephrine. Application of lidocaine abolished the neuronal responses to CCK. 2) Central N-methyl-D-aspartic acid (NMDA) receptors. To test this, NMDA receptor antagonist, dizocilpine was given systemically. Central NMDA receptor blockade reduced the CCK induced inhibition of the RVLM presympathetic neuronal discharge. The authors suggest that CCK receptors located on subdiaphragmatic vagal afferents mediate the actions of CCK on the discharge of RVLM presympathetic vasomotor neuron. Also, central NMDA receptors mediates CCK-induced sympathoinhibition. Madhan

Cholecystokinin selectively affects presympathetic vasomotor neurons and sympathetic vasomotor outflow

Daniela M. Sartor , Anthony J. M. Verberne Am J Physiol Regul Integr Comp Physiol. 2002 Apr;282(4):R1174-84. “CHOLECYSTOKININ(CCK) is a gastrointestinal hormone with actions including gastrointestinal vasodilatation reduced gut motility, gastric acid secretion, and pancreatic secretion”. CCK is an abundant neuropeptide with a central role in satiety. Its receptors are located on the vagal afferents and NTS. The authors were interested to study the effects of CCK on sympathetic vasomotor outflow on spinally projecting neurons of the RVLM. CCK was given intravenously and parameters such as mean arterial pressure, heart rate, lumbar sympathetic nerve discharge, splanchnic sympathetic nerve discharge were recorded. The authors investigated the effect of CCK on the discharge of presympathetic neurons of the RVLM to determine whether CCK could discriminate subgroups of these neurons. Also the importance of vagal afferents and involvement of specific CCK receptor subtype were examined. CCK produced differential effects on sympathetic vasomotor outflow, a dose dependent activation of LSND and inhibition of SSND. The differential effect of CCK on RVLM presympathetic neurons includes inhibition in some cells, activation in some and no changes in others. These findings suggest that vagal afferents are heterogeneous, meaning some of its population produces an inhibitory effect, some excitatory effect on sympathetic vasomotor function. In addition to these findings, the authors demonstrate that the effects of CCK are mediated by activation of CCK-A receptors. -Madhan

Hypertensive effects of central angiotensin II infusion and restraint stress are reduced with age

Benedek Erdos..and Nihal Tumer Introduction: It is well known that the risk of cardiovascular disease increases with age, and that blood pressure regulation specifically decreases with age. This deregulation can then account for age-related increases in hypertension which is correlated to morbidity and mortality. Angiotensin II is is a primary regulator in BP, through the alterations in baroreflex sensitivity and stress-induced sympathetic nervous system responses. However, the effects of age on Ang II mechanisms are unknown. It has already been shown that inhibition of Ang II type 1 (AT1) can reduce age related increases in the risk of cardiovascular disease. Here the authors look to study the effect of Ang II on BP, HR, sBRS and central oxidative stress and their differences in young and old rats. Methods: -Spontaneous baroreflex sensativity test -Restraint stress test -Western Blot -Reverse transcriptase-PCR -Electron paramagnetic resonance Results: -Resting MAP were increased in older rats with a decrease in HR and locomotor activity. After an AngII infusion in young and old rats show an increase in both cohorts in MAP, however the increase was stunted in the older rats compared to the young. Post infusion showed that HR increased in both cohorts equally. And finally varition of HR was reduced by 50% in the older cohort. -Throughout the entire study telometry systems were used to study sBRS and found that older rats had decreased sBRS compared to young rats, and after AngII infusions this SBRS was significantly reduced in the young rats. -After performing restraint stress tests, MAP, and HR responses were recorded. It was found that HR and MAP both had a rapid increase. After the addition of an AT1 inhibitor they found that stress-induced MAP elevations were reduced significantly in both groups. HR on the other had did not have any significant change after AT1 inhibition. Later, evaluation of hypothalamic tissues found decreased AT1 receptor protein and mRNA levels in older animals. ~JI

Chemoreflex Function in Heart Failure Continued

Harold Schultz and Shu-Yu Sun Central chemoreceptors, located on the central surface of the medulla, have also been found to play a role in increased activation of sympathetic activation of the chemoreflex. However, there are alternative methods suspected to lead to increased central chemoreceptor sensitivity. This main mechanisms being angiotensin two regulation mediating the alteration of baro and chemoreceptor sensitivity. Two key pieces of evidence lead to this hypothesis 1) angiotensin two levels are elevated in chronic heart failure patients and 2) Angiotensin two in the central nervous system can stimulate sympathetic ouflow, blunt baroreflexes, and stimulate breathing. It was seen that the administration of angiotensin two inhibitors in HF rabits reversed the enhanced peripheral chemoreflex function and improved baroreflex function. Ironically, the treatment they authors suggest to improve hemodynamic profiles as well as ventilatory response, decreasing the sympathetic activation in HF patients is exercise. I find this ironic because in most cases exercise will prevent HF in the first place. The reason that the authors believe exercise helps in decreasing sympathetic activation is because it increases NO synthesis which is thought to help regulate peripheral chemoreflex sensitivity. ~JI

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

Davi J. A. Moraes, Melina P. da Silva, Leni G. H. Bonagamba, Andre´ S. Mecawi, Daniel B. Zoccal,

Jose´ Antunes-Rodrigues, Wamberto A. Varanda, and Benedito H. Machado.  Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats J. Neurosci 33: 19223–19237,2013.

This paper from Benedito Machado’s group in Brazil examined RVLM presympathetic neurons and attempted to better characterize the mechanisms of respiratory related sympathetic activity also known as respiratory-sympathetic coupling.  Although it exists in most sympathetic nerve recordings, respiratory-sympathetic coupling is most easily observed when one examines both sympathetic nerve activity and phrenic nerve activity, the latter being the motor nerve that innervates the diaphragm and drives breathing.  It has been suggested that this coupling occurs at the level of the individual, spinally projecting C1 and non-C1 neuron. 

These investigators achieved this by using the in situ working heart-brainstem preparation, which has been referred to as the “half rat” preparation.  Although neither considered in vivo nor a fully intact animal, the preparation does allow the use of whole-cell patch recordings in the absence of anesthesia because the animal is decerebrated, which means the areas of the brain that perceive pain are removed.  In addition to the electrophysiology they are able to fill the recorded cells with biocytin and identify them with immunohistofluorence and stain for TH to determine if they are TH positive or not.  After recording from the cells they are able obtain the RNA from them and do single cell PCR.  In addition to being a real technical tour de force they also examine normal rats and rats that have been submitted to chronic intermittent hypoxia (CIH), a common model for sleep apnea.

The authors found that both C1 and non C1 neurons expressed several different kinds of phenotypes based on intrinsic electrophysiological properties, whether or not they had respiratory synaptic inputs, and what ionic currents they expressed.  Interestingly in this preparation all RVLM neurons had persistent sodium current-dependent pacemaker properties that persisted after synaptic blockade.  These latter data support the notion that RVLM neurons are capable of generating their own firing which could be important in states of overactivity including hypertension, heart failure and inactivity.  Also non-C1 but not C1 neurons from animals that had been exposed to CIH demonstrated enhanced excitatory input which could explain why CIH animals exhibit increased sympathetic nerve activity.

~PJM

GABAergic responses in ventrolateral medulla in spontaneously hypertensive rats

J. KEVIN SMITH AND KIRK W. BARRON

The RVLM is important for the tonic and phasic control of BP. We that the activity of these neurons is modulated by neurotransmitters. However in certain disease states the neutromitters like glutamate and GABA may have alltered effects on RVLM neurons. The RVLM receives inhibitory input from the CVLM. The purpose of this study was to dertemine whether GABAergic transmision was altered in response to hypertensive conditions. In WKYs and SHR rats GABA was injected into the RVLM. They found that the change in BP were signignantly greater in SHR compared to the WKYs. However when they  gave bicuculline into the RVLM they found that the SHR have attenuted responses compared WKYs. Finally they inhibited the CVLM to and found that the SHRs had an attentuated response compared to the WKYs. However the sensitivity to GABA in the SHR is greater compared to WKYs. These data suggest that the SHR have decrease gababergic input from CVLM compared to WKYs. This may be one potential cause for the developmentment of hypertension. 

 

-MD

 

Excitatory Amino Acids in the Rostral Ventrolateral Medulla Support Blood Pressure in Spontaneously Hypertensive Rats

 

Satoru Ito, Kazutoshi Komatsu, Kazuyoshi Tsukamoto and Alan F. Sved

The rvlm is important to tonic and phasic control of blood pressure. It has been shown in normotensive anesthetized rat that injection of kyn into RVLM has no effect on BP. However if you block CVLM and give kyn into RVLM you we see a similar decrease as if you inhibited the RVLM, suggesting that excitatory amino acids are driving both inhibition and excitation in the RVLM.  The purpose of this study was to determine the role that excitation and inhibition are playing in hypertension.  They used microinjection technique in order to address their questions. What they found was that in response to kyn into RVLM the WKYs had no response however the SHR had a decrease in bp of 40mmHG.  They then injected muscimol into CVLM and then kyn into RVLM. They found that after blockade of CVLM in both groups they saw a decrease in bp when they gave kyn into the rvlm. These data suggests that CVLM must be provide not only inhibitory input but also some non-excitatory amino acid input. Also that excitatory amino acids are playing a major role in driving rvlm activity in hypertensive animals.

-MD

Millisecond-timescale, genetically targeted optical control of neural activity.

Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Nat Neurosci. 2005 Sep;8(9):1263-8

Many people see optogenetics and Channelrhodopsin (ChR) as hot new tools to use for studying neuronal activity and connectivity, but (while I don't disagree about its hotness) it's actually not that new.  Some of the first papers published on channelrhodopsin come from 2002, in which both ChR1 and ChR2 were described.  While most people think of the Deisseroth lab when they think of ChR, these papers were actually published by two completely different groups (PMID: 12060707 and 12089443), although some of the authors later began collaborations with the Deisseroth lab.  What made Deissiroth famous, and rightly so, was this 2005 paper, in which he was the first to publish on using ChR in neurons (other groups worked in oocytes, HEK cells, etc) and recording their photoactivatable activity with whole-cell patch clamp.
In this paper, they harvested P0 rat hippocampal neurons, cultured them, and transfected them on day 7 with lentivirus in order to cause expression of ChR2-YFP.  Under voltage clamp, they found that they were able to induce inward currents with flashes of light (8-12mW/mm^2).  When they recorded in current clamp mode, they found that these currents were sufficient to cause action potentials at ~30Hz in some cases.  When they patched on to non-trasfected (non-fluorescent) cells, they were able to see excitatory and inhibitory postsynaptic potentials induced by the flashes of light that they could block with glutamatergic and gabaergic antagonists.
By showing that expression of light-gated in ion channels in neurons gives you precise temporal control of neuronal firing, this paper ushered in the age of optogenetics in neuroscience, and it is why many people think Deisseroth will eventually win the Nobel Prize.
Funny story: one of our own professors here at WSU SoM also did a lot of early work on ChR, except he thought the experiments in these papers were obvious, so he didn't do them.  He decided that since his lab works on the retina, he would use ChR in the eye - which didn't get anywhere near as much publicity when he published on using ChR to restore vision in mice suffering from photoreceptor degeration in early 2006.
-dh

Functionally different neurons are organized topographically in the rostral ventrolateral medulla of rabbits.

Ootsuka Y, Terui N. J Auton Nerv Syst. 1997 Dec 3;67(1-2):67-78.

One of the things we suspect in our lab is that the rat RVLM has a sort of viscerotopic organization that allows for differential activation of sympathetic nerves.  We believe this not only because our lab has published on it already and seen it in some of our unpublished work, but also because it has been seen in other mammals.  This paper was one in which they showed this organization exists in rabbit RVLM
In this paper, the authors examine RVLM activity in rabbits using different methods than we use.  They used a pulsed doppler flowmeter in order to measure vascular conductance.  They measured arterial pressure and conductance in the kidney, fore- and hindlimb muscles, ear skin. They also measured the heart rate via ecg. They then gave GABA microinjections in a grid-pattern within the RVLM in order to inhibit vasomotor tone and constructed contour maps demonstrating where GABA injections resulted in the greatest vascular conductance.
Their results suggest that there is a relationship between target organ and location in the RVLM, but that these regions seem to overlap.  They mention that activation of multiple regions with one injection may be partially due to their spatial resolution, which they estimate to be about 0.5mm due to the dye spots they used as markers.  This fits pretty well with the accepted formula for finding microinjection sizes, as well as our own data, and it's why I am trying to increase the resolution with my project.
One final thing I appreciated about this paper was their use of straight GABA to do their experiments.  I initially found it counterintuitive for studing RVLM activity, but their discussion pointed out that other groups using glutamate found multiple injections often resulted in to unrepeatable results, presumably due to glutamate excitotoxicity.  They chose GABA because it showed a much shorter effect than other inhibitory compounds (eg glycine and muscimol). This meant that by waiting 5 minutes in between injections, they could get repeatable results in the same animal that would not be possible using other drugs.  This brought on decreases intersubject variability and increases the data yield per animal used.

-dh

CLARITY for mapping the nervous system

Kwanghun Chung1,2 & Karl Deisseroth1–4

1Department of Bioengineering, Stanford University, Stanford, California, USA. 2CNC Program, Stanford University, Stanford, California, USA.

3Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. 4Howard Hughes Medical Institute, Stanford

University, Stanford, California, USA.

"CLARITY is a newly developed technology that can be used to transform intact biological tissue into a hybrid form in which tissue components are removed and replaced with exogenous elements for increased accessibility and functionality".

The main objective of this study is to improve the methodologies for brain mapping. Molecular phenotyping of the several layers of mammalian central nervous system is a very complex because the neural process acts as barriers for macromolecular diffusion. In the past decade, methods such as mechanical sectioning of tissue, optical clearing methods have been developed to improve tissue clearing. However, these methods were not suitable for whole-tissue molecular phenotyping. CLARITY method overcomes this issue and allows for molecular and optical interrogation of entire adult mouse brain. The cool feature of this technique is that it maintains fine structural resolution and molecular details along with macroscopic connectivity. The lipid bilayers that were hard to penetrate using other methods was replaced with a rigid and hydrogel-based infrastructure that preserves and allows access of structural and molecular information. A unique capability of CLARITY is that it preserves endogeneous biomolecules such as neurotransmitters, proteins and nucleic acids. CLARITY can also be used along with other genetic methods for identification of synapses or for labelling specific circuits. 

For more reading visit http://clarityresourcecenter.org/

For more information watch https://www.youtube.com/watch?v=c-NMfp13Uug

-Madhan

Chemoreflex function in heart failure

Harold D. Shultz and Shu-Yu Sun

There are peripheral and central chemoreceptors in the human body, located in the carotid and aortic bodies as well as the ventral surface of the medulla, that both play a role in regulating both the respiratory and sympathetic cardiovascular outflow. The peripheral receptors primarily respond to hypoxemia, where as the central receptors are most sensitive to hypercapnia.  However, both are able to influence acute ventilation and cardiovascular outflow in order to regulate O₂ and CO₂ levels in the plasma.  It was previously thought under normoxic conditions the chemoreflex played little or no role in regulating sympathetic tone, recently however it has been shown that blocking peripheral chemoreceptors activity via hyperoxima decreases sympathetic nerve activity.  This evidence leads us to believe that the chemoreflex may play a role in elevated sympathetic tone in heart failure patients, even without hypoxia. A study done with HF induced rabbits shows that peripheral chemoreflex control over renal sympathetic nerve activity and ventilation is increased, increased sensitivity of the peripheral chemoreflex therefore provided a potential mechanism for sympathetic activation in heart failure patients. After many experiments it was found that attenuated NOS activity is one of the main reasons HF patients have enhanced peripheral chemoreceptor sensitivity.

~JI

Paraventricular nucleus control of blood pressure in two-kidney, one-clip rats: effects of exercise training and resting blood pressure.

Rossi NF, Chen H, Maliszewska-Scislo M. Paraventricular nucleus control of blood pressure in two-kidney, one-clip rats: effects of exercise training and resting blood pressure. Am J Physiol Regul Integr Comp Physiol 305: R1390–R1400, 2013. First published October 2, 2013. Background: This is a paper from Dr. Rossi's laboratory which I believe is the first publication from her lab in which they look at the influence of chronic wheel running versus sedentary conditions. They examine both tonic GABA and nitric oxide signaling in the paraventricular nucleus of the hypothalamus (PVH, as opposed to the paraventricular nucleus of the thalamus). They record mean arterial pressure (MAP), plasma angiotensin II (Ang II) and renal sympathetic nerve activity (RSNA) in conscious normotensive Sprague-Dawley rats versus their two kidney, one clip model of hypertension (also Sprague-Dawleys) after 6 or 12 weeks of wheel running or sedentary conditions. The impressive thing about this study is the fact that it is done in conscious animals with RSNA and PVN microinjections in normotensive and hypertensive animals after six or 12 weeks of wheel running. A technical tour de force as they say. Results: MAP and Ang II were higher in HTN rats which were both reduced similarly by wheel running both at 6 and 12 weeks of running. HTN rats also had lower pressor and sympathoexcitatory responses to blockade of nitric oxide or GABA in the PVH which were attenuated by wheel running. Interestingly, in the most hypertensive animals (i.e. those >180 mmHg) there was very little change in response to bicuculline and in fact, in animals above 200 mmHg, decreases in arterial pressure in response to bicuculline were observed. These novel findings suggest that GABA provides a basal level of excitation through ill-defined neuroplastic changes in the most hypertensive animals. Conclusions: I thought this was not only an amazing paper technically but also very interesting to our laboratory because it involved wheel running; conscious microinjections into PVH, a brain region that projects directly to RVLM; and had measurements of conscious sympathetic nerve activity. It's a good read and a very good candidate for a future journal club if anyone with an interest in PVH, exercise and SNA. ;-) ~PJM

Manganese ions penetrate via L-type Ca2+ channels and induce contraction in high-K+ medium in ileal longitudinal muscle of guinea-pig.

Nasu T, Murase H, Shibata H.Gen Pharmacol. 1995 Mar;26(2):381-6. PMID: 7590091

Now that we do manganese-enhanced magnetic resonance imaging (MEMRI), we all know that manganese enters active cells during action potentials because it acts as a calcium surrogate and gets taken up by voltage-gated calcium channels.  We also now know which type of channels are responsible for this, because this is the paper where the authors inhibited T-type and L-type cells to show that it's the L-type channels that allow manganese to enter the cell.
In this paper, they clipped strips of ileal smooth muscle in to a strain gauge meter, and then washed the strips in a high-potassium solution to cause muscle contraction.  They found that 5mM manganese was able to block contractions, seen by a decrease in muscle tension, presumably by occupying calcium binding sites but without being able to activate them as well as calcium can.  They were also able to see that in calcium-free solutions, application of manganese could actually take the place of calcium and cause the contractions if the potassium solution was strong enough.  This was able to be blocked by the L-type calcium blockers, nifedipine and D-600, while T-type calcium channel blockers were not able to do this.  They were also able to show, using spectrophotometry, that tissue manganese content was significantly lower following L-type channel blockade, but not blockade of T-type channels.
-dh

Characterization of engineered channelrhodopsin variants with improved properties and kinetics

Lin JY, Lin MZ, Steinbach P, Tsien RY.

Biophys J. 2009 Mar 4;96(5):1803-14. PMID: 19254539

At the time this paper was being written (and even now), channelrhodopsin was growing in popularity and a number of variants were being created.  Each of variant has its own properties of conductance, inactivation, recovery time, etc. In this article, they examined the kinetics of different variants of channelrhodopsin.  What they showed was that the variants had, in some cases, vastly different properties.  For example, some were not only permeable to sodium, which is why they are useful for depolarizing neurons, but also permeable to calcium.  This is important because altering calcium homeostasis could have a number of unintended consequences on cells, up to and including death.  Most importantly, they were showed that the same preparations of cells, but using different variants of channelrhodopsin, have different abilities to follow high frequency trains of photostimulation, e.g. the ChEF variant is able to respond reasonably well to photostimulation at 50Hz, while channelrhodopsin 2 and its H134R variant can not reliably perform under even 25Hz trains.

The take-home message of this paper, in my opinion, is that there are many types of channelrhodopsin available.  Which one you decide to use should probably be based on a good analysis of its capabilities and whether or not they are suited for the type of experiments you will be doing, not just which version is available from your favorite distributor.

-dh

PP2A-B56alpha controls oncogene-induced senescence in normal and tumor human melanocytic cells

Authors: S Mannava....MA Nikiforov

Background: In most cells oncogene-induced senescence (OIS) is utilized as a tumor suppressor mechanism, preventing metastasis of mutated cells.  It is seen in malignant melanomas that cells are able to avoid OIS, allowing for proliferation.  One hypothesis is that C-MYC, a gene regulator upregulated in cancerous cells, protects oncogenic cells against OIS.  However, the mechanism in which C-MYC utilizes to suppress OIS in cancerous cells is still unknown.

Methods:

• cell proliferation assays
• cell senescence assays
• lentiviral constructs and infection
• qRT-PCR
• Immunoblotting and immunofluorescence

Results: First they were able to detect that C-MYC is elevated and maintained the level of the protein in melanoma cell lines using western blotting, qRT-PCR, and Chase half-life test.  Protein stability was then examined in cancerous cell lines, exposing the down regulation of PP2A B56-alpha, a regulator subunit that leads to the degredation of C-MYC, in cancerous cell lines.  As well, the upregulation of CIP2A, a C-MYC interacting protein that inhibits PP2A activity. It was then determined when B56-alpha was re-introduced to cancer cell lines and over expressed that the OIS phenotype was restored, with a decreased level in C-MYC protein expression not mRNA levels.  Lastly, it was shown that the depletion of B56-alpha in NHM leads to increased levels of C-MYC followed by increased resistance to OIS.

~JI

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

Tetsuya Kawabe , Kazumi Kawabe , Hreday N. Sapru

 

 

The purpose of this study was to determine the physiological effects of angiotensin (1-12) in the caudal ventrolateral medulla. They used microinjection technique in order to determine the role that ang(1-12) is playing in control of MAP and GSNA. They found that ang 1-12 caused decreases in MAP, HR, and GSNA. In response to ACE inhibitor  injection in the CVLM, the ang 1-12 response was blocked. Also that GABA receptor blockade in the RVLM, lead to blunted ang 1-12 responses. The also looked the effects of AT1R and AT2R blockade in the CVLM and they found that blockade of AT1R blocked the response to ang 1-12. The AT2R did not affect the response to ang 1-12. 

 

Conclusion: Ang 1-12  may be broken down into Ang II and ang 1-7 and these two neurotransmitters are mediating the decrease in MAP, HR, and GSNA by binding to AT1Rs in the CVLM. Then finally the GABA receptors in the RVLM are also playing role in mediating the the physiological response to Ang 1-12.

 

 

-MD

Effects of hypocretin and norepinephrine interaction in bed nucleus of the stria terminalis on arterial pressure

J.Ciriello, M.M. Caverson and Z. Li

The bed nucleus of the stria terminalis is a limbic structure that is activated stressful stimuli. The autors wanted to address whether hypocretin-1 (hct-1) and norepinephrine (NE) are playing a role in the BST to control cardiovascular responses. They used microinjection injection technique in anesthetized wistar rats to examine the question. They found that in response to glutamate (glu) there were decreases in blood presssure (BP) and heart rate (HR). I response to both NE and hct-1the BST response to glu was blunted. They also looked at whether GABAergic neurons in the BST is mediating the inhibitory responses. They injected bicuculline and phaclofen into BST.They found that Bic but not phaclophen blocked the glu responses. Next that looked at adrenergic receptor blockade with yohimibine (a2AR) and a1AR receptor. antagonist. They found that a2AR blocked Hct-1 and NE depressor responses. 

Conclusion: HCT-1 may cause the the release of NE. This in turn may activate GABAergic neurons in the BST in response to stressful stimuli.

-MD