Autonomic regulation of brown adipose tissue thermogenesis in health and disease: potential clinical applications for altering BAT thermogenesis

Autonomic regulation of brown adipose tissue thermogenesis in health and disease: potential clinical applications for altering BAT thermogenesis. Domenico Tupone, Christopher J. Madden and Shaun F. Morrison. Frontiers In Neuroscience 9:1-14, 2014 (open access) doi: 10.3389/fnins.2014.00014. This is an excellent and timely review from the standpoint of the field and in terms of our collaboration with Dr. Granneman's laboratory. Shaun Morrison has been studying brown adipose tissue or BAT for a number of years. His and his postdoc's (Chris Madden) work have really come into clinical significance with the discovery of BAT in humans and its ties to obesity. Basically BAT is a source of thermogenesis and therefore can be important in terms of burning calories to stay warm in a cold environment. Interestingly for our laboratory is the fact the BAT is innervated by the sympathetic nerves which release norepinephrine onto beta 3 receptors to activate BAT. Dr. Morrision's laboratory has worked out a lot of the central circuitry or brainstem pathways by which BAT and BAT SNA is regulated under a variety of conditions, mostly associated with cold temperature exposure and the turning on of BAT. Interestingly, BAT is one set of sympathetic nerves that are not controlled by the RVLM but by a neighboring structure in the ventral medulla, the midline raphe. There are some really beautiful figures in this review and is worth reading for anyone interested in BAT, BAT SNA, and pathwways involved in thermogenesis. In the next few weeks in fact, Madhan and Priya hope to have BAT SNA recordings up and going in the laboratory, comparing runners versus sedentary rats. Should be exciting to see it develop and be sure to keep an eye out at Experimental Biology for both Shaun Morrison's and Chris Madden's work. ~PJM

Central Command Neurons of the Sympathetic Nervous System: Basis of the Fight-or-Flight Response.

Central Command Neurons of the Sympathetic Nervous System: Basis of the Fight-or-Flight Response. Arthur S. P. Jansen, Xay Van Nguyen, Vladimir Karpitskiy,Thomas C. Mettenleiter, Arthur D. Loewy. Science 270:644-646, 1995. This is a classic paper by Arthur Loewy's group at Washington University in St. Louis in which they propose the existence of "central command" neurons. These central command neurons are hypothesized to innervate multiple sympathetic targets and be responsible for the all or none, fight or flight response that has been traditionally associated with activation of the sympathetic nervous system. One thing to keep in mind here was that this paper was published in the mid-1990s when it was still controversial whether there were neurons that controlled some or all of the sympathetic outputs, whether there existed individual neurons that controlled individual sympathetic outputs, or if both types existed (likely the reality). Unlike the Australians (i.e. McAllen and colleagues), who had just the year prior shown differential control of sympathetic outputs with very small (5ul) microinjections in the RVLM of cats, Loewy's group was trying to demonstrate the reverse idea; that is, individual neurons have the anatomical connections to control multiple sympathetic outputs. To do so, they injected rats with two different viruses to produce retrograde and transynaptic tract tracing. They put one virus in the stellate ganglion which contains the axons of sympathetic preganglionics to the heart and they put a different virus in the adrenal gland which contains the sympathetic preganglionics controlling epinephrine release. While they did show that both viruses wound up within cells of the RVLM, there were several caveats pointed out in the paper and additional ones not pointed out. First, the number of cells that did show double labeling in the RVLM were very small and if you look in the Methods sections you will see it took them hundreds of rats to get this to work out. Second, there are several technical issues in using viruses that likely preclude definitive conclusions about the double-labeled cells. In any case, it was published in Science and is often quoted as the paper that demonstrated the existence of these neurons. It also propagates the still pervasive idea that sympathetics are an all or none phenomenon. We know now that this is not the case in several instances of physiology and pathophysiology. ~PJM

Daily spontaneous running attenuated the central gain of the arterial baroreflex

Chen, Chao-Yin, Stephen E. DiCarlo, and Tadeusz J. Scislo. Daily spontaneous running attenuated the central gain of the arterial baroreflex. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H662-H669, 1995. This is the second in a series of papers by this group addressing whether altered afferent sensitivity explains why physically active animals have differences in control of sympathetic outflow. In this follow up study, Steve DiCarlo and Tadek Scislo recorded from baroreceptor afferents while changing blood pressure in animals that had been on running wheels versus those that had been sedentary for 8-13 weeks. When they measureed responses in the aortic depressor nerve of rats from each group, they were not different. It is important to note that the aortic depressor nerve in the rat is made up of almost exclusively baroreceptor afferent fibers; whereas this is not true of the carotid sinus nerve which also contains chemoreceptor information. Even more interesting, they did not find a significant difference in the central gain of the lumbar sympathetic nerve whereas this group had shown that renal sympathetic nerve activity was different in rabbits and a Brazilian group (Negrao et al., 1993) had shown renal was also different in rats. This supports our contention that physically activity versus inactivity affects sympathetic outflow differently. Ultimately, though, since afferent activity of the aortic depressor nerve was not different these data further support the contention that changes in control of sympathetic outflow are not due to changes in the peripheral baroreceptors but due to changes in the brain. Again, this and their previous papers would be important to quote in a student's thesis on alteratons in control of sympathetic outflow in physically active verus sedentary rats (hint, hint: Mary, Dan, and Judy!). ~PJM

Daily spontaneous running did not alter vagal afferent reactivity

Scislo, Tadeusz J., Stephen E. DiCarlo and Heidi L. Collins. Daily spontaneous running did not alter vagal afferent reactivity. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): Hl564-H1570, 1993. This is an important paper in our field since it is one of two by this group to address whether altered control of sympathetic outflow is due to peripheral adaptations in afferent sensitivity. In the present study, Tadek Scislo and Steve DiCarlo examined the sensivity of cardiopulmonary receptor afferents in wheel running rats and sedentary rats. They did so by recording directly from these afferents and producing two different types of stimuli to the animals. Since these afferents can be sensitive to stretch (i.e. low pressure volume receptors) they tested their sensitivity to increases in left atrial pressure. Since these afferents are also be sensitive to chemical stimuli during that produced during hypoxia, for example, they tested their responsiveness to the 5HT receptor agonist phenylbiguanide. The results were that spontaneously running rats had no difference in their afferent sensitivity to either stimuli compared to sedentary controls. These data suggested that alterations in cardiopulmonary reflex control were due to central adaptations (i.e. in the brain) rather than in the periphery at the level of the afferents themselves. This and the next paper would be good for students to quote in their thesis since it provides evidence of changes in the brain being responsible for difference in sympathetic output in physically active versus sedentary animals. ~PJM

Functional imaging of the human brainstem during somatosensory input and autonomic output.

Functional imaging of the human brainstem during somatosensory input and autonomic output. Luke A. Henderson and Vaughan G. Macefield. Frontier in Human Neuroscience. Published as open access: September 17 Vol 7: 1-8, 2013 doi: 10.3389/fnhum.2013.00569. Very cool paper in which functional magnetic resonance imaging (fMRI) was used in humans to image brainstem sites believed to be involved in pain and autonomic cardiovascular control. Images were compared at 3T and 7T and shown quite distinguished differences. Muscle sympathetic nerve activity (MSNA) was used to correlate to brain areas that were activated mainly in the NTS, CVLM, and RVLM. I'm pretty sure someone did this paper for journal club but I didn't see that it had been blogged so here it is. ;-) ~PJM

Glial cells modulate the synaptic transmission of NTS

Glial cells modulate the synaptic transmission of NTS neurons sending projections to ventral medulla of Wistar rats. Daniela Accorsi-Mendonca, Daniel B. Zoccal, Leni G. H. Bonagamba & Benedito H. Machado. Physiol Rep, 1 (4), 2013, e00080, doi: 10.1002/phy2.80 This is a really cool paper by a group in Brazil that used whole cell patch clamp (i.e. recordings from individual neurons) in a brain slice preparation from teh NTS. They also recorded from atrocytes and used inhibitors of glial metabolism to show that they contribute to excitatory neurotransmission. They also showed that ATP was involved in excitatory neurotransmission by the use of a purinergic antagonist which reduced excitatory transmission. In combination they showed that glial cells were the source of the ATP released after activation of the afferents that drive excitatory transmission in the NTS. In the end, they showed that ATP released from glia activated presynaptic P2X receptors which facilitated glutamate release and enhanced excitation. This study is relevant to our because they specifically looked at ventrolateral medullary projecting neurons (i.e. RVLM likely). Pretty cool. ~PJM

Differential Content of Vesicular Glutamate Transporters in Subsets of Vagal Afferents Projecting to the Nucleus Tractus Solitarii in the Rat

Differential Content of Vesicular Glutamate Transporters in Subsets of Vagal Afferents Projecting to the Nucleus Tractus Solitarii in the Rat. Sam M. Hermes, James F. Colbert, and Sue A. Aicher* The Journal of Comparative Neurology 522:642–653 (2014). Vagal afferents are sensory nerves that originate from a variety of areas in the body and send information about the periphery to the brain. The first place they stop or what is called their primary termination site is the nucleus tractus solitariius or NTS. Hopefully the NTS is familiar to everyone as it is also the primary termination site of the arterial baroreceptor afferents. Both vagal (i.e. cardiopulmonary) and arterial baroreceptor afferents are thought to release glutamate as their primary neurotransmitter. Anatomically these afferents can be identified as glutamatergic if they contain one of three types of glutamate transporters (VGLUT1, VGLUT2, or VGLUT3) which help to package and release glutamate and produce primarily excitatory neurotransmission. It has been generally thought that the VGLUTs can identify different populations of neurons in the CNS since they're distribution tends to differ. In the present study, the authors wished to determine if myelinated and non-myelinated vagal afferents could be identified by their VGLUT expression. They did so by utilizing two different tracers: one being cholera toxin B subunit, better know as the CTB we also use in the lab. They also used a isolectin B4 or IB4 which seemed to have some selectivity for unmyelinated nerves in other systems. The authors injected both tracers into the vagus nerve and used confocal microsocopy of the NTS to examine the possibility of labeling these two different populations of fibers. In addition, they used electron microscopy to look at ultrastructural differences. The results demonstrated that CTb was found in both types of afferents (i.e. myelinated and unmyelinated) as well as on nerve terminals in the NTS. In contrast IB4 was observed only in unmyelinated afferents. At the ultrastructural level these tracers were found on primarily excitatory (i.e. glutamatergic) synapses as observed by the presence of asymmetrical post-synaptic densities on the electron micrographs. Interestingly, when they examined the VGLUT distributions, they were different on CTB vs. IB4 labeled neurons. CTb-labeled afferents had mostly VGLUT2 (83%), but IB4-labeled afferents had low levels of vesicular transporters, VGLUT1 (5%) or VGLUT2 (21%). These findings suggest the possibility that glutamate release from unmyelinated vagal afferents are regulated by a distinct, non-VGLUT, mechanism. This would be relevant to our studies since glutamate is the primary excitatory transmitter that not only drives RVLM neurons, but is also used by RVLM neurons to tonically excite sympathetic preganglionic neurons in the spinal cord. No one knows however if the RVLM neurons that control different sympathetic outputs contain different populations of VGLUTs (only because it hasn't been done to my knowledge). ~PJM

Blood pressure regulation: every adaptation is an integration?

Blood pressure regulation: every adaptation is an integration? Michael J. Joyner • Jacqueline K. Limberg. Eur J Appl Physiol 2013 Apr 5. [Epub ahead of print] DOI 10.1007/s00421-013-2636-5. This is the first in a series of review articles examining blood pressure regulation from a number of different viewpoints. This one in particular explores how we view "normal" blood pressure and the misconception that it is always supposed to 120/80. In other words, "normal" blood pressure varies throughout the day, under a variety of conditions, both physiological and pathophysiological. A number of helpful examples are used including acute exercise, resumption of normal upright posture, and sleep. The other interesting question addressed in this review is whether it is blood pressure per se that is the regulated variable. Rather, the authors ask the reader to consider the possibility that it is not blood pressure that is regulated but delivery of oxygen. The classic example is when we sleep, our heart rate and brain activity lowers relative to the awake state and thus oxygen demand is lowered. Blood pressure also goes down because the heart and brain don't require as much oxygen delivery. So in this case it's not blood pressure that's being maintained it's oxygen delivery. I think this a really good article to read to keep us open-minded about the concepts we take for granted. ~PJM

Physical inactivity is a disease synonymous for a non-permissive brain disorder

Physical inactivity is a disease synonymous for a non-permissive brain disorder. Leo Pruimboom. Medical Hypotheses 77 (2011) 708–713. This another interesting paper on inactivity and along with agreeing that physical inactivity is a major cause of chronic disease, it puts a new twist on this idea. The premise is that people already know that exercise is good for them but many choose not to lead an active lifestyle. Therefore physical inactivity belongs in a category called "sickness behavior" which includes depressive, facilities energy conservation but seems to project against injury and social conflict. If progressive then self-defeating copying styles and a conversion to non-permissive behaviors. They also propose that inactivity produces a "reptile phenotype" that includes a low metabolic rate and hypothermia. -PJM

Lack of Exercise Is a Major Cause of Chronic Diseases

Lack of Exercise Is a Major Cause of Chronic Diseases by Frank W. Booth, Christian K. Roberts, and Matthew J. Laye. Compr Physiol 2:1143-1211, 2012. This is a major review of the evidence for inactivity and its relationship to chronic disease by Frank Booth from the University of Missouri. He and his colleagues have painstakenly gone through a lot of literature to produce this 70 page review with over 575 references. The review is broken into three parts major parts each with subsections related to the detrimental effects of inactivity versus the beneficial effects of exercise. Some of the more interesting sections include how mechanisms of physical activity may different from mechanisms of inactivity. Additionally, physical activity is examined as a primary prevention against 35 chronic conditions. Lastly the article ends with some additional interesting and timely topics including clinical consequences of inactivity in childhood/adolescence and public policy issues. Although a fairly extensive review it could be one worth looking at as a dissertation reference, for example. There are also several figures that could be used in seminars and/or informal presentations. -PJM

The ventrolateral medulla and sympathetic regulation of arterial pressure.

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

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

Posted by Pat

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

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?

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