RVLM: Selective Projections to the Thoracic Autonomic Cell Column from the Region Containing C1 Adrenaline Neurons

By Christopher A. Ross and Donald J. Reis

The purpose of this study was to determine the organization of projections from the RVLM to the thoracic spinal cord and to compare these to the distribution of PNMT-containing terminals in the intermediolateral column (IML).  This was done by first staining for TH and PNMT. Next, some animals had horseradish peroxidase injected into their spinal cord or adrenal medulla to act as a retrograde tracer. Additionally, some animals had HRP injected into their RVLM to act as an anterograde tracer.

The results showed that a large proportion of the projections from the RVLM to the IML are made up of PNMT-containing C1 neurons. This finding, along with previous findings that demonstrated that stimulation of the RVLM results in pressor responses and that lesions of the C1 regions cause a drop in MAP, leads us to believe that the C1 neurons play an important role in blood pressure regulation.

-BH

PNMT-containing terminals synapse directly on sympathetic preganglionic neurons in the rat

by. Teresa A. Milner, Shaun F. Morrison, Cory Abate and Donald J. Reis

The purpose of this study was to determine whether PNMT-containing terminals in the intermediolateral (IML) nucleus influence sympathetic nerve discharge through synapses directly on sympathetic preganglionic neurons (SPNs). This was done by using the peroxidase anti-peroxidase method to localize PNMT-containing terminals. Additionally, horseradish peroxidase (HRP) retrograde identification of SPNs was used to determine whether or not PNMT-containing terminals made direct contact with SPNs. The results showed that PNMT-containing terminals synapse directly on SPNs. This allows us to classify C1s as premotor neurons of the sympathetic nervous system.

Another interesting finding from this study is that PNMT-containing terminals form both asymmetric (excitatory) and symmetric (inhibitory) synapses with dendrites in the IML.

-BH

Rostral ventrolateral medulla: a source of the glutamatergic innervation of the sympathetic intermediolateral nucleus

by Shaun F. Morrison, Janie Callaway, Teresa A. Milner and Donald J. Reis

The purpose of this study was to demonstrate whether or not neurons in the RVLM contribute to the glutamatergic innervation of the intermediolateral (IML) nucleus in the spinal cord. This was done by injecting PHA-L (anterograde label) into the RVLM and subsequently treating the IML with PAP to detect PHA-L. The results confirmed that the axon terminals of RVLM neurons that project to the IML contain glutamate-like immunoreactivity and make excitatory contacts upon local dendrites.

Short but sweet!

-BH

Lesions in Rostral Ventromedial or RVLM Block Neurogenic Hypertension

by Kurt J. Varner, Elisardo C. Vasquez and Michael J. Brody

This study used NMDA-induced lesions of the RVLM or RVMM to determine whether neurons in either region are involved in the acute hypertension and tachycardia produced by sinoaortic deafferentiation (SAD). SAD is a technique used to induce hypertension by surgically interrupting peripheral baroreceptor afferent nerves.

First, NMDA was injected into the RVLM and RVMM. As the name would suggest, NMDA binds to NMDA-receptors, which are the glutamatergic receptors on neurons in the RVLM. However, NMDA does not have the same excitatory effects that glutamate does. Instead, it is an excitotoxin which means that it kills neurons by overexciting them. Following NMDA-lesion, SAD was performed. There were 4 groups used in this experiment: sham lesion (saline injection into RVLM or RVMM), NMDA lesion (lesion of sites rostral to RVLM or RVMM), RVLM lesion and RVMM lesion.

MAP decreased in both RVLM and RVMM conditions. RVLM lesioned animals showed the lowest MAP in both post-lesion and post-lesion + SAD conditions. The lack of hypertensive response likely reflects the loss of baroreceptor-sensitive sympathoexcitatory neurons in these regions. This study shows that both the RVLM and RVMM are involved in the development of neurogenic hypertension under these conditions.

-BH

Medullary GABA Receptors and the Regulation of Blood Pressure in the Rat

by Robert N. Willette et al.

The purpose of this study was to determine the cardiovascular effect of a GABAergic agonist and antagonist upon vasopressor and vasodepressor motoneuron pools in the ventrolateral medulla. Rats underwent microinjection of muscimol (GABA receptor agonist) to vasopressor (VLDA) and vasodepressor (VLPA) sites in the ventrolateral medulla.

Muscimol injection to VLDA sites consistently caused an increase in BP, HR and pulse pressure. Pretreatment with alpha adrenergic blockers abolished the pressor effect of muscimol. It should be noted that increases in HR were resistant to this blockade. Muscimol injection to VLPA sites caused a fall in BP, HR and pulse pressure. Additionally, injection of biuculline (GABA receptor antagonist) reversed the effects of muscimol in both VLDA and VLPA sites.

These results indicate that VLPA and VLDA sites are tonically active in maintaining BP. The results also suggest that a GABAergic system may be involved with the modulation of neural activity in these areas.

-BH

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

Many previous studies have shown that sedentary conditions can result in enhanced nerve activity following activation of the rostral ventrolateral medulla (RVLM). This means that there is some type of mechanism that differs between active and sedentary animals in terms of the excitation and inhibition that occurs in the RVLM. The RVLM is primarily regulated by glutamate, an excitatory neurotransmitter, and GABA, an inhibitory neurotransmitter. This study was trying to determine how the glutamate could directly excite the RVLM, and the effects that blocking the GABA pathway may have on the response to glutamate. The hypothesis was that sedentary conditions would enhance sympthoexcitatory response to direct activation of the RVLM, with the responses increasing further by blocking tonic GABAergic transmission.

The methods of the experiment were similar to our microinjection protocols in terms of exposing the RVLM and recording nerve activity and blood pressure. The first protocol involved injection 1, 10, and 100mM concentrations of glutamate into the RVLM while recording the blood pressure, heart rate, and lumbar sympathetic nerve activity (LSNA). The second protocol administered a GABA receptor blocker called bicuculline before glutamate injections were performed. The glutamate was injected 5, 10, 15, 30, and 45 minutes after the bicuculline was administered to investigate the changes in nerve activity after certain periods of time. The third protocol was a control section in which glutamate response was tested in the presence and absence of artificial cerebral spinal fluid.

In protocol one, responses to varying concentrations of direct glutamate administration did not differ between the sedentary and active animals. This is because heart rate responses were small and increases in LSNA also did not differ significantly between the active and sedentary animals. In protocol two, the initial injections of bicuculline increased the baseline blood pressure, heart rate, and LSNA in both the active and sedentary animals prior to any glutamate being injected. The increases did not differ significantly between the two groups. The glutamate injection responses at 5 and 15 minutes after the bicuculline was administered were greatly enhanced in the sedentary animals compared to the physically active animals. These enhancements were observed as increases in mean arterial pressure, however, were no longer observed by the 30- and 45-minute marks. Increases in LSNA were enhanced at the 5-, and 15-minute marks as well, however there was not a significant difference between the sedentary and active groups.  Once again, these effects were no longer observed at 30 and 45 minutes. This suggests that there was full recovery from bicuculline within the time course mentioned. Protocol three showed that responses to glutamate injections at different time points after injection of the bicuculline vehicle (aCSF) did not differ significantly from the control microinjections. In addition, responses to the repetitive microinjections did not differ significantly between the active and sedentary groups.

Overall, there were results that both confirmed and opposed the hypothesis. The hypothesis was confirmed in that there was enhancement in the response to glutamate in sedentary animals after the GABA receptor block was administered in the RVLM.  However, the LSNA responses did not differ between groups under direct glutamate activation nor under GABA receptor blockade conditions.

Based on the results, the paper determined several new findings that helped distinguish the effects of sedentary verses active conditions on nerve activity and blood pressure. First, sedentary conditions enhance GABAergic control of glutamate-sensitive neurons in the RVLM that regulate blood pressure. Second, sedentary conditions increase nerve activity when glutamate is administered in the absence of GABAergic modulation. Lastly, LSNA does not control the responses recorded in animals after GABA receptor blockers were administered.

This study helps us to possibly think about what other protocols we may want to add to the microinjection experiments. We could find other drugs that block glutamate or GABA responses in the RVLM and then see what effects present themselves when glutamate and GABA doses are given in the active and sedentary animals.

 

-Lyndsey M.

 

Blockade of Rostral Ventrolateral Medulla Apelin Receptors Does Not Attenuate Arterial Pressure in SHR and L-NAME induced Hypertensive Rats

Philip R. Griffiths, Stephen J. Lolait, Louise E. Pearce, Fiona D. McBryde, Julian F.R. Paton, Anne-Marie O’Carroll. Frontiers in Physiology (October 2018)

Apelin is a neuropeptide found in many organs, including the brain. It is thought to regulate the intake of food, water, and the release of vasopressin. Previous studies have shown that apelin receptors play a role in heart failure, hypertension, and heart diseases.  Apelin has been shown to be increased in the Rostral Ventrolateral Medulla (RVLM) in hypertensive rats, which is the main control center for sympathetic activity. This study focuses on if the apelin receptor plays a role in the progression of hypertension in two different models and if it is also part of the development stage of hypertension.

Thirty-six Wistar Kyoto (WKY) rats, sixteen spontaneous hypertensive rats (SHRs), and twenty younger SHRs were used in these studies. To generate the L-NAME model of hypertension, six WKY rats were given a daily dose of a nitric oxide synthase inhibitor, which increased sympathoexcitation and leads to hypertension. Lentiviruses with an apelin receptor gene knockout were created and injected into the RVLM. Control lentiviruses without the knockout were also created and injected directly into the RVLM. Injections of an apelin receptor agonist were used to test the knockout of the gene and then glutamate injections confirmed RVLM location.

Researchers first looked at the micropunches of the RVLM and found that the protein levels of apelin and the receptor gene were significantly higher in SHRs, when compared to the control rats. When observing cardiovascular effects, researchers started by measuring the baseline blood pressure and observed it was significantly higher in the SHRs. Microinjections of apelin-13 were then given directly into the RVLM, which exhibited a significant increase in MABP and systolic blood pressure in both SHRs and the controls. When rats were injected with a saline solution in the place of the apelin-13, no increases were observed. The increases in blood pressure were much greater in the SHRs compared to the controls. When an apelin receptor antagonist was injected, only the increases from the apelin-13 cancelled out, but no further decrease occurred. Researchers then used a lentivirus vector to knockout the apelin receptor. The lentivirus delivery was confirmed using immunofluorescence and observing the expression within the RVLM 25 days after they were injected. The lentivirus was shown to decrease expression of the receptor by about 65% at day 25. Apelin-13 was then injected into the knockout and control rats. The control lentivirus rats exhibited a significant increase in blood pressure, while the knockout rats had no increase. Using injections of glutamate to also confirm the pipette was in the correct place, similar blood pressure increases in the lentivirus knockout and the control rats were observed. 

There were no significant differences in heart rate or blood pressure in the normotensive control rats given the lentivirus knockout or lentivirus control. Similarly, the blood pressure drops caused by an injection of hexamethonium exhibited no significant difference. The SHRs and the L-NAME treated rats exhibited higher blood pressures at the beginning of the experiments, which is expected with those models, though the respiration rate and the heart rate were similar to the normotensive rats. SHRs and the L-NAME treated models exhibited no significant difference in heart rate, blood pressure, hexamethonium- induced blood pressure drop, and body weight between the lentivirus knockout and lentivirus control rats. When the RVLM was injected with apelin-13 25 days after the lentivirus delivery, SHRs and L-NAME treated rats with the knockout exhibited a decrease in the apelin receptor gene and blood pressure, when compared to the rats treated with the lentivirus control.

Researchers then tested if a SHR was injected with apelin-13 when it was still young, would hypertension would develop? In SHRs that only received the lentivirus knockout or control, blood pressure remained very similar after 10 weeks. In SHRs that received the lentivirus and an injection of apelin-13, blood pressure was observed to slightly decrease in rats given the lentivirus knockout and increase in rats with the lentivirus control.

In conclusion, although the mechanisms are still not fully understood, it appears that the apelin receptor does not have a role in the development of high blood pressure or hypertension. These results also suggest that if the apelin receptor gene is knocked out in SHRs, the onset of hypertension is not slowed or prevented. Injections of the receptor agonist were shown to increase blood pressure and when the agonist and a receptor antagonist were given after one another, the agonist induced increases were eliminated. When just the antagonist was given to the SHRs, there was no change in blood pressure, which suggest these receptors do not play a role in the regulation of high blood pressure in hypertensive rat models.

-Paul M