The sympathetic role of glutamatergic paraventricular nucleus neurons in blood pressure regulation

Brief summary

Hypertension is among the most powerful modifiable risk factors for cardiovascular and cerebrovascular diseases. Although the underlying mechanism is complex and still under investigation, it is well‐accepted that the sympathetic nervous system probably plays a central role. A vast network of neurons, including the hypothalamic paraventricular nucleus (PVN) and regions of the brainstem are crucially involved in autonomic control of blood pressure (BP). PVN circuitry provides tonic input to the rostral ventrolateral medulla (RVLM) controlling sympathetic outflow through an array of glutamatergic and gamma‐aminobutyric acid (GABA)‐ergic neurons. However, it is still unknown how PVN glutamatergic neurons contribute to BP regulation. In a recent issue of The Journal of Physiology, Basting and colleagues (2018) hypothesized that overexcitation of the PVN‐RVLM glutamatergic interneuronal population leads to excess sympathetic outflow, evoking an increase in baseline BP and contributing to hypertension.

To address the unknown role of PVN‐RVLM excitatory glutamatergic interneurons in the development of hypertension through overactivation of the sympathetic nervous system, Basting et al. (2018) completed a set of experiments in mouse models that involved unilateral and bilateral manipulation of PVN‐RVLM excitatory glutamatergic interneurons, and assessed their role in both RVLM activity and BP. First, the researchers conducted an in vivo optogenetic procedure in conscious mice to investigate the effects of PVN‐RVLM glutamatergic interneuronal activation on BP using channelrhodopsin‐2 (ChR2) under the calcium/calmodulin‐dependent protein kinase type Iiα (CaMKIIα) promoter (AAV‐CamKIIα‐ChR2‐eYFP sero‐type 2) in a selective manner such that they would activate these neurons unilaterally. This approach demonstrated causally the role of PVN‐RVLM excitatory glutamatergic interneurons in increasing BP to hypertensive levels. Second, PVN‐RVLM excitatory glutamatergic interneuronal lesioning using AAV‐flex‐taCasp3‐TEVp in a bilateral manner in vesicular glutamate2 Cre‐recombinase (vGlut2‐Cre) mice was used to test the effects of complete disruption of excitatory signalling between the PVN and RVLM on downstream sympathetic outflow and BP. For further investigation, the researchers used the deoxycorticosterone acetate (DOCA)‐salt model to investigate the role of PVN‐RVLM excitatory glutamatergic interneurons in the development of hypertension through gain‐ and loss‐of‐function procedures. This study supported their hypothesis by demonstrating that disrupting a portion of the PVN‐RVLM excitatory glutamatergic interneurons neuronal population led to a loss of both BP regulation and sympathetic outflow, contributing to the manifestation of hypertension.

Interpretation of results

Using optogenetic stimulation of the PVN‐RVLM excitatory glutamatergic interneurons, Basting and colleagues (2018) showed that there is a clear and immediate positive relationship between the frequency of stimulation and increase in BP. The proposed mechanism for the observed frequency‐dependent BP increase is due to increased excitation of the RVLM, specifically through PVN‐RVLM excitatory interneurons. This supports the concept that hypothalamic inputs to the brainstem play an important role in BP control and modulating downstream responses (Guyenet, 2006).

Bilateral PVN‐RVLM excitatory glutamatergic interneuronal lesions were completed using Cre‐dependent caspase to disrupt PVN‐RVLM excitatory glutamatergic interneurons. The results showed diminished BP modulation in the lesioned group in comparison to controls during DOCA‐salt‐induced hypertension. Furthermore, lesioning of the PVN‐RVLM excitatory glutamatergic interneurons in non‐DOCA animals unexpectedly raised noradrenaline (NA; norepinephrine) levels, which may demonstrate an inhibitory role for PVN to release NA under normal conditions. It is possible that PVN‐RVLM excitatory glutamatergic interneurons control BP through largely renal mechanisms (e.g. elevated angiotensin II, sodium retention, elevated renin), while the nucleus tractus solitarius (NTS)‐RVLM interneurons largely influence vascular NA release. If this was the case, this may explain the elevation in NA during hypotension induced by PVN‐RVLM excitatory glutamatergic interneuronal lesioning, as the baroreflex would be unloaded. Regarding the significant increase in GluR1 mRNA expression (P = 0.017) and a small but non‐significant increase (P = 0.052) in GAD67 mRNA expression within the RVLM in the lesioned animals, a possible mechanism for this may be upregulation of RVLM glutamate α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in NTS‐RVLM projections and other unknown excitatory inputs in the RVLM.

With the multifactorial pathology of hypertension, this paper sheds light on the necessity of PVN‐RVLM excitatory glutamatergic interneurons in appropriate autonomic function. The results highlight the crucial role of excitatory neuronal populations within midline brain structures in the regulation of sympathetic outflow and autonomic function.

Experimental strengths and limitations

Strengths:

1. Using selective optogenetic stimulation approach, the investigators were able to activate the PVN‐RVLM excitatory glutamatergic interneurons in conscious animals using a full range of physiological firing levels.

2. Inhibition of the PVN‐RVLM excitatory glutamatergic interneurons was also performed using selective lesioning approaches. Bilaterally injecting flex‐taCasp3‐TEVp resulting in partial lesioning of the PVN‐RVLM excitatory glutamatergic neurons was sufficient to increase sympathetic activity and blunt the rise in BP in the DOCA‐salt‐treated animals.

3. Another strength is the authors’ decision to address a potential role for the endocrine system in the BP responses to PVN‐RVLM excitatory glutamatergic interneuron lesioning. The unchanged levels of AVP between control and lesioned groups indicate that increased sympathetic drive was due to RVLM‐mediated descending pathways, as opposed to through endocrine pathways.

Limitations:

1. Some outcome measures might have benefited from increasing the sample size. The observed differences in GAD67 expression may have reached statistical significance with additional animals. However, we do acknowledge the complex experimental design limits the feasible number of animals that can be included in this stream of the study.

2. The lesions produced a 39.3% decrease in PVN‐RVLM excitatory glutamatergic interneurons. It is likely that more significant differences in BP and sympathetic activity would be observed if a higher density of PVN‐RVLM excitatory glutamatergic neurons were lesioned.

Despite these limitations, the injection of ChR2 vectors and the utilization of in vivo photostimulation is a unique technique that expands our understanding of the role PVN‐RVLM excitatory glutamatergic interneurons in hypertension. Such an approach can be applied to explore interactions up‐ and downstream of the PVN, as well as GABA downregulation and glutamatergic neuroplasticity.

Future directions and clinical implications

The influence of afferent inputs (i.e. subfornical organ (SFO)) and efferent outputs (i.e. RVLM) to and from the PVN are clearly essential to clarify the role of PVN in neurogenic hypertension. For example, the RVLM glutamate AMPA receptors may have played a compensatory role due to the loss of PVN‐RVLM excitatory glutamatergic interneurons. However, RVLM glutamate AMPA receptors were unable to compensate for the reduction of baroreceptor modulation. A future direction would be to evaluate if there is compensatory activation of RVLM glutamatergic AMPA neurons.

PVN‐RVLM GABAergic interneurons may have an equally important role to that of PVN‐RVLM excitatory glutamatergic interneurons. Yet no study has investigated the possibility of GABA downregulation in PVN‐RVLM excitatory glutamatergic interneurons. Thus, the role of PVN‐RVLM GABAergic inhibitory interneurons should also be explored in the downregulation of PVN‐RVLM excitatory glutamatergic interneurons, homeostatic mechanisms and firing rate activities. Such findings could lead to novel therapeutic approaches in the treatment of chronic drug‐resistant hypertension.

Finally, the data from the current study also indicates an immediate BP rise following stimulation of the PVN‐RVLM excitatory glutamatergic interneurons. The possibility of applying these findings in the clinical setting is promising (Phillips & Krassioukov, 2015). For instance, spinal cord injury patients suffer frequently from dramatic BP instability (e.g. orthostatic hypotension (OH)) which leads to cardiac and cerebral complications (Phillips et al. 2016). Better understanding of the PVN‐RVLM excitatory glutamatergic interneuronal circuitry and communication relays may reveal new potential therapies. It would also be informative for understanding the PVN‐locus coeruleus (LC) interneuronal circuitry in more detail, as the LC provides perivascular input to the cerebrovasculature and over/under activation of this pathway may be an additional mechanism underlying the greater risk of stroke in people with spinal cord injury, hypertension and low resting BP (Phillips et al. 2017).

Conclusion

This interesting study provides evidence supporting a role for glutamatergic PVN‐RVLM excitatory glutamatergic interneurons in BP regulation within the context of elevated BP, innovatively using optogenetic techniques to manipulate highly specific brain circuits in conscious animals. From a clinical perspective, high BP is a leading cause of death and this research provides insights into the underlying mechanisms.

Additional information

Competing interests

None declared.

Author contributions

All authors contributed equally in drafting/revising, analysis or interpretation of data in this manuscript. J.E.S. prepared the final draft of the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

None.