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

Lateralisation of projections from the rostral ventrolateral medulla to sympathetic preganglionic neurons in the rat Elizabeth A. Moon, Ann K. Goodchild, Paul M. Pilowsky Hypertension and Stroke Research Laboratories, Departments of Physiology and Neurosurgery, University of Sydney, Block 3 Ground Floor, Royal North Shore Hospital, St. Leonards, 2065 Sydney, NSW, Australia This study contains three different techniques: anterograde tract-tracing (using Phaseolus vulgaris leucoagglutinin, PHA-L), retrograde tract-tracing(using CTB) and electrophysiology. Spinally projecting neurons (SPNs) that project to the adrenal gland or to the superior cervical ganglia were labelled retrogradely. The retrograde labeling was paired with anterograde tract-tracing from the RVLM to see if lateralization in the bulbospinal projection to SPN innervating the adrenal gland or superior cervical ganglia could be identified. Unilateral injections of the spinal cord were made with a retrograde tracer in order to determine the amount of lateralization between the upper and lower thoracic spinal cord. The last experiment they did was stimulate the RVLM with glutamate and record from the left cervical sympathetic and left adrenal nerve to test for functional lateralization. They found that the SPNs controlling the SCG were primarily bilateral, meaning that both the ipsilateral and contralateral sides were nearly equally labelled following anterograde labeling. The SPNs controlling the adrenal medulla were almost exclusively labelled on the ipsilateral side, suggesting ipsilateral control. Next in the retrograde tracing studies, they showed that CTB injected unilaterally at T2 and T8 both showed about 66%-75% ipsilateral projections to 25%-33% contralateral projections. The electrophysiological studies showed that following glutamatergic stimulation of RVLM, there are no differences in the ratio of responses to either the cervical sympathetic truck or adrenal nerve, suggesting that while there are differences in anatomical connections, the functional contribution of each RVLM to the respective nerve activity may very well be the same under the given conditions. This paper will provide helpful anatomical knowledge for my presentation where I will present my differential control data. My results corroborate with their anatomy data for the adrenal nerve, demonstrating that under normotensive conditions, that the adrenal nerve responds the primarily ipsilateral to a given injection of glutamate. It would be interesting to do a similar tract tracing study to see if the nature of the ipsilateral/contralateral connections might be altered following physical (in)-activity. -MTL

Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats

Wang, Yang-Kai, et al. "Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats." American Journal of Physiology-Heart and Circulatory Physiology (2014). This article was focused on the role of ACE2 in the RVLM and its effects on hypertension. Overexpression of ACE2 was achieved using microinjections of lentivirus, containing a GFP tag expressed upon transcription, into the RVLM. WKY, SHR, SHR-leniGFP, and SHR-lentiACE2 were observed for 6 weeks and measured for BP, HR, Norepinephrine excretion, and glutamate concentrations within the RVLM. Acute microinjection studies were also done with Kyn to look at responses to glutamatergic inhibition in the RVLM. It was found that three weeks following injections, BP and levels of NE excreation were significantly decreased from baseline in lentivirus-ACE2 positive animals but not in lenti-shams, SHR, or WKYs. It was also observed after acute microinjecitons of Kyn into the RVLM that SHR-ACE2 animals had decreased RSNA activity, and an attenuated decrease in blood pressure compared to the SHR-GFP animals. Lastly, it was discovered that with an upregulation of ACE2/Ang(1-7) there was a corresponding downregulation of the AT1R, NMDA receptor 1 protein, and GluR5/6/7 proteins. Contrarily, there was an upregulation of Mas receptor that Ang(1-7) is known to innervate. Based on this evidence the authors concluded that ACE2 attenuates tonically active glutamatergic inputs to the RVLm in SHR. They discussed possible mechanisms being through the upregulation of Mas interfering with glutamatergic neurotranmission at the level of the pre-synaptic cleft. However, further research is needed in order to characterize the exact mechanism in which ACE2 is attenuating glutamatergic input. Conclusively, this study provides evidence for the possible use of ACE2 as a therapeutic agent for hypertension. ~JI

Tonic glutamate-mediated control of rostral ventrolateral medulla and sympathetic vasomotor tone.

Full cite: Ito S, Sved AF. 1997. Tonic glutamate-mediated control of rostral ventrolateral medulla and sympathetic vasomotor tone. Am J Physiol 273:R487-R494. Department of Neuroscience, University of Pittsburgh, Pennsylvania 15260, USA. This paper was one of the first that demonstrated that EAA is likely driving tonic GABAergic input by some indirect connection. It is either that EAA are driving GABAergic input, or that EAA input is only revealed following the removal of GABAergic inhibition. In this particular paper, they injected muscimol into CVLM to illicit a positive pressor response, similar to how we inject bicuculline into RVLM. In addition to this, they injected kynurenic acid into RVLM to decrease BP. It is important to note that they did not look at SNA, only BP in this paper. It believed that the BP decrease following injection of Kyn is due to the fact that glutamate is playing some kind of excitatory role only after removal of GABA. Something interesting out of this paper is that Sved suggests that the RVLM is feeding back to CVLM via some kind of interneuron. In addition to this, he suggests that the CVLM has some excitatory projections to RVLM and that they are not EAA-related. Something important to note is that they were not able to see any differences in responses due to the anesthetic used in the experiments (urethane v. chloralose). Similar to Nick’s 2012 paper, once the GABAergic input into RVLM is blocked, pressor responses to glutamate are increased, suggesting that GABA is limiting the amount of glutamate excitability under normotensive conditions. This paper is one of (if not the first) that demonstrates that injection of kyn alone has no particular effect on BP, suggesting that kyn under baseline conditions either has no effect, or that there are competing glutamatergic mechanisms that makes it seem like kyn as no effect. This paper is the primary pieces of background for my study looking at tonic glutamatergic/GABAergic input into RVLM and is one of the first that suggests that there may be some kind of interneuron activity in RVLM, whose activity is undetermined. -MTL

Central circuits regulating the sympathetic outflow to lumbar muscles in spinally transected mice by retrograde transsynaptic transport.

Xiang HB, Liu C, Liu TT, Xiong J. Int J Clin Exp Pathol. 2014 May 15;7(6):2987-97. I gave this paper a look because I’d been thinking about differential control of nerves and how the RVLM plays in to this in some species. In this paper, they injected the lumbar epaxial muscles (near L5 vertebrae) of mice with a pseudorabies virus (PRV) with a red fluorescent protein reporter in order to map which regions of the CNS control lumbar sympathetic nerve activity. The mice were also given spinal transections at L2, caudal to where the sympathetic nerve exits so that they could “cut out” any PRV transport through motor neurons. They tracked expression time in different regions and found IML labeling by day 3, and that some animals began to show labeling in RVLM and PVN by day 3 as well, with one animal showing labeling in the raphe pallidus (RPa). By day 4 labeling in those areas was much more widespread, covering those areas as well as the medullary and pontine reticular regions, the A5, the locus coeruleus and the ventromedial hypothalamus. By day 5 and 6, labeling began to show up all over, e.g. in the dorsal brainstem, midbrain, cortex, etc. They also did a count of cells that were labeled with PRV and also expressed TH and TPH. I’m somewhat surprised that they DIDN’T see the most TH-positive neurons in the RVLM (they saw the most in the A5, which makes sense), but they DID see a lot of TPH-positive neurons in the RVLM. So LSNA is controlled by serotonergic presympathetic neurons? I think that for their next trick they should label different nerves with PRV that will report different colors and see what colocalizes – that would be a messy study that would be hard to interpret, but the presence or lack of coexpression would still suggest a lot one way or the other. -DH

The nucleus of the solitary tract and the coordination of respiratory and sympathetic activities.

Zoccal DB, Furuya WI, Bassi M, Colombari DS, Colombari E. Front Physiol. 2014 Jun 25;5:238 I read this review paper because I wanted to learn a little bit more about the inputs to the RVLM, and this paper covered a lot of how the NTS plays a role in regulating sympathetic activity. They talk about how sympathetic nerve activity shows rhythmic activity even after vagotomy and decerebration, presumably because the ventral respiratory column (BotC, pre-BotC, rVRG, and cVRT) either activate or inactivate rhythmicity via the RVLM or CVLM. However, since different nerves (like the phrenic and assorted sympatheticnerves) can have their rhythmicity decoupled by different stimuli, other areas are involved in the pattern generation, like the NTS. The NTS is primarily regulated by a combination of glu and ATP, resulting in changes in SNA. However, NTS microinjection of ACh will increase phrenic nerve activity, but not SNA – suggesting that its control of different groups is regulated by different neurotransmitters. It’s even more complicated in that the NTS has subdivisions that will receive input from some regions but not others, even though some inputs go to multiple subdivisions. To bring it all back to neurogenic hypertension, they did mention that in SHR rats, removal of the carotid chemoreceptor or lesioning the caudal NTS will reduce SNA, suggesting that somehow the chemoreceptor induces the NTS to increase sympathetic tone, maybe through activation of the RVLM? -DH

Rapid swim-stress reduces GABAA receptor α1 subunit mRNAs in the mouse hippocampus

Pascale Montipied, Avraham Weizman, Ronit Weizman, Karin A. Kook A. Leslie Morrow, and Steven M. Paul. Molecular Brain Research, 18 (1993)267-272. They investigated how swim training would affect GABAA receptor subunits mRNA expression in mice. They used a northern blot in order to quantify GABA receptor subunit mRNA expression in the hippocampus. They found that swim stress (or maybe training!!!) for 14 days significantly reduced GABA A receptor α1 subunit expression in the hippocampus. They also looked at GAD expression and β-actin and that was unchanged by 7 and 14 days of swim stress. What I thought was interesting was that the mentioned that adrenectomy that don’t see these changes in GABA A receptor α1 subunit expression. These data suggest that the adrenal glands may be playing a role in modulation of GABAA receptor α1 subunit expression. This could be due to release of adrenal steroids that are can alter GABA A receptor expression indirectly after a long exposure. Based on the finding from this paper, I think the adrenal nerve activity may be key to elucidating the mechanism that leads to changes in glutamate and gaba neurotransmission in the RVLM.-MD

Effects of exercise training on dendritic morphology in the cardiorespiratory and locomotor centers of the mature rat brain

Nelson, Amanda J., et al. "Effects of exercise training on dendritic morphology in the cardiorespiratory and locomotor centers of the mature rat brain." Journal of Applied Physiology 108.6 (2010): 1582-1590. This study was very similar to Nick's structural study in that it examined morphological plasticity of neurons involved with cardiac sympathetic modulation. However, this study looked at respiratory and locomotor brain regions as well. Another distinguishable difference between the two studies was the age at which the animals began exercising. This study was interested in examining neural plasticity in adult rats unrelated to development. To do this they started exercise training the rats only after the animals were 91 days old, and maintained spontaneous free-running for 50 days following initiation. The reason I was interested in this paper particularly was because we are having trouble imaging the smaller rats (~99g), and in order to do a longitudinal study it is imperative we collect reliable data at each time point. I thought this may be helpful because we have not looked at plasticity in rats older than ~18 weeks of age 11-13wks running. These rats were 13 weeks old when the exercise training began and ~20 weeks old when the sholl analysis was done looking at dendritic arborization. Unfortunately, no differences were found between exercising and sedentary animals dendritic branching within the RVLM at 20 weeks of age. Based on these results I would avoid prolonging my longitudinal study looking at neuronal activity plasticity to an older subset of rats if at all possible, which I believe is. They did, however,see structural plasticity in other brain regions at this stage in life, including in the NTS, posterior hypothalamus, periaqueductal gray, and the cuneiform nucleus. In the majority of these brain regions though, they found that the effect size was not as great in older rats as it was in the younger rats. One of the major limitations to this study was that they were not able to differentiate if the decreasing plasticity with age was due to increases in age, or decreases in running because older rats naturally run less than their younger counter parts. I wondered if they looked at a correlation between individual animals running and plasticity because this show evidence for one possible versus the other. ~JI

c-fos identifies GABA-synthesizing barosensitive neurons in caudal ventrolateral medulla

Jane B. Minson, Ida J. Llewellyn-Smith, John P. Chalmers, Paul M. Pilowsky and Leonard F. Arnold. NeuroReport8, 3015-3021(1997). We know that CVLM provides barosensitive and non-barosensitive GABAergic input to RVLM. This study provides anatomical along with functional support for the previous statement. They microinjected CTB into the RVLM of Wistar rats and infused with PE for 60min. Following the infusion of Phenylephrine, the rats were perfused and used for immunohistochemistry. They looked at 3 things in this study 1.) Barosensitive neurons in the CVLM 2.) Examined whether these barosensitive neurons in CVLM project to the RVLM and 3.) Determined if the barosensitive neurons that were GABAergic or glutamatergic. The results showed that PE increased the BP of the rats and lead to increase Fos expression in CVLM and in the NTS but not in the RVLM. Fos expressing and RVLM projecting neurons were identified and located in the more rostral areas of CVLM. Most of these neurons were located rostral to obex. Next the phenotype of the neurons was investigated, in order to determine if the neurons expressed GAD, PAG and TH. There were no TH and GAD neurons identified. About 40% of Fos positive neurons were also GAD positive. Most of these neurons were located in the rostral portion of the CVLM. There was no difference between fos/ PAG and Fos/ TH when PE infused animals were compared to the saline group. The Fos/ PAG/ TH neurons are located in the caudal regions of the CVLM. The most interesting finding in my opinion is that they were able to identify PAG/ CTB and GAD/ CTB neurons in the CVLM. This suggests that CVLM provides both glutamatergic and GABAergic inputs to RVLM. Even though the PAG/CTB neurons were not Fos positive, maybe CVLM provides a tonic level of glutamate to RVLM. Is this driving the activity of spinally projecting RVLM neurons? Also could this glutamatergic drive be altered in SEDs and WRs? These are questions that we could potentially look into. -MD

Patterning of somatosympathetic reflexes reveals nonuniform organization of presympathetic drive from C1 and non-C1 RVLM neurons

Full cite: Burke, P. G. R., Neale, J. K. W. S., and McMullan, S. G. A. K. (2011). Patterning of somatosympathetic reflexes reveals nonuniform organization of presympathetic drive from C1 and non-C1 RVLM neurons. Am. J. Physiol. 301, R1112–R1122. doi: 10.1152/ajpregu.00131.2011 Peter G. R. Burke, Jemima Neale, Willian S. Korim, Simon McMullan, and Ann K. Goodchild Australian School of Advanced Medicine, Macquarie University, Sydney, Australia Submitted 16 March 2011; accepted in final form 20 July 2011 This paper is looking at the makeup/organization of C1 and non-C1 neurons within the RVLM. The method that was used to differentiate between these two subgroups was by recording sciatic nerve responses (SN) from several different afferent inputs. These inputs were activated at graded intensities by utilizing different intensities of SN they were able to excite different nerve fiber afferents specifically: A-fiber (low intensity), and A- and C- fiber (high intensity). In addition to this, they looked at low-intensity SN stimulation, they examined cervical somatosympathetic reflex (SSR) following RVLM microinjection of somatostatin. By using intraspinal injections of anti-dopamine-β-hydroxylase-saporin (anti-DβH-SAP) they were able to selectively lesion C1 neurons. As a result of the study, they determined that use of anti-DβH-SAP abolished the activity of RVLM neurons with slow conduction velocities and sparred RVLM neurons with fast velocities. They concluded that axons projecting from C1 neurons are likely unmylenated, whereas the axons from non-C1 neurons are likely mylenated, allowing for higher conduction velocities. If there was some means by which we could selectively lyse the non-C1 neurons in the RVLM, similar to how anti-DβH-SAP removes C1 neurons, by utilizing the fact that non-C1 neurons are myelinated, then we could prove whether or not non-C1 neurons are maintaining basal sympathetic outflow. In addition to this, I believe that looking at graded action potentials while pairing with immunohistochemistry may be the means by which we will be able to figure out where the excitation of RVLM neurons is coming from. If we are able to either expose some kind of excitatory projection to RVLM neurons that are not glutamatergic via immunohistochemistry, or characterize the neurons electrochemically to another similar known group of neurons, we might be able to determine the tonic excitatory drive. - MTL

The orexinergic neurons receive synaptic input from C1 cells in rats.

Bochorishvili G, Nguyen T, Coates MB, Viar KE, Stornetta RL, Guyenet PG. J Comp Neurol. 2014 Jul 1 In this paper, they wanted to see if there was a C1-mediated activation of hypothalamic orexinergic cells. The idea was that even though C1s can directly activate the sympathetic system, they are also suspected to be able to activate the hypothalamic-pituitary axis to reinforce their effects. To test for C1 hypothalamic connections, they used a prsx8-chr2-mcherry vector (to cause mcherry expression preferentially in C1 neurons) to see innervation of C1 in to orexinergic cells of the hypothalamus. They also confirmed the prsx8 vector by using TH-cre rats and injecting them with a virus carrying a cre-dependent reporter and saw the same thing. They found many unmyelinated PNMT axons in the region throughout the hypothalamus including in the region of the PVN. They found that, of the synaptic terminals they could identify, 77% of them had terminals that synapsed mostly on to dendrites of orexinergic cells. All of these terminals had dense core vesicles AND small clear vesicles. The clear vesicles are evidence that they release glutamate to activate the orexinergic neurons, but dense core vesicles usually contain polypeptides. They didn't discuss what that would mean in this instance… maybe norepinephrine or epinephrine? -DH

Central command dysfunction in rats with heart failure is mediated by brain oxidative stress and normalised by exercise training.

Koba S, Hisatome I, Watanabe T. J Physiol. 2014 Jun 27. The authors wanted to investigate the role of oxidative stress (OS) in the RVLM of rats with chronic heart failure (CHF) because it is believed that RVLM OS in CHF rats potentiates sympathoexcitation, and that this potentiation can be suppressed by exercise training. They then looked at the RNSA in CHF rats after an induced myocardial infarction (MI) and electrical stimulation of the mesencephalic locomotor region (MLR) compared to the same stimulation in controls and sham-MI CHF rats. The stimulation of the MLR was to simulate exercise training done in humans with MI and CHF, because many of these patients wind up with exercise intolerance due to the enhanced SNA. They found that injection of Tempol, which scavenges reactive oxygen species (ROS), in to the RVLM of control and CHF rats had no effect on RNSA following the stimulus, but did reduce the increased RNSA and MAP in MI CHF rats. They also stained the tissue with dihydroethidium to confirm that MI rats generated increased ROS as confirmation that they were actually seeing changes in ROS between groups specifically in the RVLM. What was interesting here was that their experiments were done after neuromuscular blockade, so this probably isn’t due to signals coming from the muscles, but may be due to collaterals coming from locomotor brain regions that project to RVLM or some area controlling it. Also, Mueller 2007 was referenced in this paper for its RVLM microinjection approach, so that’s cool. -DH

Modulation of Bulbospinal Rostral Ventral Lateral Medulla Neurons by Hypoxia/Hypercapnia but Not Medullary Respiratory Activity

Carie R. Boychuk, Amanda L. Woerman, David Mendelowitz. Hypertension. 2012; 60:1491-1497 Published online before print October 29, 2012, doi: 10.1161/HYPERTENSIONAHA.112.197954. There is respiratory modulation of sympathetic nerve activity but the sites that are a responsible for this are unknown. The rvlm is located near respiratory neurons. The purpose of this study was to determine whether respiratory network pathways can drive rvlm activity. Also they tested whether hypoxia/ hypercapnia can modulate RVLM activity in brainstem slices. They injected CTB into the spinal cord and allowed the rats to recover for 3 to 5 days. They then euthanized the rats and they used bulbospinal neurons that were TH and CTB positive. They then recorded rhythmic inspiratory- related hypoglossal activity and spontaneous synaptic events in the RVLM bulbospinal neurons for 2 to 4 minutes. Then slices were infused with hypoxic /hypercapnic acsf for 10 minutes and then switched back to the control bath for 20 minutes. They showed that none of the bulbospinal rvlm had changes in glycinergic, GABAergic and glutamatergic events. However, in response to hypoxia/hypercapnia the slow firing increased their rate of firing and stayed elevated during the control period. The fast firing had a reduction in their firing rate after the hypoxic/hypercapnic event and returned to baseline firing during the control time period. In order to determine whether hypoxia/hypercapnic changes in rvlm neuronal activity were dependent on GABAergic and glycinergic neurotransmission, gabazine and strychnine were applied to slices. Applying gabazine and strychnine did not alter the neuronal activity alone. However, hypoxic/ hypercapnic events where blocked in the slow firing cells. From this study they concluded that inspiratory events do not affect RVLM neuronal activity in young rats. Also they showed that hypoxic /hypercapnic events can reverse gabaergic and glycinergic neurotransmission leading to increases in firing rate in C1 bulbospinal neurons slow firing in the RVLM. Finally, that fast firing c1 neurons in the rvlm response to hypoxia/hypercapnia are not due to modulation of gabaergic and glycinergic neurotransmission.-MD

Altered c-fos in Rostral Medulla and Spinal Cord of Spontaneously Hypertensive Rats

Jane Minson, Leonard Arnolda, Ida Llewellyn-Smith, Paul Pilowsky, John Chalmers. Hypertension. 1996; 27: 433-441 doi: 10.1161/01.HYP.27.3.433. Hypertension leads to elevated sympathetic nerve activity. We know that the RVM is important for tonic control of blood pressure and sympathetic nerve activity. The neurons located in the RVM are tonically active. C-fos is an early gene that is expressed in response to stimuli. In this particular study they wanted to investigate RVM neuronal activity in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) using c-fos. They also wanted to phenotype the cells by staining for serotonin and catecholamine. First they instrumented 16 to 18 week old WKY and SHR rats so they could record blood pressure and give an infusion of nitroprusside through femoral artery and vein, respectfully. Prior to the nitroprusside infusion, at 12 to 14 weeks CTB-gold was injected into the adrenal medulla and superior cervical ganglion. As for the controls there were two groups WKY and SHR rats that were just implanted with arterial and venous catheters but were not subject to surgical interventions. The infusion of nitroprusside leads to a fall in blood pressure in both groups along with reflex tachycardia. Obviously the saline did not cause a change in blood pressure and heart rate. The SHRs had an enhanced response to the nitroprusside infusion when compared to the WKYs. They then perfused the animal following the infusion and 60minute rest period. The prepared the tissue for immunostaining. Fos immunoreactivity was significantly higher in the nitroprusside group when compared to the saline WKYs. However, the SHRs had similar fos production between the saline and the nitroprusside group. But the SHR group still demonstrated higher fos immunoreactivity than the WKYs. They found that 50% of the TH positive neurons where positive for fos also in the nitroprusside group in the WKYs. As for the saline group, only a small amount was double labelled for fos and TH positive in WKYs. They also looked at serotonin neurons in the RVM. Fos immunoreactivity was not as prevalent in the serotonin population as it was in the TH population. They also looked at fos in the spinal cord. Both groups had a significant amount of fos in the T1-L1 segments s of the spinal cord. As for retrogradely labelled sympathetic preganglionic neurons, they found fos positive sympathetic preganglionic neurons projecting to the sympathoadrenal neurons in the SHRs. They were located in segments T7-T11 and accounted for 39% of all fos neurons in the SHRs. As for sympathetic preganglionic neurons projecting to the superior cervical ganglion there was a limited number found in the SHRs. In the WKYs, they found sympathetic preganglionic neurons projecting to the sympathoadrenal neurons in the t1-t13 region of the spinal cord. After nitroprusside 71% of the fos positive neurons were sympathetic preganglionic neurons projecting to the sympathoadrenal neurons in WKYs. As for the superior cervical ganglion, retrogradely labelled sympathetic preganglionic neurons were found in the T1-T5 region but now were immunoreactive for Fos after nitroprusside. The conclusion that was drawn from the studies at resting level in the SHRs, RVLM neuronal activity is elevated. This increased activity is driving the sympathoadrenal neurons mostly, and these neurons are contributing to the hypertension that is observed in the SHRs.-Mary

Running Throughout Middle-Age Improves Memory Function, Hippocampal Neurogenesis, and BDNF Levels in Female C57BI/6J Mice

Marlatt, Michael W., et al. "Running throughout middle‐age improves memory function, hippocampal neurogenesis, and BDNF levels in female C57BL/6J mice." Developmental neurobiology 72.6 (2012): 943-952. I found this article to be relevant because I am interested in comparing BDNF levels within the RVLM of sedentary vs physically active animals. I am also interested in reading more about effects of exercise later in life than the "adolescent phase" due to the great technical difficulties we are facing imaging younger rats for MeMRI. Specifically, this article discusses the beneficial effects seen from chronic running during later stages in life. The model they used was a nine month old mouse model that exercised freely for 1 month prior to the first set of experiments and then another 5 months prior to the second set of experiments. As stated, at both 1 month and 6 months of exercising anxiety, memory, and motor tests were performed. Then at 8 months post exercise each of the animals were sacrificed and BDNF levels and BrDu positive neurons were examined. They found that after one month of exercising, mice exhibited less anxiety with increased central area during an arena test, as well as increased distance traveled. They also exhibited a tend toward increased latency between falls in a rotarod performance test measuring motor abilities. It was then seen that after 6 months of exercise animals showed increased social memory by increasing the amount of time within the target quadrant in a Morris water maze. These animals also trended towards a decreased number of falls during the rotarod test. To support these findings animals that had chronically run had increased levels of mature BDNF peptide within the hippocampus after 8 months. Exercising animals also had a greater number of BrDu labeled cells in the dentate gyrus, along with an increase in the total number of new neurons. Concluding, this study was able to show that chronic running later in life (9 months) improved spacial memory, motor skills, as well as decreased anxiety. This may be due to increased neurogenesis and neurotrophins in the hippocampus after 8 months of free exercise. I think it would be informative to compare BDNF levels, as well as neurogenesis within the RVLM between sedentary and physically active population. ~JI

Selective enhancement of glutamate-mediated pressor responses after GABAA receptor blockade in the RVLM of sedentary versus spontaneous wheel running rats

Patrick J. Mueller* and Nicholas A. Mischel Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA I needed an excuse to read this paper again……. In this study we looked at the response of WRs and SEDs to exogenous glutamate following the removal of GABAergic tone via bicuculline. This paper is an attempt to build on Nick’s 2011 paper which looked at sympathoexcitation following a dose of exogenous glutamate and looked at the blood pressure and splanchnic sympathetic nervous activity response. This paper is examining lumbar sympathetic nervous activity and blood pressure response to different doses of glutamate, with the inclusion of pre-bic and post-bic protocols. Interestingly enough he was not able to duplicate the same dose response curve of the 2011 paper for lumbar nerve activity, suggesting that SEDS/WRs do not have different sympathoexcitatory responses to solely exogenous glutamate in regards to lumbar nerve. They were able to show sympathoexcitatory differences in the post-bic protocols 5’ and 15’ minutes after the injection of bic, suggesting that GABAergic transmission is hiding the sympathoexcitatory differences between WRs and SEDs in regards to lumbar and blood pressure. It is important to consider that bicuculline was injected unilaterally so there could have been some kind of compensation from the contralateral RVLM. I would like to see what the glutamate responses would be if the RVLMs were blocked bilaterally, that being said, the blood pressure might raise too high (190 mmHg) with a bilateral injection of bic. It would also be interesting to see if there were any differences in glutamate responses with the ipsilateral RVLM completely disinhibited with bic and the contralateral RVLM excitated with glutamate. Venturing to what I have been looking at with my endogenous input experiments, it would be important to see if there were differences in the nerve activity responses to bicuculline in regards to the lumbar nerve, or these nerves (splanchnic and lumbar) might be differentially inhibited by GABA. -M.T.L.

Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo

Full cite: Abbott, S.B., Holloway, B.B., Viar, K.E. & Guyenet, P.G. (2013)Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo. Eur. J. Neurosci., 39, 98-106. Stephen B. G. Abbott,1 Benjamin B. Holloway,1 Kenneth E. Viar1 and Patrice G. Guyenet1,2 1Department of Pharmacology, University of Virginia, Charlottesville, VA, USA 2University of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Avenue, Charlottesville, VA 22908-0735, USA Our lab and others have looked at the RVLM’s role in reflex responses to different homeostatic challenges such as hypotension, hypoxia, and hypoglycemia. The general belief is that the RVLM utilizes glutamate as the primary transmitter in response to these homeostatic challenges; however, this has never been proven in-vivo. Guyenet’s laboratory utilized a combination of optogenetics, immunohistochemistry, and multi-cell recordings in gene knockout mice in an attempt to confirm that these responses are glutamate-mediated. In particular, they were looking at the role of a glutamate transporter VGLUT2. They used DβHCre/0 mice which mark noradrenergic and adrenergic neurons with Cre-recombinase. In addition to this they paired the DβHCre/0 with rats that knock out the VGLUT-2 gene, called cKO mice. By utilizing DβHCre/0 and cKO mice using optogenetics, the team was able to determine the role of glutamate and VGLUT-2 in these responses. They found that VGLUT-2 deletion has no effect on the number or morphology of C1 neurons in RVLM, but completely eliminated several homeostatic responses. The acute respiratory and parasympathetic activation produced via optogenetic stimulation was abolished following removal of VGLUT-2. It would be interesting to do this same study, but incorporate the non-C1 neurons via some other technique. Most importantly though, this once again plays into Guyenet’s belief that C1 neurons function to maintain homeostasis following different physiologic challenges, that C1 neurons are the body’s EMTs and glutamate is the main means by which the RVLM affects the periphery. At some point we could look at adding optogenetics into our experiments, perhaps even with MEMRI. -M.T.L.

The entry of manganese ions into the brain is accelerated by the activation of N-methyl-D-aspartate receptors

Itoh, K., et al. "The entry of manganese ions into the brain is accelerated by the activation of< i> N-methyl-d-aspartate receptors." Neuroscience 154.2 (2008): 732-740. There are many advantages of using MeMRI to study in vivo neuronal activity. However, there is the disadvantage of having to anesthetize the animal during the imaging which has an effect on overall brain functioning. Previous studies done by Lin/Koretsky and Aoki have demonstrated that manganese enhancement in MR images is dampened the deeper an animal is in anesthesia. That being said, no studies had been done looking at effects of manganese enhancement across different methods of anesthesia until Itoh in this study in 2008. They found that most anesthetics including isoflurane, urethane, and pentabarbitol did not have significant affects on manganese enhancement. Where as the use of ketamine significantly decreased manganese enhanced contrast. Unlike the other anesthetic that act by potentiating GABAergic pathways, ketamine acts as a partial antagonist for NMDA receptors. An NMDAR under natural conditions is acted upon by glutamate to then activate glutamatergic neurons via the influx of calcium. The observation of decreased manganese enhancement in the presence of ketamine then led to further questions about the entry of systemic manganese into the cerebral spinal fluid and eventually brain tissue. Using various NMDA, GABAa, and AMPA agonists and antagonists this study found that NMDAR mediated glutamatergic excitation plays a major role in influencing manganese enhancement in MRI. Both NMDAR antagonist significantly decreased manganese enhancement within the ventricles (other brain regions did not show differences because images were only taken up to three hours post MnCl2 injections). Oppositely, NMDAR agonist produced significantly increased manganese enhancement compared to controls. AMPA antagonist did not appear to change manganese enhancement, while GABAa antagonist also produced increased manganese enhancement. A limitation to this study is that they did not address effects resulting from changes in the NMDARs within brain tissue itself, which is important to our studies looking at plasticity in neuronal activity in the RVLM of sedentary and physically active rats. I would hypothesized by extrapolating from this study however, that sinoaortic dennervated rats will show an increase in manganese enhancement within the RVLM when compared to the non-dennervated control animals. ~JI