Ventrolateral Medulla AT1 Receptors Support Arterial Pressure in Dahl Salt-Sensitive Rats

By Saturo Ito and Alan Sved

Angiotensin II acting in the brain has been implicated in the pathogenesis of hypertension. The site at which angiotensin acts to maintain increased AP in hypertensive rats is unknown (at the time this paper was written). It is thought that the RVLM could play a role for a number of reasons, one of which is the high concentration of angiotensin receptors (AT1 receptors). The RVLM may also play a role in the effects of changes in dietary salt intake on cardiovascular regulation.

This study tested the hypothesis that activation of RVLM AT1 receptors contributes to the increased AP in rats fed a diet high in sodium. The role of the PVN in the maintenance of resting AP was also examined.

All rats were initially fed a diet containing 0.3% NaCl for at least 4 weeks and then some were switched to a high salt diet (8% NaCl) 4 weeks prior to experiments. Rats were anesthetized and prepared for measuring AP and HR. The RVLM was identified with glutamate injections before ANG II injections followed by valsartan (binds to AT1 receptors and inhibits ANG II action) injections. For PVN experiments, rats were first tested with injections of bicuculline (GABA receptor antagonist) followed by injections of muscimol (GABA receptor agonist).

It was found that injection of valsartan into the RVLM or injection of muscimol into the PVN produced a significant decrease in MAP in the high-salt diet hypertensive rats, whereas these treatments had little effect on MAP in low-salt diet rats. These results suggest that RVLM AT1 receptors are tonically activated in hypertensive rats. Additionally, they suggest that the PVN may contribute to the maintenance of baseline AP by way of a tonically active angiotensin-mediated input to the RVLM.

-BH

Acute sympathoexcitatory action of angiotensin II in conscious baroreceptor-denervated rats

By Ling Xu and Alan F. Sved

Angiotensin II (ANG II) levels have two competing influences on sympathetic outflow. ANG II seems to increase SNA. However, it also acts as a vasoconstrictor to increases MAP, which in turn stimulates baroreceptors, thereby inhibiting SNA. This makes it hard to determine the direct actions of ANG II. In order to determine what it directly effects, this experiment administered ANG II to sinoaortic-denervated rats (no baroreflex). 

Baseline MAP was higher in SAD than control rats. In control rats, infusion of ANG II rapidly increased MAP, which was accompanied by bradychardia and sympathoinhibition. Over time, HR and LSNA slowly returned to normal by the end of the infusion period. In contrast, denervated rats had a larger initial increase in MAP upon ANG II injection. However, the increase in MAP was accompanied by increased HR and LSNA, rather than the decrease seen in the control group. This indicates that ANG II can produce rapid sympathoexcitation. There are several sites at which ANG II could act to increase SNA and HR. It has been shown to act directly on the heart in high concentrations. Alternatively, in might increase HR by increasing sympathetic neural activity to the heart and/or decreasing parasympathetic neural activity to the heart. ANG II may act on areas of the CNS lacking a blood brain barrier, such as the area postrema. I will expand on its effects on the CNS in future blog posts.

PNMT-containing neurons of C1 cell group express c-fos in response to changes in baroreceptor input

by Alan F. Sved, Dara L. Mancini, Jennifer C. Graham, Ann M. Schreihofer and Gloria E Hoffman

This study used the expression of c-fos as a marker of neuronal stimulation to determine whether decreased baroreceptor afferent activity could activate PNMT-containing (C1) neurons.

Baroreceptor afferents were decreased using two treatments: hydralazine injection (vasodilatoràhypotension) or surgical denervation of the carotid sinus and aortic baroreceptors (artificial hypotension). Control rats received saline injections or sham denervation, respectively. Some rats received Fluorogold injections to retrogradely label bulbospinal RVLM neurons. Finally, neurons were stained for Fos, the protein produced by the c-fos gene. Separate groups of rats had catheters placed in the right femoral artery to record MAP and heart rate in response to hydralazine or sinoaortic denervation.  Stained neurons were counted via light microscopy.

In hydralazine-injected rats, approximately 80% of the PNMT-positive neurons in the RVLM were immunoreactive for Fos. This was true of the entire population of PNMT-positive neurons as well as the specific group of PNMT-positive neurons that were also labeled with Fluorogold. Approximately 45% of the Fluorogold-positive neurons contained PNMT in the hydralazine- and saline-injected groups. Approximately 40% of the PNMT-positive neurons were also label with Fluorogold. If 80% of the spinally projecting C1 neurons are affected by baroreceptor afferent input, and C1 neurons comprise 40% of the bulbospinal RVLM neurons, then greater than one-third of the bulbospinal RVLM neurons that are sensitive to baroreceptor afferent input must be C1 neurons. Neurons in the RVLM express Fos in response to sinoaortic denervation as well as hydralazine injection.

These results are central to a lot of the work we do in our lab.

-BH

Organization of Central Adrenergic Pathways: I. Relationships of Ventrolateral Medullary Projections to the Hypothalamus and Spinal Cord

By Diane Tucker, Clifford Saper, David Ruggiero and Donald Reis

This experiment had multiple purposes including:

- Determining whether PNMT-positive C1 cells contain other marker of catecholamine biosynthesis

- To determine the extent to which C1 neurons in the VLM send collaterals to both the spinal cord and the hypothalamus

- To examine the collateralization of VLM neurons of the A1 cell group that project to PVH and median preoptic nucleus (MnPO)

This summary will only focus on objective #2, since the others are not the relevant to what I am doing.

Dye was injected into rat spinal cord and hypothalamus. Rats were perfused, sacrificed, and their brain tissue was obtained and immunohistochemically processed. Cell counts were performed of:

- Total TH and PNMT positive cells

- The number of cells retrogradely labeled with each fluorescent dye

- The number of cells retrogradely labeled with both fluorescent dyes

- The number of cells labeled with each fluorescent dye that showed immunoreactivity for TH or PNMT

- The number of cells labeled with both fluorescent dyes that showed immunoreactivity for both TH and PNMT

Results showed that about half of the spinal projection cells in the RVLM were PNMT-positive. Catecholaminergic neurons throughout the length of the VLM project to the hypothalamus. The PVH also receives catecholaminergic afferents from the entire VLM. While there is sometimes considerable spatial overlap between cells with different projections (spinal cord, hypothalamus, PVH), very few VLM neurons are found to innervate more than one region.

There was no evidence for noradrenergic projection from the MVLM or CVLM to the spinal cord.

It should be noted that about half of the neurons in the RVLM that project to the spinal cord did not stain for PNMT. These other cell populations may play a role in blood pressure modulation as well.

-BH

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