A novel functional neuron group for respiratory rhythm generation in the ventral medulla.

Onimaru H, Homma I. J Neurosci. 2003 Feb 15;23(4):1478-86. This paper looked at respiratory-related neurons in the RVLM. What they wanted to know was how the neuronal activity was related to the different phases of respiratory activity. To examine this, they used a voltage sensitive dye in order to visually observe increases in activity, and compared it to the phases of respiration. By using the increase in activity in the motor inspiratory nerve as a trigger, they found that they were able to "see" action potentials occurring approximately 500ms before inspiration occurred. The voltage-induced signal increased until just before inspiration (rat brainstem spinal cord preparation), and decreased after that. It seems that the signal was initiated in the region of the RVLM just ventrolateral to the facial nucleus and near the ventral surface. After that, the increase in dye signal spread caudal and medial to the point of initiation. The other paper I blogged this week studied cells that were modulated by respiratory activity, but this one looks at cells that seem to generate respiratory activity. I can't guarantee it right now, but I'm pretty sure that if I were to look at my recordings, I've seen some cells that have their bursts cut short by the ventilator, and others that burst at other phases. I don't know how physiologically relevant those are, since being on a ventilator is not exactly natural, but it does reinforce that the RVLM is pretty heterogeneous and we should keep that in mind when we plan experiments and look at our data. -DH

Electrophysiological properties of rostral ventrolateral medulla presympathetic neurons modulated by the respiratory network in rats.

Moraes DJ, da Silva MP, Bonagamba LG, Mecawi AS, Zoccal DB, Antunes-Rodrigues J, Varanda WA, Machado BH. J Neurosci. 2013 Dec 4;33(49):19223-37 In this paper, they looked at presympathetic cells and how some of them are modulated by respiratory activity. They started by looking at rats that had been exposed to chronic intermittent hpoxia (CIH) and found that CIH caused increases in arterial pressure, abdominal electromyography, and abdominal and thoracic nerve activity. They next used whole cell blind patch clamp in the working rat heart-brainstem preparation. They identified presympathetic neurons by position, inhibiinhibitory response to baroreflex, and antidromic action potentials caused by electrical stimuli at T8-12. What they found was that presympathetic neurons were "noisy," which means that that, in addition to spontaneous APs, there were many transient changes in membrane potential that were related to respiration, seemingly due to EPSPs. They identified 4 different groups of presympathetic neurons - (1) Neurons with very regular discharge that were not modulated by respiratory activity, (2) irregular neurons with inspiratory modulation, (3) irregular neurons with post-inspiratory modulation, and (4) regular with inspiratory inhibition. Their results were pretty interesting in that all the C1 neurons they identified (by post-test immuno and single cell qRT-PCR using the cytoplasm which was aspirated after patch recording) fell in to groups 1 and 2, but were NOT different between normal and CIH groups. Group 4 consisted of non-C1 neurons that did not differ between groups. Group 3, however, consisted of non-C1 neurons that WERE different between groups. Apparently, CIH causes some non-c1 presympathetic neurons to fire faster in a way that is NOT due to any changes in their own membrane properties (spikes per current injection, input resistance), but rather through changes in their input (Dare we say that it might be due to changes in their sensitivity to excitatory input?) Anyway, they found evidence that some of the changes may be due to differences in pacemaker current activity, which seemed to be due to INaP, which is a persistent inactivating sodium current. This TTX-sensitive current is known to generate subthreshold oscillations in membrane potential, which can change neuronal excitability. So I guess the moral of this story is that if you can't exercise, make sure you get plenty of oxygen all the time, or else you're in serious trouble. -DH

Increasing Angiotensin (1-7) Levels in the Brain Attenuates Metabolic Syndrome related risks in Fructose-Fed Rats

Priscila S. Guimaraes, Mariana F. Oliveira, Janaína F. Braga, Ana Paula Nadu, Ann Schreihofer, Robson A.S. Santos and Maria Jose Campagnole-Santos Hypertension. published online February 10, 2014; In this study they investigated fructose fed diet and the role of angiotensin (1-7) in the brain. In this study they had a group that received intracerebroventricular infusion of Ang (1-7) FF+A7, and a fructose fed group that received an introcerebroventricular infusion of 0.9% saline (FF). Both groups received the infusions for 4 weeks. They looked at the baroreceptor reflex responses for arterial pressure, cardiac sympathethetic/ parasymthetic tone, and heart rate. They found that in the FF+A7 group that the arterial pressure and cardiac sympathetic tone are significantly reduced compared to the FF group. They also measured metabolic parameters and found that Glucose, insulin, and insulin resistance is significantly reduced in FF+A7 compared to FF. they also found that Leptin, HDL and triglycerides were significantly elevated compared to control group. They also measured glycogen in the liver and found that it was significantly higher in the FF+A7 compared to the FF. however the glycogen stores in the muscle was significantly lower in the FF+A7 compared to the FF. Blood glucose was lower in the FF+A7 compared to the FF. They also looked the expression of the Mas receptor, nNOS and NR1/NMDAr in the hypothalamus and dorsomedial medulla and they found that the mRNA expression of NR1 and nNOS was significantly reduced in the FF+A7 compared to FF in both the hypothalamus and the dorsomedial medulla. These findings suggest that Ang (1-7) may improve metabolic parameters along with blood pressure and cardiovascular responses to fructose diet induced changes in the body.-MD

Stimulation of a2-adrenergic receptors in nucleus tractus solitarius is required for the baroreceptor reflex

Alan F. Sved, Kazuyoshi Tsukamoto and Ann M. Schreihofer Brain Research, 576 (1992) 297-303 This study was investigating the role of α2 adrenergic receptors in the NTS, specifically if they are involved in baroreceptor reflex. In rats they gave yohimbine and idazoxan (both are αs adrenergic receptor blockers) in a dose dependent matter into the NTS. They both caused an increase in arterial pressure. Stimulation of the aortic depressor nerve (ADN) resulted in decreases in arterial pressure and HR. They next gave the doses of yohimbine and idazoxan that caused the maximum response in arterial pressure blocked the decrease in arterial and heart rate when the ADN stimulated. The results suggests that α2 adrenergic receptors are important for mediating the baroreceptor reflex response in the NTS.-MD

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

Effects of voluntary exercise on synaptic plasticity and gene expression in the dentate gyrus of adult male Sprague-Dawley rats in vivo.

Neuroscience. 2004;124(1):71-9. Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH, Christie BR. “Voluntary exercise produces enhanced neurogenesis and long-term potentiation (LTP) in the dentate gyrus (DG) of mice in vitro”. In the present study, the authors investigated the mechanisms by which voluntary activity increased neurogenesis. First they confirmed that voluntary exercise increased neurogenesis and LTP in the DG of rats in vivo. Second they showed LTP could be easily achieved in rats performing voluntary exercise and finally they suggested that the functional changes might be mediated through increased expression of BDNF, NR2B and GLUR5 mRNA at cellular level in voluntary running animals.-Madhan

Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.

Nat Neurosci. 1999 Mar;2(3):266-70. van Praag H, Kempermann G, Gage FH. “Exposure to an enriched environment increases neurogenesis in the dentate gyrus of adult rodents”. In this study the authors exposed the animals to various factors such as enriched environment, hidden-platform water maze learning, forced exercise, voluntary exercise and control housing conditions and tested their effects on adult hippocampal cell proliferation and neurogenesis. The authors injected BrdU intraperitoneally to study the proliferation of living hippocampal neurons. They found that mice that were allowed to run voluntarily had increased cell proliferation and number of newborn cells in the dendate gyrus was approximately doubled under voluntary activity and enrichment. On the other hand, mice that were trained in water maze or swim control does not show neurogenesis in adult hippocampal neurons. This study suggests that voluntary running enhances cell proliferation, survival and differentiation in adult hippocampal neurons.-Madhan

Manganese-enhanced magnetic resonance imaging (MEMRI): methodological and practical considerations

Silva, Afonso C., et al. "Manganese‐enhanced magnetic resonance imaging (MEMRI): methodological and practical considerations." NMR in Biomedicine 17.8 (2004): 532-543. The development of manganese-enhanced magnetic resonance imaging (MeMRI) has led to three different functional applications including systemic MnCl2 accumulation for anatomical mapping, activity-induced manganese-enhanced MRI (AIM-MRI), and manganese-enhancement neuronal tract tracing. The third of these techniques, MeMRI neuronal tract tracing, is particularly useful for visualizing neural circuits in vivo. In recent years it has been shown that manganese is not only transported throughout a neuron, but can also cross synapses as well (Pautler et al.). In Pautler’s experiment 2.4 M MnCl2 solutions were injected into the nose and eyes of mice and imaged 48 hours. Significant traces of manganese were then found in the olfactory bulb as well as the olfactory cortex, indicating the manganese is able to go trans-synaptic. It has also been proven that acute injections into the brain can produce significant visualization of neural connections. So far small doses of manganese chloride (10-1000nl of 5mM-.8M) have been injected into the brains of birds, monkeys, and mice. As promising as these results seem for future neuronal tract tracing techniques, it has also been shown that manganese does not cross synapses along other pathways, such as the visual circuit. Unfortunately, these results suggest that manganese behaves differently amongst different neural pathways and needs to be further examined. It is promising however, that with direct applications of manganese, much smaller concentrations can be used to produce significant results. The smaller the amount of manganese needed, the more likely that MeMRI can be used as a diagnostic tool in humans. ~JI

Voltage-dependent calcium currents in bulbospinal neurons of neonatal rat rostral ventrolateral medulla: modulation by alpha2-adrenergic receptors.

Li YW, Guyenet PG, Bayliss DA. J Neurophysiol. 1998 Feb;79(2):583-94 I picked this paper because we're currently using manganese to probe for activity-dependent MRI contrast enhancment in the RVLM. Manganese is known to enter neurons during action potential, primarily through L-type voltage-gated calcium channels. I wanted to know a little more about the presence of these channels on the neurons we study, and this paper has a breakdown of of different calcium channels' relative contributions to the overall calcium currents. Neonatal rats were given retrograde tracers in the spinal cord in order to label the bulbospinal neurons of the RVLM. 3-5 days later, 150um slices of their brainstems were taken and labeled cells were recorded by patch-clamp. Calcium currents were recorded under voltage clamp, and the different types of channels were drugged out in order to identify channel type contributions. What they found was that N-type channels made up 38%, P/Q-type made up 30%, and our favorite L-type only made up 8% So the big question I'm left with, is that 8% enough for us to see? I guess I should have faith in the fact that 150um slices may not have quite as many calcium channels as whole cells in vivo. -DH

Evaluation of manganese uptake and toxicity in mouse brain during continuous MnCl2 administration using osmotic pumps.

Contrast Media Mol Imaging. 2012 Jul-Aug;7(4):426-34. doi: 10.1002/cmmi.1469. Sepúlveda MR, Dresselaers T, Vangheluwe P, Everaerts W, Himmelreich U, Mata AM, Wuytack F. Following up on my post from last week, where a group compared injections of manganese with slow infusion via osmotic minipump, this paper is from a group that looked at the levels of manganese uptake between different brain regions following implantation of a minipump. Here, they implanted the pumps subcutaneously under the back skin of mice, to deliver 30mg/kg/day,and looked at T1 relaxation times (signal increase) after 3 weeks and found significant reductions in a number of regions. They also implanted the pumps for 12 days, before removing them and looking for changes in signal before and after removal, finding that some recovery can be seen in as little as 3 days. Interestingly, they found that tissues high in the SPCA1 Mn/Ca ATP-ase seemed to be the same areas which had higher Mn accumulation, suggesting that this pump may be responsible for sequestering Mn in the golgi apparatus. A couple of drawbacks that they saw after using minipumps were that mice treated for 3 weeks showed small but significant changes in their step sizes, as well as behavioral changes which suggested neurological damage may have occurred. -DH

Nucleus tractus solitarius and control of blood pressure in chronic sinoaortic denervated rats

ANN M. SCHREIHOFER AND ALAN F. SVED Department of Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Am J Physiol. 1992 Aug;263(2 Pt 2):R258-66. This study was done in order to determine whether the NTS was still driving changes in AP following chronic sinoaortic denervation (SAD). In this study they first they gave mus or lidocaine bilaterally in the NTS of either control or chronic SAD rats. They looked at AP in response to the drugs given into the NTS. They found that AP went up in response to mus and lidocaine into the NTS in the control rats however in the chronic SAD rats there was no change in AP to the same drugs in the NTS. Next they looked at bilateral NTS lesion and whether this would lead to changes in AP. So they lesioned the NTS in control and chronic SAD rats under anesthesia and then allowed the rats to wake and recovery for 1hr and then they recorded AP. They found that the AP was elevated in the control but the chronic SAD rats maintain a similar AP as before the NTS was lesioned. These data demonstrate that the NTS is not proving any control over AP in chronic SAD rats.-MD

Glutamatergic inputs to the CVLM independent of the NTS promote tonic inhibition of sympathetic vasomotor tone in rats

Daniel A. Mandel , Ann M. Schreihofer American Journal of Physiology - Heart and Circulatory PhysiologyPublished 1 October 2008Vol. 295no. H1772-H1779DOI: 10.1152/ajpheart.216.2008 In this study they wanted to determine if the NTS was providing baro independent glutamatergic input to CVLM. They also wanted to investigate whether glutamate was driving baroreceptor into to CVLM. Finally they wanted to look at the role respiratory neurons in controlling CVLM neuronal activity. In male S-D rats, they recorded sSNA and AP. They also exposed the brainstem in order to microinjections into NTS and CVLM. So they infused SNP in order to lower AP below baroreceptor reflex threshold. Then they bilaterally blocked the NTS with muscimol. They found that AP did not change however they gave mus into CVLM following NTS blockade and found that they got an increase in AP and sSNA. Next they gave only mus bilaterally into NTS and then they gave either mus or kyn into CVLM. They found that after NTS blockade there was an increase in AP and sSNA and when the CVLM activity was blocked by either giving mus or kyn they saw an increase that was significantly greater than NTS blockade alone. Finally they looked how central respiratory drive would affect CVLM neuronal activity. They found that in the presence of SNP and kyn into the CVLM sSNA was significantly enhanced in response to elevated pCO2. The conclusion based on the data from this article suggests that glutamate is playing a major role in driving the activity of CVLM and that it is not only coming from NTS. Also that NTS glutamergic input to CVLM is being driven by the baroreceptor reflex. Finally the activity of CVLM is also being modulated by central respiratory areas. -MD

Glutamatergic receptor activation in the rostral ventrolateral medulla mediates the sympathoexcitatory response to hyperinsulinemia.

Hypertension. 2010 Feb;55(2):284-90. Bardgett ME, McCarthy JJ, Stocker SD. “Hyperinsulinemia increases sympathetic nerve activity (SNA) and has been linked to cardiovascular morbidity in obesity. The rostral ventrolateral medulla (RVLM) plays a key role in the regulation of SNA and arterial blood pressure (ABP)”. The authors tested the hypothesis whether glutamatergic receptor activation in the RVLM mediate sympathoexcitatory response to hyperinsulinemia. Microinjection of kynurenic acid, a glutamate receptor antagonist, into the RVLM significantly reduced lumbar sympathetic nerve activity during hyperinsulinemic euglycemic clamps in rats. On the other hand, insulin microinjection directly into the RVLM did not alter lumbar sympathetic nerve activity. Taken together these findings provided insight into the potential neural pathways and signaling mechanisms involved in insulin mediated increases in central sympathetic outflow and suggest that an excitatory glutamatergic pathway to the brainstem may be involved in insulin stimulated sympatho-excitation.- Madhan

Expression in Escherichia coli of chemiacally synthesized genes for human insulin

Goeddel, David V., et al. "Expression in Escherichia coli of chemically synthesized genes for human insulin." Proceedings of the National Academy of Sciences 76.1 (1979): 106-110. I actually had to read this paper for my lab report that I am doing this week so it does not have to do with RVLM, but still fun to know. This was actually one of the first studies to utilize protein recombination, and they did it in order to synthesize human insulin. At the time insulin was being harvested from animals at slow rates with some complication. Protein recombination allowed for cheap, fast, and large amounts of insulin production. In order to create the two insulin chains, a pIB1 plasmid and pIA1 plasmid were transformed into E. K-12 coli strain 294. After growth of the transformed bacteria, each colony was induced with IPTG for maximal expression of the insulin protein. The bacteria was then lysed and the mixture was S-sulfunated for the purification of the insulin chains. After purification a radioimmun assay and DNA sequencing was done for verification of the plasmid products. It was shown that human insulin had indeed been synthesized and was ready for wide spread use. ~JI

Excess dietary salt alters angiotensinergic regulation of neurons in the rostral ventrolateral medulla.

Hypertension. 2008 Nov;52(5):932-7. Adams JM, McCarthy JJ, Stocker SD. “Excess dietary salt intake contributes to or exacerbates some forms of hypertension by increasing sympathetic nerve activity (SNA) and arterial blood pressure (ABP) through angiotensin II (Ang II) type 1 receptor activation in the rostral ventrolateral medulla (RVLM)”. The authors tested the hypothesis whether dietary salt alone alters angiotensinergic regulation of neurosn in the RVLM. Microinjection of Ang II into the RVLM significantly increased renal sympathetic nerve activity and mean arterial blood pressure in rats that we allowed to drink water with 0.9% NaCl for 14 days. Blockade of Ang II type 1 receptors in the RVLM significantly reduced renal, splanchnic SNA and mean arterial blood pressure suggesting that neurons in the RVLM that regulate angiotensinergic mechanisms are altered by excess dietary salt.- Madhan

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

Monosynaptic excitatory connection from the rostral ventrolateral medulla to sympathetic preganglionic neurons revealed by simultaneous recordings.

Oshima N, Kumagai H, Onimaru H, Kawai A, Pilowsky PM, Iigaya K, Takimoto C, Hayashi K, Saruta T, Itoh H. Hypertens Res. 2008 Jul;31(7):1445-54 Before this paper, it was known that the rostral ventrolateral medulla (RVLM) projected to the intermediolateral cell column (IML), and it was known that changes in RVLM activity also corresponded to changes in the activity of sympathetic preganglionic neurons (SPNs) and those corresponded with postganglionic effects. Apparently nobody had been able to demonstrate that it was actually the RVLM neurons that drove the activation of SPNs through monosynaptic connections. It seems like a simple thing that should have been done early on, but after reading the paper, I can see how technically challenging it was to show that the two regions are functionally connected. To show how the RVLM drives the sympathetic nervous system, they combined extracellular recording in the RVLM with whole cell recording of SPNs in the IML at the level of the second thoracic vertebra in a brainstem-spinal cord preparation. They verified SPNs by stimulating afferent preganglionic fiber bundles and observing induced antidromic action potentials. They experiment involved stimulating presympathetic neurons in the RVLM and also their afferent fibers in the cervical region and observing excitatory postsynaptic potentials (EPSPs) and action potentials. Their results found 3 types of SPNs, one of which did not seem to respond to stimuli in the RVLM (which is interesting in that some area other than the RVLM must be driving them). They also found that application of antiotensin II (Ang II) to only the brainstem caused an increase in firing rate among PSNs. Through averaging 200 sweeps of paired neurons, they found that Ang II-induced spikes in the RVLM resulted in EPSPs in the SPNs with a latency similar to the known values for conduction velocity. So there you have it, the RVLM really does drive the activity of the sympathetic nervous system, and this paper shows that it is through monosynaptic connections to SPNs. -DH

Mapping of functional brain activity in freely behaving rats during voluntary running using manganese-enhanced MRI: implication for longitudinal studies.

Eschenko O, Canals S, Simanova I, Beyerlein M, Murayama Y, Logothetis NK. Neuroimage. 2010 Feb 1;49(3):2544-55 I picked this paper because we’ve been running in to some confusion with our MeMRI studies. Of late, we’ve been taking a step back and trying to answer some basic questions about the technique and how to apply it to our area. This paper is interesting because it answers a question or two we were wondering about and we might be able to work in to our future studies. In this paper the authors wanted to look at different routes of manganese (Mn) administration and see how they affected the physical activity of rats. Why is this important? Because if you’re studying something like exercise, you probably want to do things in a way that won’t stop the animals from exercising. It turns out that both subcutaneous and intraperitoneal injections of Mn, which cause observable lethargy in rats, also cause significant dose-related decreases in spontaneous wheel-running. Since Mn is taken up over the course of hours dependent on physical activity, decreases in physical activity were seen to significantly limit activity-dependent Mn uptake. They looked at an alternative route of administration, slow infusion over days by using an osmotic minipump. They found that this method did not decrease physical activity or food/water intake, but still allowed for enhanced physical activity-dependent contrast after MRI imaging. -DH

In vivo detection of neuroarchitecture in the rodent brain using manganese-enhanced MRI

Aoki, Ichio, et al. "In vivo detection of neuroarchitecture in the rodent brain using manganese-enhanced MRI." Neuroimage 22.3 (2004): 1046-1059. Manganese-enhanced magnetic resonance imaging has been developed for multiple uses over the past couple decades including functional MRI and neuronal tract tracing. In this paper Aoki's laboratory examined how MeMRI could be used to visualize cytoarchitecture in the brain in order to further understand brain structures. Several brain regions in particular were reviewed including the olfactory bulb, cortex, ventricles, hippocampus, brainstem, and cerebellum. Manganese chloride was given intravenously at a rate of 1.8 ml/hr with a final concentration of 884.3 micromols/kg. Rats were broken into four groups and imaged at time points 0-2hrs, 1 day, 4 days, and 14 days post manganese chloride administration. After the analysis, most brain regions of interest had peak signal intensity 24 hours post injection such as the hippocampus, coritcal areas, subcortical areas, cerebellum, and the brainstem. However some brain regions closely associated with the ventricles showed peak manganese accumulation after only 2 hours. These brain regions include the ventricles themselves, the pituitary, pineal gland, and the medium eminence. More importantly, this paper was one of the first to distinguish between different sub-layers of certain brain regions. For example three separate molecular cell layers were distinguishable within the cerebellum. The same goes for the olfactory bulb where six distinguished layers were found with the MRI signal. These findings are important for future longitudinal studies focusing on architecture under different conditions such as development or drug abuse. ~JI

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

Domitila A. Huber and Ann M. Schreihofer May 1, 2010 The Journal of Physiology, 588, 1515-1525. doi: 10.1113/jphysiol.2009.186 In previous study they showed that obese zucker rats have impaired baroreflexes. This study was done in order to determine whether this impairment is due to altered afferent input to the CNS. They recorded aortic depressor nerve activity (ADNA) along with arterial pressure (AP) and splanchnic sympathetic nerve activity (sSNA) in obese and lean zucker rats. They basically recorded ADN responses along with AP and sSNA in response to phenylephrine. They looked at baseline sSNA but for the PE challenge they gave a ganglionic blocker to prevent any changes in SNA in response to AP changes. So they found that the change in AP and ADNA in both groups were similar. Suggesting that ADNA in response changes in systemic AP is not altered in obese zucker rats. However, in another set of experiments, when they stimulated the ADN and looked at AP and sSNA responses, they found that the obese zucker rats had attenuated sSNA and AP to different AND stimulation frequencies. However when changes in AP where clamped, they still saw attenuation in sSNA response in the obese zucker rat compared to the lean zucker rat. However there was no difference in AP between the two groups. These data suggest that the impairment of the baroreceptor reflex in obese zucker rats is due to altered signaling in the CNS pathway.-MD

Impairment of sympathetic baroreceptor reflexes in obese Zucker rats

Ann M. Schreihofer, Daniel A. Mandel , Susan C. Mobley , David W. Stepp American Journal of Physiology - Heart and Circulatory Physiology Published 1 October 2007 Vol. 293 no. H2543-H2549 DOI: 10.1152/ajpheart.01201.2006 In this article they looked at baroreceptor reflexes in obese and lean zucker rats that were 7 or 14 weeks old. They instrumented the animals so they could record arterial pressure (AP), splanchnic nerve activity (sSNA) and they also looked at heart rate (HR) changes. In the 7 week old rats they found that there was no difference between the obese and lean zucker rats in responses to phenylephrine and nitroprusside. However when they did sigmoidal analysis of the PE and SNP responses, they found that the baroreflex gain was significantly blunted. As for the 14 week old rats the obese zucker rats had blunted responses to nitroprusside and phenylephrine challenges when compared to the lean zucker rats. The also showed that the baseline weight and AP was significantly elevated in the 14 week old obese zucker rats when compared the lean zucker rats. As for the 7 week old rats the obese rats were significantly heavier compared to the lean rats. These data suggest that impaired baroreflex compensation occurs in adult rats after they become obese not in juvenile rats.-MD

Exercise-induced activation of NMDA receptor promotes motor unit development and survival in a type 2 spinal muscular atrophy model mouse.

J Neurosci. 2008 Jan 23;28(4):953-62. Biondi O, Grondard C, Lécolle S, Deforges S, Pariset C, Lopes P, Cifuentes-Diaz C, Li H, della Gaspera B, Chanoine C, Charbonnier F. “Spinal muscular atrophy (SMA) is an inborn neuromuscular disorder caused by low levels of survival motor neuron protein, and for which no efficient therapy exists”. The authors have previously shown that running enhanced motor neuron function and increased life span in type 2 SMA-like mice. In the present study, the authors investigated whether there is a direct relationship between maturation state of a motor neurons and resistance to neuronal cell death. Further they report the signaling pathway by which exercise provides neuroprotection in SMA-like mice. They report their findings after performing a series of experiments in a knock-out transgenic SMA-like mice. The authors report that exercise leads to a delay in motor-neuron death, which is independent of the rate of postnatal motor-unit maturation. In the spinal cord motor neurons of neonatal SMA-like mice the authors observed a defective expression of NR2A subunit. In addition, the expression of NR2A subunit is enhanced in the trained SMA-like mice suggesting that this could contribute to NMDA-receptor activation in type 2 SMA-like mice. Further, the authors inhibited NMDA-receptor activity to show that exercise-induced benefits in the trained type 2 SMA-like mice were suppressed. The authors conclude that restoring the function of NMDA receptor could be a potential treatment for SMA.- Madhan

In vivo NMDA receptor activation accelerates motor unit maturation, protects spinal motor neurons, and enhances SMN2 gene expression in severe spinal muscular atrophy mice.

J Neurosci. 2010 Aug 25;30(34):11288-99. Biondi O, Branchu J, Sanchez G, Lancelin C, Deforges S, Lopes P, Pariset C, Lécolle S, Côté J, Chanoine C, Charbonnier F. “Spinal muscular atrophy (SMA), a lethal neurodegenerative disease that occurs in childhood, is caused by the misexpression of the survival of motor neuron (SMN) protein in motor neurons”. In the present study, the authors evaluated whether activation of NMDA receptor in a type 2 SMA mouse affects SMN expression and motor neuron survival in SMA spinal cord. The authors reported the level at which NMDA receptor gets activated could provide beneficial or detrimental effects. At adequate level it could accelerate motor unit maturation, prevention of the spinal motor neurons from apoptosis and modification of SMN2 expression. At higher levels of activation, it could provide opposite effects. Further activation of NMDA receptor lead to reactivation of downstream signaling pathway enhancing SMN expression suggesting that activation of NMDA receptor at adequate levels could be used in enhancing SMN expression and reducing motor neuron death in SMA spinal cord. - Madhan

In vivo tract tracing using manganese-enhanced magnetic resonance imaging

In vivo tract tracing using manganese-enhanced magnetic resonance imaging By: R.G Pautler et. al, A. P. Koretsky This was one of the first studies validating the use of manganese-enhanced magnetic resonance imaging (MeMRI) as a tract tracer. In order for an agent to function as a tract tracer it must have the ability to cross from one neuronal synapse to another, and it must also be detectable while in the cell. A tract tracer can be defined as a retrograde tracer, anterograde tracer, or both. Prior to manganese, other tracing agents utilized included biotinylated dextran, horseradish peroxidase, fluorogold, and HSV. However, all of these agents must be in fixed tissue in order to be detected, making the techniques fatal to the subjects. Pautler and Koretsky were able to prove manganese was not only a tract tracing agent, but could be utilized in vivo after they successfully defined the olfactory and visual pathways in conscious rats using detectable MRI. This discovery, along with the established characteristic of manganese to accumulate in neurons in an activity dependent manner, makes MeMRI a powerful and feasible technique for neurological studies, structural as well as functional. ~A very tired and forgetful JI.

Noninvasive and simultaneous imaging of layer-specific retinal functional adaptation by manganese-enhanced MRI

Berkowitz, Bruce A., et al. "Noninvasive and simultaneous imaging of layer-specific retinal functional adaptation by manganese-enhanced MRI."Investigative ophthalmology & visual science 47.6 (2006): 2668-2674. In order t measure the cellular demand for ions in light/dark adaptation environments in both the inner and outer layers of the retina Dr. Berkowitz utilized functional manganese-enhanced MRI. Although his studies were focused in the eye, his toxicity studies still give us some insight on manganese affects in vivo. It was after extensive studies examining blood retinal barrier integrity and comparative histology that it was established single I.P doses of manganese at 44mg/kg produced no toxic effects. Specifically, the rats were injected with manganese chloride and then examined one month later for ocular health finding no significant differences between control and manganese injected rats histology slices, blood retinal function, and retinal thickness. After toxicity studies, Dr Berkowitz was also able to show as expected retinal layer differences in ion utilization under both light and dark adaptations. ~JI

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

Regional Specificity of Manganese accumulation and clearance in the mouse brain: implications for manganese-enhanced MRI

Grünecker, B., et al. "Regional specificity of manganese accumulation and clearance in the mouse brain: implications for manganese‐enhanced MRI." NMR in Biomedicine (2012). Introduction: Similar to what we are trying to do now, this study was done mostly to establish MeMRI as a technique that the authors will now be able to use in order to study the hippocampus. Although the hippocampus is not the brain region we are interested, this study did look at multiple brain regions with the intent of better understanding manganese accumulation, clearance, structural affects, and degradation to the brain. They found similar results to what we are finding in that manganese accumulation and clearance rates can vary from brain region to brain region. These factors are important for multiple reasons when trying to plan out an experimental design in a new brain region, such as the RVLM. Design: • For the accumulation study 90 rats were separated into nine groups. One group was the control group and the rest of the groups received 1 dose of manganese and each group consecutively received an additional dose of manganese, so that group 8 had received 8 daily doses of manganese. Each group receiving a daily dose of 30 mg/kg manganese. Each group was then imaged 24 hours after the final injection. • For the clearance study 50 rats were broken up into five groups. Group one was imaged one week following injections, Group two was imaged two weeks following injections, Group three was imaged four weeks following injections, Group four was imaged eight weeks following injections, and Group five was imaged twelve weeks following injections. • Forty animals were then used to test hippocampus-dependent learning before and after injections of manganese. Animals were broken up into a control group and manganese group, and were tested for contextual and conditioned fear before and after injections. Conclusion: • Based off a logarithmic analysis this study found that manganese accumulation positively correlated with the number of dose injections given. This result is not surprising, however what was surprising was that between day 7 and day 8 injections a significant difference was seen in signal intensities. Meaning that even after 8 days of injections totaling a dose of 240mg/kg saturation of the hippocampus was not seen. Also important, it was seen that different brain regions accumulate manganese at different rates. There seemed to be a correlation between signal intensity and relative location to the brain ventricles. The brainstem was found to have a much slower rate of uptake compared to other regions such as the cerebral cortex. • Efflux rates were found to correlate well with accumulation rates, in that brain regions with faster uptake had faster efflux rates and brain regions with slower uptake had slower efflux rates. Unlike previous studies, the half life of manganese was found to be much shorter in this study. In the brain stem in particular the average half life was seen to be about 7 days. However, with that said, the signal intensities of manganese were not found to drop back to control levels until about 8 weeks post injection. • The learning test performed observing contextual and conditioned fear before and after manganese injections found that there was no significant difference pre versus post injection. This is important because it is evidence that even at doses of 120 mg/kg applied over a four day period the animals were not suffering from any learning disabilities that would insinuate brain damage to the hippocampus. ~JI

Contribution of AMPA/kainate receptors in the rostral ventrolateral medulla to the hypotensive and sympathoinhibitory effects of clonidine.

Wang WZ, Wang LG, Gao L, Wang W. Am J Physiol Regul Integr Comp Physiol. 2007 Sep;293(3):R1232-8 In this paper, they wanted to look at the how clonidine injections (IV or in to the RVLM) have a sympatholytic effect, so they took the simple and obvious course of action - use a glutamate receptor inhibitor in the RVLM to block the inhibitory effect of clonidine. Yes, that was a fun bit of sarcasm because this paper was not easy to understand. They were able to use microinjections of CNQX, an AMPA type glutamate receptor antagonist to block the inhibitory effect of clonidine, which is apparently known to induce GABA release in the RVLM. It seems that the effect of clonidine here is not actually on presympathetic neurons, but possibly on presynaptic GABAergic terminals, which is something that is possible. It's strange, because clonidine is not known to be an AMPA agonist, so I guess that blockade of AMPA receptors on presynaptic gabaergic fibers removes just enough excitation to counteract the effect of clonidine through the receptors it does act on. And of course, it is safe to assume that CNQX microinjections in to the RVLM also blocked the AMPA receptors on presympathetic neurons, but we already know that this can have little to no effect on sympathetic tone (which is what they also were able to show in this study). So it's a little hard to figure out what's going on, but the results speak for themselves. -DH

Synaptic and extrasynaptic transmission of kidney-related neurons in the rostral ventrolateral medulla.

Gao H, Derbenev AV. J Neurophysiol. 2013 Dec;110(11):2637-47 In this paper, they wanted to look at the possibility of tonic excitatory and inhibitory currents in the rvlm that would regulate activity. To do this, they applied pseudorabies with a GFP reporter to the kidney cortex, then approximately 4 days later, obtained brainstem slices containing the RVLM from infected rats. They could then do whole cell recording on fluorescent cells, confident that they were examining kidney-related presympathetic neurons. What they found was that the cells exhibited spontaneous excitatory currents that were sensitive to AP-5 and CNQX, meaning that both NMDA and AMPA channels are spontaneously active. They also saw that glutamate mediates a persistent and tonic current when examining the cells under current-clamp. When they looked at GABA-A-mediated currents, they found that tonic and phasic currents were sensitive to different antagonists, with phasic inhibition being due mostly to gabazine sensitive currents, while tonic inhibition was due mostly to bicuculline-sensitive currents. The interesting part of this paper is that glutamate seems to be causing tonic excitation through ionotropic channels, an effect I haven't really heard of before. They don't know where it comes from or if it's synaptic or extrasynaptic receptors, but they know it's not from presynaptic release of glutamate because TTX (which blocks action potential-induced neurotransmitter release) didn't affect the excitatory currents. They pointed out the interesting possibility that this tonic excitation may be due to glial cell releasing glutamate on to extrasynaptic receptors, which is not what people usually think of when they think of glutamate excitation. -DH

Effect of aging on treadmill exercise induced theta power in the rat

Terry B. J. Kuo & Jia-Yi Li & Sandy Shen-Yu Hsieh & Jin-Jong Chen & Ching-Yao Tsai & Cheryl C. H. Yang AGE (2010) 32:297–308 DOI 10.1007/s11357-010-9143-y This study investigates how age effects treadmill changes in EEG and electromyogram signals. In 8 week and 60 week old Wistar rats they implanted electrodes for the occipital EEG and nuchal EMG. Then the animals were started on a treadmill training protocol. The animals were allowed to run for 30 minutes during the actual experiment. EEG and EMG were recorded 80minutes during dark period and the animals were allowed to run for 30 minutes doing 13m/min which is considered moderate exercise. They analyzed the EEG spectra in order to see if there were any differences. They found that during EEG levels during waking were lower in aged rats compared to younger rats. They also found that during exercise the older rats had less EEG activity compared to the younger rats. The most interesting thing about the EEG data is that the older rats only had an elevation in the theta power however this response was still significantly less than the younger rats. The main conclusion is that aging leads to attenuated activation of brain areas that are activate in younger rats.-MD

An increase in adenosine-5’-triphosphate (ATP) content in rostral ventrolateral medulla is engaged in the high fructose diet-induced hypertension

Authors: Kay LH Wu*, Chun-Ying Hung, Julie YH Chan and Chih-Wei Wu j of biomed sci 2014 jan 27 21(1)8 Doi 10.1186/1423-0127-21-8 The purpose of this article was to determine if fructose was being utilized as an energy source in RVLM and whether fructose lead to enhanced sympathetic system activity. This study is very interest because they did both in vitro and in vivo studies along with multiple techniques (western blot, immunofluorescence, Gene silencing and etc.) in order to address their questions. Basically high fructose feed rats had higher systemic arterial pressure control rats. They found that ATP was elevated along with protein expression of ketohexokinase, Glut 2 and Glut . They then wanted to show that blocking ATP production would lead to decreases sympathetic activation. They found that infusion of an ATP synthase inhibitor (oligomyocin) they had blunted pressor responses in the high fructose group, as for the normal diet rats they found pressor responses were only slightly blunted. They then did a series of experiments in cell culture. They used the N2a cells and found that in response to 72hr exposure to fructose the cells had higher ATP, and dopamine content. Also there was higher pyruvate content, glut 2, glut 5 and ketohexokinase expression. They were able to show using gene silencing that if ketohexokinase is inhibited that ATP and pyruvate and dopamine release is decreased compared to control. Conclusion: They showed that RVLM neurons are capable of metabolizing fructose and using for energy. The ATP generated from fructose breakdown leads to increase in blood pressure. Therefore, high fructose diet leads to increased sympathetic activation due to increased ATP production in the RVLM. -MD J Biomed Sci. 2014 Jan 27;21(1):8. doi: 10.1186/1423-0127-21-8. J Biomed Sci. 2014 Jan 27;21(1):8. doi: 10.1186/1423-0127-21-8.

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