Rhythmic activity of neurons in the rostral ventrolateral medulla of conscious cats: effect of removal of vestibular inputs.

Barman SM, Sugiyama Y, Suzuki T, Cotter LA, DeStefino VJ, Reighard DA, Cass SP, Yates BJ. Am J Physiol Regul Integr Comp Physiol. 2011 Oct;301(4):R937-46 In most electrophysiological studies of RVLM neurons, animals are either anesthetized (like we do) or decerebrated (like in the half-rat prep). In this paper, they went ahead and examined neurons in conscious/behaving cats, and correlated their activity with carotid blood flow. They found that around 8% of RVLM neurons showed their spiking to be most likely to occur during periods of decreased blood flow (in between heart beats). Their previous work showed that the vestibular system normally suppresses RVLM responses to body rotation in intact/awake rats (think like the Dampney paper where they inhibited the colliculi in order to get sympathetic activation following normally non-stimulating cues). So in this paper, they looked at what removal of vestibular inputs would do the cardiac-linked RVLM activity and found that vestibular removal caused them to have an increased likelihood of finding neurons that showed cardiac related activity. What I find most interesting though, is that their data came in bursts - some days they didn't get any neurons with flow-related activity, while other days up to 60% of the neurons they recorded had it. Maybe that's the problem with my recordings - I just keep picking the wrong days to do experiments... -DH

Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats

Barman, Susan M., and Hakan S. Orer. "Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 299.5 (2010): R1269-R1278. Previous to this study, work by Dr. Barman and her laboratory had shown that the medullary lateral tegmental field (LTF) neurons set the pattern for resting sympathetic nerve activity (SNA), not the NTS, CVLM, or RVLM. She was very open in stating that sympathetic tone to most of the vasculature was not driven by the RVLM, and that the RVLM, CVLM, and NTS were not the only neuronal pathways that contributed to SNA modulation resulting from the baroreflex, chemoreflex, or vagal afferents. However, these hypotheses were based upon various microinjecitons into directly into the LTF while recording SNA, so this study looked to further characterize the role the LTF plays in the sympatho-sympathetic reflex in response to afferent stimulation. After the separate microinjection of 1) non-NMDA receptor antagonist (NBQX) 2) NMDA receptor antagonist (D-AP5) 3) muscimol into the LTF there were no differences seen in sympathoexcitatory response recorded from the right inferior cardiac nerve, following stimulation of either the left inferior cardiac afferent nerve (CN) or sympathetic afferent nerve (SN). This same protocol was then repeated in the RVLM and injections of NBQX reduced the sympatho-sympathetic excitatory response to ~25% from baseline levels, as well as increased the onset latency following the electrical stimulation to the CN and SN. Like in the LTF, microinjections of D-AP5 produced no significant differences in the sympatho-sympathetic excitatory response. Muscimol, however, like NBQX significantly reduced the responses to CN and SN stimulation. In most cases the response seen was negligible, with little to no excitation. Conclusively, there were no changes in the excitatory responses following identical microinjections into the NTS and CVLM. Results from this study disproved the original hypothesis and instead showed that the RVLM is a critical region involved in sympatho-sympathetic cardiovascular responses. Dr. Barman also goes on to hypothesize that non-NMDA receptors play a primary role in this reflex compared to NMDA receptors. ~JI

Exaggerated Cardiovascular Stress Responses and Impaired β-Adrenergic–Mediated Pressor Recovery in Obese Zucker Rats

Gerard D’Angelo, James D. Mintz, John E. Tidwell, Ann M. Schreihofer,David M. Pollock, David W. Stepp.Hypertension. 2006;48:1109-1115.Obesity is a major risk factor for development of metabolic disorders. Besides the increased risk for metabolic disorders there increase in the pressor response that results from mental, physical and acute stress. Since α and β adrenergic receptors are important for determining vascular tone, changes in either receptor expression or sensitivity to norepinephrine or epinephrine. In a previous study this laboratory demonstrated that pressor responses to ganglionic blockade was not different between obese and lean zucker rats. They also demonstrated that isolated mesenteric resistance arteries responses to exogenous norepinephrine is attenuated when compared to lean zucker rats. They concluded from this data that there is elevated peripheral vascular resistance. Furthermore, it may be due suppressed β-adrenergic-mediated vasodilation. In this study they hypothesized that pressor responses to environmental stressors would be greater in the obese versus the lean zucker rats. The elevated pressor response in the obese zucker will be most likely due to attenuated vasodilation as a result of impaired β- adrenergic mechanisms. Obese and lean zucker rats ( and LZR, respectively)were implanted with telemetry probes in order to record blood pressure under conscience conditions. Following recovery animals were brought to a soundproof room and then the animals were put into restrainers and a baseline blood pressure and heart rate were monitored for fifteen minutes prior to the air jet stress. In a separate group of obese and lean zucker rats, following the fifteen minute control period propranolol was administered and then the air jet stress was initiated. They also instrument animals so they could determine blow flow to the mesenteric arteries and aorta.They also looked at cardiac output along with blood glucose, plasma levels of cholesterol and triglycerides. They also looked at thyroid hormones T3/T4.They found that OZR had higher baseline BP and HR variability. In response to stress the OZR the area under the curve (AUC) for blood pressure was significantly higher in obese untreated when compared to the LZR. the AUC 20 minutes post stress was significatly greater in the OZR when compared to LZR. There was also a significant increase in AUC following treatment with propranolol in the LZR. They further analyzed the role of β-adrenergic receptors following ganglionic blockade. prior to ganglionic blockade, β-adrenergic receptor blockade resulted concentration dependent decreases in MAP in both groups, however the OZR had attentuated response when compared to LZR. Following ganglionic blockade resulted in a similar trend. They also looked at hindlimb and mesenteric conductance. There were no differences in hindlimb conductance between OZR and LZR ganglionic blockade in response to β- adrenergic blockade. As for mesenteric conductance both group exhibit increases in conductance, however only at 0.05,0.1, 0.5 μmoles of isoproterenol is there an attenuation in the OZR response when compared to LZR. As for cardiac output, there is an increase in response to isoproterenol in both group however the OZR had attenuated changes in CO at 0.05 and 0.5 μmoles. These data suggest that Obesity may lead to attenuated β- adrenergic mediated vasodilation. This β- adrenergic mediated vasodilation is playing an important role in the recovery period for stress.-MD

Increased reactive oxygen species in rostral ventrolateral medulla contribute to neural mechanisms of hypertension in stroke-prone spontaneously hypertensive rats.

Kishi T, Hirooka Y, Kimura Y, Ito K, Shimokawa H, Takeshita A. Circulation. 2004 May 18;109(19):2357-62. Epub 2004 Apr 26. In this paper, the authors wanted to look at how baroreflex sensitivity (BRS) is impaired in cardiovascular disease (CVD) models, and how it can be linked to changes in reactive oxygen species (ROS). Their previous work had shown that by causing increased expression of manganese superoxide dismutase (MnSOD), they could reduce ROS and cause sympathoinhibition in hypertensive (HT) rats. However, that was kind of an acute study, so this time they wanted to look at BRS in HT rats, using a chronic redution in ROS via MnSOD overexpression. To do this, they injected the RVLM of spontaneously hypertensive rats (SHRs) with a construct causing expression of MnSOD. They found strong reductions in MAP and HR 5-9 days after injection, and a strong reduction in urinary norepinephrine at day 7. For the BRS, they found that the SHRs started with lower sensitivity than WKY-MnSOD controls, but became more sensitive after transfection (days 5-8). WKY-MnSODs did not demonstrate this increase in sensitivity, nor did LacZ-SHR controls. I have a bit of confusion about this paper... I'm honestly not sure exactly how they measured BRS, partially because I didn't look up the two papers describing how BRS is calculated. I admit, not knowing this is kind of a big hole in my interpretation of the paper. In my defense, the two papers they referenced on the technique seem to be calculating BRS in humans, and they never referenced any papers showing how it was done in rats, so I can't yet be really confident about the technique. I dug up a couple of this group's other papers and didn't see it in those ones either, so I might have to go look in to this one more, or consult our resident expert. I'm also a little confused about how all of the effects peaked at one week before returning to baseline levels, as soon as day 9. I know that viral expression isn't always permament, but is 7 days "chronic?" Is there a combined effect of the transfection failing AND some kind of compensation happening to maintain the MAP/HR/BRS? The levels of MnSOD were only shown at days 0,7, and 21... I'd like to know if their expression graphs match the effects - that might answer part of this question. -DH

Blood pressure is maintained during dehydration by hypothalamic paraventricular nucleus-driven tonic sympathetic nerve activity.

Holbein WW, Bardgett ME, Toney GM. J Physiol. 2014 Jun 27. In this paper, they wanted to look at how what happens during dehydration that allows the blood pressure to be maintained. To do this they looked at the PVN , because previous papers suggested that it might be the region controlling th effect. Their first experiment was to look at the differences in SSNA between dehydrated (DH) and euhydrated (EH) rats. They found that MAP and phrenic nerve activity (PNA) that wasn't different between groups, but DH rats had a much higher baseline SSNA. When they gave injections of muscimol in to the PVN to block its activity, they saw almost no effect in the EH rats, but in the DH rats they a strong and rapid reduction MAP and SSNA. For PNA, both groups showed only a slight decrease in burst frequency, but not amplitude, after the injection of GABA. They also noted that, even though comparison of SSNA between animals is touchy, the post-inhibition SSNA was not different between EH and DH rats, suggesting that the entire process may be mediated by the PVN. This also suggests that the splanchnic nerve is critical to maintaining blood pressure during dehydration, though they have yet to examine nerves leading to other vascular beds. -DH

Efficacy of an L- and N-type calcium channel blocker in hypertensive patients with neurovascular compression of the rostral ventrolateral medulla

Aota, Yasuko, et al. "Efficacy of an L-and N-type calcium channel blocker in hypertensive patients with neurovascular compression of the rostral ventrolateral medulla." Hypertension Research 32.8 (2009): 700-705. It has been previously shown in clinical studies that essential hypertension may be linked to neurovascular compression (NVC), and that NVC maybe be mediating hypertension through increased levels of sympathetic nerve activity. Neurovascular decompression studies have been shown to decrease blood pressure in patient with essential hypertension, but requires a rather intricate invasive surgery. The purpose of this study was to examine the effects of non-invasive, sympatholytic, L- and N-type calcium channel antagonist (Cilnidipine) for the use of a treatment for essential hypertension mediated by NVC. In this trial, 46 patient with essential hypertension (22 –NVC, 24 +NVC) were treated with Cilnidipine for 16 weeks, with clinical follow ups at 0, 8, and 16 weeks. At 8 and 16 weeks, systolic and diastolic blood pressure in both groups was significantly reduced from baseline levels. Correspondingly, systolic and diastolic pressures were significantly reduced in the +NVC group, compared to the –NVC group. To examine SNA, norepinephrine levels were taken at each time point, revealing that baseline levels of norephinephrine were significantly higher in the +NVC group than the –NVCs. That being said, at 16 there was a significant decrease in NE levels in the +NVC, but not –NVC group. With no difference in NE levels found between the groups. Finally, after measuring left ventricular mass index (LVMI) at each time point, it was observed that LVMI decreased in the +NVC, but not the –NVCs. Conclusively, this study was able to show the use of Cilnidipine by patients with EH mediated by NVC may be a non-invasive antihypertensive treatment. However, because Cilnidipine is not only an N-type calcium channel antagonist, but also an L-type vascular calcium channel antagonist more studies are being done to distinguish if the results seen were due to sympatholic or vascular effects. ~JI

The sympathetic control of blood pressure

Patrice G. Guyenet Nat Rev Neurosci. 2006 May; 7(5):335-46 I’ve been writing my introduction and figured this paper would be a great resource, so I read it *again*…………… The overall purpose of this review is to examine sympathoregulation and how altered sympathoexcitation can result in hypertension and other cardiovascular diseases. It is also important to mention that while SNA has been correlated to CVD, there are likely other mechanisms involved in the development of CVD, including hypertension. Apparently renal SNA is selectively regulated by volume receptors whereas the other sympathetic nerves are not. Also the rostral ventromedial medulla (RVMM) and medullary raphe are involved in cutaneous circulation, whereas the rostral ventrolateral medulla (RVLM) is not. I also did not know that the C1 neurons were only one of three clusters of adrenaline-synthesizing cells in the CNS, which is particularly interesting because we still don’t particularly know the role that adrenaline synthesis is even having in the RVLM, aside from being a good marker. Here are C1 cells that are non-barosensitive that control adrenaline-releasing chromaffin cells. Guyenet mentions that the baroreflex can be set (towards a higher level) in the case of different activities that require heightened sympathetic tone, exercise for example. I do not know the mechanism by which the baroreflex can be altered; I know that reflexes like the muscle metaboreflex exist and different types of hypoxia can raise blood pressure above baroreflex regulation. Something interesting, which is slightly unrelated (however mentioned in the review), is that obstructive sleep apnea (OSA) increases SNA throughout the waking period of the day, which I thought was surprising because I figured it would only have an effect during the night. Something that I found particularly interesting (as well as something that I don’t necessarily agree with) is the remark that “sympathetic efferents that innervate the kidneys are commonly presented as the only ones that are capable of influencing 24-h average BP.” I don’t think that renal controlling neurons are the only neurons involved in tonic cardiovascular regulation, I think that looking at only the neurons controlling the kidneys is totally short sighted. It would be interesting to look at a review with a similar approach as this, but considering all of the papers presented after 2006 as well. -MTL

Olfactory exposure to males, including men, causes stress and related analgesia in rodents

Sorge, R.E., Martin, L.J., Isbester, K.A., Sotocinal, S.G., Rosen, S., Tuttle, A.H., Wieskopf, J.S., (...), Mogil, J.S. Olfactory exposure to males, including men, causes stress and related analgesia in rodents (2014) Nature Methods, 11 (6), pp. 629-632. doi: 10.1038/nmeth.2935 Dr. Berkowitz had mentioned this paper…………………… The goal of this paper is to look at the responses of male and female mice to different stimuli which are intended to gauge the stress or withdrawal response to the presence or perceived presence of a male or female researcher or animal. The first experiment consisted of an injection of zymosan, an inflammatory agent. The researchers studied the facial grimacing of mice in the presence or absence of an experimenter (male or female, seated at a distance of ~0.5m). All of the four male observers elicited significant changes in grimaces, while all four of the women did not. They conducted the same study, but instead of using the researcher, they placed a shirt worn the previous night by the male or female experimenters ~0.5 away from the mice. They found that they were able to see the same trend of responses. Interesting enough, the rats did not show the response if both the male and female shirts were placed together. Next, they placed the mice in different bedding materials from either unfamiliar male mice, nonpredator male guinea pigs, rats, cats, and dogs, and once again saw the same decreases in facial grimacing. Castrated male mice, cats, and dogs did not produce the same effect in mice, suggesting that this response is likely in large part due to androgens. A longitudinal study was performed to examine their previous experiments and look at the baseline and peak responses of mice, and found that mice tested by male experimenters displayed lower baseline pain sensitivity. This stress induced analgesia in the mice is shown only when in the presence of a male. The stress caused by male experimenters is short lasting, however, it is something to consider when conducting animal research, particularly when interested in different stress responses. -MTL

Developmental changes in GABAergic neurotransmission to presympathetic and cardiac parasympathetic neurons in the brainstem

Olga Dergacheva , Carie R. Boychuk , David MendelowitzJournal of Neurophysiology Published 1 August 2013Vol. 110no. 672-679DOI: 10.1152/jn.01054.2012. This article was interesting because it investigated how postnatal development altered gabaergic neurotransmission specifically, the effects on parasympathetic cardiac and presympathetic neurons in the brainstem. This group was interested in how hypoxia and hypercapnia (H/H) may play role in this. They used retrograde labelling with CTB injected in the T2-T4 region of the spinal cord. Then at certain days postnatal p5, p20, p30 and they looked at GABAergic neurotransmission and how it was altered by hypoxia and hypercapnia. In the parasympathetic cardiac vagal neurons there was IPSCs in response to strychnine in P5, P20, and P30. However, the P20 had increased frequency and amplitude of IPSCs than the other groups. As for the cardiac parasympathetic neurons on the P20 frequency was altered in under control conditions when compared to the P5 and P30. In response to H/H in the P5 and P30 the IPSCs were reversed however in the P20 frequency and amplitude IPSC not altered for presympathetic neurons. H/H decreased IPSC frequency in P5,P20, and P30. In only decreased the amplitude in the P20. These data demonstrate P20 is an important developmental stage for parasympathetic and sympathetic development. The reason why this study is so important is because this age in rats is equivalent to the age when children tend to develop SIDS. This increased sensitivity to hypoxia/ hypercapnia may be a possible reason why SIDS. -MD

Exercise Training Lowers the Enhanced Tonically Active Glutamatergic Input to the Rostral Ventrolateral Medulla in Hypertensive Rats

Yan-Ping Zha, Yang-Kai Wang, Yu Deng, Ru-Wen Zhang, Xing Tan, Wen-Jun Yuan, Xiao-Ming Deng & Wei-Zhong Wang. doi: 10.1111/cns.12065 The study investigated how exercise can modulate glutamate neurotransmission. Using WKYs and SHRs that were sedentary (sed) or exercise trained (ext) they looked basal blood pressure (BP), which was significantly reduced in the SHRs-ext when compared to the SHRs-sed. HR was also significantly reduced in the SHR-ext when compared to the SHR-sed. Similarly, they saw that exercise also reduced the decrease in BP and HR that is caused by giving a glutamate receptor blocker. Kyn had little affect ion bp and HR responses in the WKYs. In order to determine whether the concentration of glutamate is altered they used HPLC and found that SHR-sed had a higher concentration of glutamate when compared to WKYs(both sed and ext) and also SHR-ext. Western blot was done in order to examine the amount of vglut2 protein being expressed in the RVLM . Results showed that the SHR-SED had more vglut2 compared to the other groups. Finally they wanted to determine where this glutamatergic input is originating from. Western blot was done on punches from the NTS, PVN and PRF for glutaminase2. They found a significant increase in NTS and PVN in SHR-seds for glutaminase2 when compared to SHR-ext. Overall message, exercise reduces glutamatergic input via reduction in concentration of glutamate in rvlm and also a reduction in glutamatergic inputs from NTS and PVN. Maybe we should start looking at NTS also because it could be providing some glutamatergic input to rvlm.-MD

Discharge of RVLM vasomotor neurons is not increased in anesthetized angiotensin II-salt hypertensive rats.

Pedrino GR, Calderon AS, Andrade MA, Cravo SL, Toney GM. Am J Physiol Heart Circ Physiol. 2013 Dec;305(12):H1781-9. I might get in trouble for saying this... but I kind of dislike this paper for personal reasons. These people are also interested in differences in RVLM neuron firing rates between normotensive and hypertensive rats. They looked at the difference in RVLM unit activity between normotensive (NT) rats and rats that had become hypertensive (HT) due to three weeks of AngII infusion and consumption of a high-salt diet. Neurons were confirmed to be presympathetic and spinally projecting via baroinhibition and antidromic action potentials. They noted that the neurons fired one spike per heart beat, which was likely to fire during mid-late systole, which was not different between groups. The discharge rate at resting MAP was similar between groups, so they looked at barosensitivity and found that HT rats required a higher increase in MAP before they would stop firing. They also used nitroprusside to unload baroreceptors and found that both groups had similar maximum discharge rates and requisite change in MAP needed to achieve these max rates, but within a certain range of MAPs (120-150mmHg) neurons in HT rats were more likely to be firing at a higher rate. This says that just like we would expect to see in our exercise vs inactivity model, unhealthy rats are reluctant to decrease a high sympathetic tone even when there is no physiologic need for it. They also did juxtacellular of some cells found that in NT rats, 3 were C1 (PNMT positive) and 4 were non-C1. In HT rats, 6 were C1 and 6 were non-C1. -DH

Disinhibition of the midbrain colliculi unmasks coordinated autonomic, respiratory and somatomotor responses to auditory and visual stimuli.

Müller-Ribeiro FC, Dampney RA, McMullan S, Fontes MA, Goodchild AK Am J Physiol Regul Integr Comp Physiol. 2014 Aug 6. Previous work has shown that if you cause blockade of GABA-A receptors in the colliculi of awake rats, the rats will show defensive/escape responses to stimuli that normally wouldn't bother them. This suggested that the colliculi are involved in activation of the sympathetic nervous system, but that the neurons are usually under tonic GABAergic inhibition. Based on that idea, this paper studies the effect of GABA-A blockade in the superior and inferior colliculi and its effect on splanchnic, sciatic, and phrenic nerve activity in anesthetized rats. Normal, anesthetized rats should have no increases in nerve activity or MAP after the rat is exposed to clapping, flashes of light, and paw-pinches. That's exactly what they saw in their preparation. They then blocked GABA-A receptors with picrotoxin (which rarely caused changes in baseline nerve activity), and gave the rats the same stimuli. After blockade, they saw that all three of those stimuli could cause synchronized increases in nerve activity among all three of the nerves. This is strong proof that the colliculi are relay points for autonomic activity that are normally kept under GABAergic inhibition. They followed this up by trying to figure out where this GABA might be coming from - to do this they removed the forebrain and saw the exact same effects. Because they saw the complete response in decerebrate rats, they can now say that GABA is coming from either the midbrain or the brainstem, tonicically inhibiting the colliculi's activation of nerve activity, and also that whatever signal is driving that GABAergic tone is ALSO located in the midbrain or brainstem. -DH

Role of voltage-gated L-type calcium channel isoforms for brain function

Striessnig, J., et al. "Role of voltage-gated L-type Ca2+ channel isoforms for brain function." Biochemical Society Transactions 34.Pt 5 (2006): 903-909. This is a more detailed study examining the role of both L-type calcium channel (LTCC) isoforms 1.2 and 1.3 within the brain. Like discussed previously in my last blog, because the LTCC agonist and antagonist do not show specific binding to either 1.2 or 1.3, it has been difficult to differentiate the roles each plays. However, more recently discovered, two groups of genetically modified mice have enabled scientist to conduct much needed studies better characterizing these two calcium channels. The first mouse group Cav1.3-/- has a selective knockout for all Cav1.3 channels. Whereas the second modified mouse group Cav1.2-/- has a modified α1 subunit that inhibits the binding of agonists and antagonists from the channel. With the creation of these mice, creative studies can now selectively examine either isoform 1.2 or 1.3 depending on which pharmacological substances are utilized. Recent studies have now found that homologous knockouts of isoform 1.3 causes complete deafness, as well as sinoatrial node dysfunction. These results lend to the hypothesis that 1.3 specific calcium channels play a vital role in cochlear hair and sinoatrial node cell signaling transduction. Similarly, experiments utilizing Cav1.2-/- have revealed the importance in Ca2+ influx through Cav1.2 in regulating smooth muscle and cardiac contractility. This study also reiterates the importance of both isoforms in synaptic plasticity pertaining to memory, learning, and the storage of fear induced memories. Interestingly, when sensitivity to direct Ca2+ channel activators was inhibited in Cav1.2-/-, the efflux of glutamate from neurons in the ventral striatum was completely abolished, providing evidence for the importance of Cav1.2 in neurotransmission of glutamate. Again, I think this paper provides plausible evidence that L-type calcium channels may play an important role in synaptic plasticity within the RVLM. ~JI

Role of rostral ventrolateral medulla centrally mediated pressor responses

Full cite: Kiely JM, Gordon FJ. Role of rostral ventrolateral medulla centrally mediated pressor responses. Am J Physiol Heart Circ Physiol 267:H1549–H1556, 1994. Role of rostral ventrolateral medulla centrally mediated pressor responses James M. Kiely and Frank J. Gordon Department of Pharmacology, Emory University of Medicine, Atlanta, Georgia 30322 This paper is one of the first to look at how the RVLM mediates different central pressor responses, in particular how EAA receptors play a role in the proliferation of these responses. In this particular experiment they used female Sprague-Dawley rats, recorded BP, and instrumented for sciatic nerve stimulation (SPR). Electrodes were positioned into the periventricular nucleus (*which I thought was pretty cool*). Then a head surgery was performed to expose the brainstem, similar to how we prepare for our microinjection experiments. Kyn was injected bilaterally and there was no BP response, similar to in our experiments. When Kyn was injected in the RVLM, the hypothalamus’s pressor responses were intact. Injection of kainic acid increased BP for a short period of time (= 10s), after that point BP fell around 35 mmHg (~5 min). SPR and PFH responses were attenuated, PVN and AH responses were intact. PFH and PVN responses were reduced following injection of muscimol; lidocaine into RVLM abolished SPR, and greatly reduced the PFH and PVN responses. This paper is likely one of the first to demonstrate that the PVN and PFH have spinal projections of their own and are able to function at least somewhat independently from RVLM. Also it seems that SPR are almost exclusively mediated through the RVLM. It would be interesting to see how the activity of PVN and PFH might be altered during an RVLM knockout or blockade. I also do not know whether or not PVN or PFH are barosensitive. I think that at some point we are either going to have to integrate PVN into our experiments or knock it out in some way, because it may very well be playing a role in our responses. MTL

The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain functions

Berger, Stefan M., and Dusan Bartsch. "The role of L-type voltage-gated calcium channels Cav1. 2 and Cav1. 3 in normal and pathological brain function." Cell and tissue research 357.2 (2014): 463-476. There are multiple types of voltage-gated calcium channels (VGCCs) including L-type, T-type, P/Q-type, R-type, and N-type that are defined by their pharmacological responses. Each channel is made up of five subunits, with the main pore forming subunit being alpha1. For this discussion I will mainly be focusing on the L-type calcium channels(1.1-1.4) which are believed to be the channels that allow manganese entry into a depolarized cell when using manganese enhanced MRI. More specifically, I will be focusing on 1.2 and 1.3 which are expressed by in large in the body compared to the more restricted 1.1 and 1.4. Cav1.2 and Cav1.3 can be found on multiple organs in the periphery including the hear, smooth muscle, pancreases, and adrenal glands, but most importantly for my studies, both of these channels are also expressed on neurons in the brain. With some cells expressing both 1.2 and 1.3, leading to the hypothesis that each channel may have its own specific function and importance. Some studies using radioreceptor assays have suggested that 89% of all Cav isoforms in the brain are 1.2, where as only 11% are 1.3. However, other studies utilizing western blotting have stated that within neurons in the hippocampus, cerebral cortex, and cerebellum 1.2 and 1.3 isoforms are equally abundant. As for location within a cell, it appears 1.2 are mostly found on the post synaptic dendrites, compared to the 1.3 which is most dense around the cell body. Electrophysiologically, 1.3 isoform channels are activated more rapidly and in more hyperpolarized membranes. They are also inactivated with a slower current than 1.2 isoforms. In both channels calmodulin acts as an imperative calcium sensor that 1) initiates the inactivation of the calcium channel preventing intracellular calcium toxicity 2) causes phosphorylation of the channel which increases the probability of an open state during repeated or prolonged activation and 3) enables the expression of calcium dependent genes within the cell. Unfortunately, there still is not a well established antibody for the isoform 1.3 so these studies may be less reliable then more recent studies being undertaken using Cre recombinase knockout mice. Using genetically modified mice, pharmacological experiments have showed that LTCCs play a role in synaptic plasticity involving learning and memory. Interestingly, injections of LTCC antagonists into the hippocampus have shown increases in acquisition and retention of spatial reference and working memory. Correspondingly, chronic injections into older animals has shown prevention of age-related hippocampal-dependent memory loss. It is believed this change is linked to the loss of NMDA-receptor-independent form of late long-term potentiation. Related, it has also been shown using LTCC antagonist injections into the amygdala that blocking LTCC also blocks the formation of fear memories. Finally, other studies have shown a link between LTCC's and the modulation of the mesoccumbal dopamine signaling pathway, which plays a major role in the reward system and addiction. Compared to animal studies, few human studies have been done analyzing LTCCs. However, in the past few years the hypothesis that LTCC's play an important role in psychiatric diseases is becoming more and more accepted. Stemming from patients with Timothy Syndrome, upregulated LTCC activation leads to the upregulation of tyrosine hydroxylase expression, causing increased concentrations of norepinephrine and dopamine. They have also shown that stimulation of TS-mutated calcium channels 1.2 cells led to dendritic retraction. Since the GWA the CACNA1C gene, associated with the alpha1 subunit on Cav1.2, was identified as a common risk factor allele for bipolar disorder, schizophrenia, and major depression. As for physiological defects in the isoform 1.3, recent studies are showing Cav1.3-mediated vulnerability of the dopaminergic neurons affected by Parkinson's disease. Specifically, increases in calcium entry increases alpha-synuclein aggregates present in Parkinson's disease. Conclusively, very few studies have been done examining the role of VLCC's in normal human cognition up to date. I think studies examining the role of L-type calcium channel activity within the RVLM would be interesting, and may shed light on differences in neuroplasticity between sedentary and physically active animals. ~JI

Role of presympathetic C1 neurons in the sympatholytic and hypotensive effects of clonidine in rats

Ann M. Schreihofer and Patrice G. Guyenet . American Journal of Physiology - Regulatory, Integrative and Comparative Physiology Published 1 November 2000Vol. 279no. R1753-R1762. The rostral ventrolateral medulla (rvlm) is important to control of blood pressure (BP) and sympathetic nerve activity (SNA). Clonidine is an antihypertensive medication that acts as an agonist for α1 adrenergic receptor on axons and terminals of rvlm neurons. The purpose of this paper was to determine whether clonidine acted on spinally projecting c1 or no c1 in the rvlm in order to lower SNA and BP. What they found was that after injecting DβH- saporin the response to clonidine. This finding along with previous data from other labs that showed that injections of clonidine into the nucleus tractus solitarius (NTS) and in the intermediolateral cell column (IML) demonstrates that clonidine may act on several areas in the brain in order to have the antihypertensive affect that is known for.-MD

Stress-induced elevations of y-aminobutyric acid type A receptor-active steroids in the rat brain

ROBERT H. PURDY, A. LESLIE MORROW, PERRY H. MOORE, JR., AND STEVEN M. PAUL. Proc. Nati. Acad. Sci. USA. Vol. 88, pp. 4553-4557, May 1991 Neurobiology. They investigated the effects of stress on the production of pregestrone metabolites in the brain. They used adrenectomized and non-adrenalectomized rats in order to determine how csf levels of progesterone, allopregnanolone and pregnanolone in rats were altered in response to swim stress. They used a couple of techniques such as chromatography, and RIA or Radioimmuno assay. What they found was that in the adrenalectomized, adrenalectomized stressed and controls had undetectable amounts of allotetrahydroDOC in plasma and cortex, however the stressed rats had significant enhanced levels of allotetrahydroDOC in both cortex and plasma. As for progesterone was not altered in response to stress when compared to the control group however the adrenalectomized rats had significantly lower progesterone when compared to the control rats. As for the allopregnanolone, in response to swim stress the level was significantly higher when compared to control animals in the cortex and plasma. Another interesting finding is that the in the adrenalectomized rats there was reduced allopregnanolone. The most important point I got from this article is that allopregnanolane and allotetrahydroDOC have the ability to bind to the benzodiazepine site on the GABA receptor, thus potentiating the effects of GABA. Since it seems that doing a adrenalectomy, abolishes the increase in these steroid metabolites, adrenal gland may be important to modulating GABAergic mechanisms in the brain. -MD

Somatic nerve stimulation evokes qualitatively different somatosympathetic responses in the cervical and splanchnic sympathetic nerves in the rat

Full cite: S. McMullan, et al. Somatic nerve stimulation evokes qualitatively different somatosympathetic responses in the cervical and splanchnic sympathetic nerves in the rat. Brain Res., 1217 (2008), pp. 139–147 Simon McMullan, Karrnan Pathmanandavel, Paul M. Pilowsky, Ann K. Goodchild Australian School of Advanced Medicine, Macquarie University, Australia University of Sydney This study is examining the reflex responses of two different sympathetic nerves in response to somatic pressor response– from the sciatic nerve. The two sympathetic nerves that they are looking at is the splanchnic and cervical nerve. The primary technique used is this study is electrophysiology. They stimulated from the aortic depressor nerve, using antidromic action potentials to confirm that the neurons that they were recording from in RVLM were barosensitive. They found that the splanchnic nerve had a primarily biphasic response to sciatic nerve stimulation, whereas the cervical nerve was primarily monophasic. This suggests qualitatively different responses between the sympathetic nerves given the same stimulus, suggesting a difference in the pathways controlling these nerves. It was determined that the first peak in both cervical and splanchnic were mediated throughout the same pathway, however, the second peak of the splanchnic –which was not present in the cervical– was due to an additional response in the splanchnic to the stimulus. This additional response to the splanchnic might be likely due to some input from unmyelinated afferents, that are selectively absent in the cervical control. The splanchnic receives both A- and C- fibers, whereas the cervical only receives A- fibers, suggesting a non-uniform organizational scheme when it comes to the control of RVLM. This paper provides a little bit of additional evidence into the complexity of RVLM’s control of the sympathetic nervous system, and that each sympathetic nerve has different properties to its control and function. There are likely different neurons controlling the different nerve activities, but also different afferents projecting to these nerves. My presentation and the study paired with it is another piece of evidence to RVLM’s sympathetic control, showing that adrenal and splanchnic control are mediated differentially by GABA following baroreflex unloading. -MTL

Development of attenuated baroreflexes in obese Zucker rats coincides with impaired activation of nucleus tractus solitarius.

Guimaraes PS, Huber DA, Campagnole-Santos MJ, Schreihofer AM. Am J Physiol Regul Integr Comp Physiol. 2014 May;306(9):R681-92. In this paper, they wanted to look at some of the changes that might be responsible for increases in SNA and MAP, and the blunted baroreflex seen in obese zucker rats (OZR) compared to lean zucker rats (LZR). They pharmacologically lowered MAP and saw that while LZR and juvenile OZR (jOZR) had an immediate tachycardia, this response was attenuated in OZR for the first five minutes, but it eventually caught up with the other two grops. They looked at c-Fos expression in the RVLM, and didn't see any differences between the groups. However, when they increased MAP with injections of PE, they saw that OZR had attenuated bradycardia and increases in MAP, possibly due to the fact that they also showed less c-Fos in the NTS. They then moved on to doing microinjections and found that injections of glu in to the CVLM induced similar decreases in SNA, HR, and MAP between all three groups, but injections in to the NTS produced attenuated responses in OZR. The conclusion here was that changes in sensitivity in the NTS, but not the CVLM or the RVLM, seem to be responsible for the systemic effects seen in adult OZR. However, they did also find that jOZR showed greater c-FOS in the NTS than juvenile LZR after injection of PE, which suggests that the changes start occuring in the brainstem early, but don't have systemic effects until the rats become adults. - DH

Magnetic Resonance Imaging of Cortical Connectivity

Canals, S., et al. "Magnetic resonance imaging of cortical connectivity< i> in vivo." Neuroimage 40.2 (2008): 458-472. Understanding the vast potential of manganese-enhanced MRI as a neuronal tract tracing tool for examining complex neural connections, this study by Canals et. al looked to characterize direct injections of manganese into the cerebral cortex, as well as better define efferent connectivity from of the somatosensory and motor regions. Specifically, a variety of manganese concentrations (0.05-0.80M) and volumes (10-200nl) were injected into the somatosensory and motor cortex using an osmotic pump and cannulae. Following cranial injections (multiple protocols), immunohistochemistry and fluorescence microscopy were performed to determine the extent of neuronal damage/toxicity. An initial study using injections of 200nl .4M and .8M MnCl2 in water revealed that injections of 200nl .8M MnCl2 produced extensive cell death and astrogliosis. This is important because cell viability is critical when performing longitudinal studies. Also, damaged tissue compromises the spread and uptake of acute manganese, effecting acute studies as well. A second protocol using multiple injections with varying manganese concentrations with an array of pH's demonstrated that injections of higher concentrated manganese and greater acidity produced larger neuronal lesions and increased astrogliosis. On the basis of the characterization results, this study proclaimed infusion of 100mM MnCl2 in 80nl at a rate of .5nl/min produced the most optimal results with the lowest toxicity. The later half of the study was then designed to investigate the efferent projections from the somatosensory and motor cortex using the previously defined manganese solution against non-optimal solutions (acidic, hypertonic,and concentrated). Importantly, the results from this second study showed better signal enhancement following an injection of 8nmol 100mM MnCl2 pH 7.3 than 8nmol 800mM MnCl2 pH 5.5 in multiple brain regions the somatosensory cortex projects to, including the thalamus and S2. Also, using the optimal manganese solution also allowed for the visualization of trans-synaptic tracing. With an appropriate manganese solution identified, they then examined the possibility of enhancing multiple neuronal pathways with multiple injection sites. Conclusively, it was determined using 3D analysis that it is possible to not only image multiple neuronal pathways, but also to image and analyze the overlap between these pathways using statistical connectivity maps. Interestingly, they were also able to study and quantify connectivity strength though the corpus callosum using this same 3D statistical mapping analysis. Conclusively, the final study revealed that using a microosmotic pump to slowly infuse large amounts of manganese (24nmol .25ul/hr compared to the 8nmol .5nl/min)allowed for visualization of brain regions previously unidentified using the initial faster injection. However, there are still experiments being done to mearue teh time course of transport and effective injection site viability. Overall, this was a large descriptive study that examined many factors of direct injections of MnCl2 into the brain, that will be an extremely helpful reference for our future studies. ~JI

Neurosteroid modulation of arterial baroreflex function in the rostral ventrolateral medulla

Cheryl M. Heesch. Autonomic Neuroscience Volume 161, Issues 1–2, 26 April 2011, Pages 28–33 DOI: 10.1016/j.autneu.2010.10.002This article was interesting because it showed that a microinjection of 3α-hydroxy-dihdroprogesterone may alter the GABAA receptor sensitivity. They microinjected of 3α-hydroxy-dihdroprogesterone into the RVLM of female virgin rats and looked at blood pressure and renal sympathetic nerve activity (rsna). They found that 15 minutes following the microinjection of 3α-hydroxy-dihdroprogesterone significantly reduced the baroreflex rsna gain in response to hypotension. However, when the inactive isomer of 3β-hydroxy-dihdroprogesterone was microinjected into the rvlm it did not alter the baroreflex. These data suggests that 3α-hydroxy-dihdroprogesterone may alter the activity of the GABAA receptor in the RVLM. Since 3α-hydroxy-dihdroprogesterone can be synthesized in the brain it may be possible that the synthesis of this metabolite could be altered under different pathophysiological conditions. I think that this relevant to us because we don’t know how production of neurosteroids in the brain is altered in exercise and this could be another future potential avenue that may be pursued.-MD