4-year PhD Studentship: Chemogenetic modulation of neuronal activity as a refined alternative to physiological stress

High blood pressure is one of the biggest risk factors for cardiovascular disease (1). The hypothalamus regulates neuroendocrine homeostasis and helps to maintain metabolic processes such as body weight, fluid balance and blood pressure (2). Secretion of the neuropeptide hormone arginine vasopressin (AVP) is a neuroendocrine response to maintain fluid and blood pressure homeostasis. AVP is synthesised by hypothalamic magnocellular neurons located predominantly in the supraoptic (SON) and paraventricular (PVN) nuclei and is transported via axons projecting to the posterior pituitary (PP) gland where it is stored (3).

Traditionally, water restriction experiments have been used to stimulate AVP secretions and study its physiological effects (4). Water restriction causes distress to an animal, and periods longer than a few days are fatal, making it unsuitable for long term studies.

Chemogenetics is an emerging and increasingly popular tool in neuroscience (5). This technique allows neurones to be activated (using activating hM3Dq receptors) or silenced (using inhibitory hM4Di receptors) in defined temporal windows by delivery of the inert designer drug, clozapine N-oxide (CNO) (6). This offers the possibility to manipulate AVP expression long term, in awake animals behaving in a natural way without the need for harmful fluid or dietary changes.

Aims and Objectives

We aim to bring together the latest advances in chemogenetics, molecular biology and imaging to improve animal welfare. We intend to deliver three methodological refinements during this project that, in combination will enable us to stimulate AVP neurons in-vivo by the least invasive and stressful means possible: 1) we will replace the use of dehydration protocols by developing chemogenetics to study the AVP system, 2) we will refine methods of adeno-associated virus (AAV) administration to deliver hM3Dq and hM4Di receptors to the brain, and 3) we will improve and refine the administration of CNO.

Methodology

We will develop retrograde AAVs to express hM3Dq in the SON and PVN under the control of the AVP promoter (AAV AVP-hM3Dq-mCherry), using several molecular biology techniques and virus production approaches. The PP provides a unique route, consisting of distal axons of hypothalamic magnocellular neurons, that allows to deliver hM3Dq to the SON and PVN by performing a single stereotaxic injection to the PP using retrograde AAVs. The expression of the AAV reporter will then by mapped using the optical clearing technique CLARITY and 3D immunohistochemistry with lightsheet microscopy.

Next, we will confirm effectivity of AAV AVP-hM3Dq-mCherry following the delivery of CNO by assessing expression of the immediate early gene c-fos (an indicator of neuronal activity) in brain by immunohistochemistry and measuring secretion of AVP into the bloodstream by ELISA.

Finally, we will assess the effects of chronically activated AVP neurones using osmotic pumps for chronic deliver of CNO. Delivery pumps reduce the need for multiple daily injections of CNO, reducing animal stress. After implantation of pumps, AVP plasma levels will be measured from blood, and we will perform RNA sequencing to compare changes in gene expression in the PVN and the SON with our datasets from chronic dehydration (7).

Keywords

Vasopressin, chemogenetics, neuroendocrinology, animal welfare, refinement, reduction

How to apply for this project

This project will be based in Bristol Medical School - Translational Health Sciences in the Faculty of Health Sciences at the University of Bristol.

Please visit the Faculty of Health Sciences website for details of how to apply