The hypothalamic-neurohypophyseal system: from genome to physiology

Abstract

The elucidation of the genomes of a large number of mammalian species has produced a huge amount of data on which to base physiological studies. These endeavours have also produced surprises, not least of which has been the revelation that the number of protein coding genes needed to make a mammal is only 22 333 (give or take). However, this small number belies an unanticipated complexity that has only recently been revealed as a result of genomic studies. This complexity is evident at a number of levels: (i) cis-regulatory sequences; (ii) noncoding and antisense mRNAs, most of which have no known function; (iii) alternative splicing that results in the generation of multiple, subtly different mature mRNAs from the precursor transcript encoded by a single gene; and (iv) post-translational processing and modification. In this review, we examine the steps being taken to decipher genome complexity in the context of gene expression, regulation and function in the hypothalamic-neurohypophyseal system (HNS). Five unique stories explain: (i) the use of transcriptomics to identify genes involved in the response to physiological (dehydration) and pathological (hypertension) cues; (ii) the use of mass spectrometry for single-cell level identification of biological active peptides in the HNS, and to measure in vitro release; (iii) the use of transgenic lines that express fusion transgenes enabling (by cross-breeding) the generation of double transgenic lines that can be used to study vasopressin (AVP) and oxytocin (OXT) neurones in the HNS, as well as their neuroanatomy, electrophysiology and activation upon exposure to any given stimulus; (iv) the use of viral vectors to demonstrate that somato-dendritically released AVP plays an important role in cardiovascular homeostasis by binding to V1a receptors on local somata and dendrites; and (v) the use of virally-mediated optogenetics to dissect the role of OXT and AVP in the modulation of a wide variety of behaviours.

Fig.1. A pyramid of complexity in genome expression

There are thought to be approximately 22,333 protein coding genes in the mammalian genome, but though processes such as alternative slicing and RNA editing, this small number can give rise to perhaps millions of protein isoforms, which are then subject to a whole range of post-translational modifications that can alter their activity. Proteins feedback to the genome and transcriptome to control gene expression and transcript maturation. In addition, it is now recognised that the genome hosts the information that give rise to millions on ncRNAs, transcripts that do not code for protein. These ncRNAs impose their regulatory will at all levels of the gene expression pathway (represented by the yellow splodge). It remains to be determined how this pyramid of complexity imposes physiological order.

Fig. 2

Approach used for collecting peptide release in vitro from a hypothalamic brain slice. These results demonstrate the complexity of peptides released even from a defined brain region such as the SCN. (A) Sample collection and preparation scheme. (B) MS-based characterization of SCN releasates. (Inset: a zoomed mass range.) Labeled analytes are as follows: (a) AVP, (b) proSomatostatin 89–100, (c) substance P, (d) PENK 219–229, (e) melanotropin a, (f) somatostatin 14, (g) pyro-glu neurotensin, (h) big LEN, (i) little SAAS, (j) unknown 2028.02 m/z, (k) PEN, (l) unknown 2380.10 m/z, (m) unknowns 2481.26/2481.77 m/z, (n) galanin, and (o) thymosin β-4. Work adapted with permission from PNAS (79).

Fig. 3

Four transgenic rats that express AVP-eGFP, OXT-mRFP, c-fos-eGFP and c-fos-mRFP were generated and can be bred to create informative double transgenic rats. AVP-eGFP and OXT-mRFP are cytosolic fusion proteins, whereas c-fos-eGFP and c-fos-mRFP are nuclear.

Fig. 4

By utilising the facile monitoring of different fluorescent proteins under the control of different promoters, the temporal pathway of a physiological response to a stimulus can be readily monitored in identified neuronal populations of the HNS.

Fig. 5

Schematic illustration of pharmacological (A) and transgenic (B) approach for studying receptor function. Adenoviral transfection in PVN induces more selective and longer-lasting changes of V1a receptor function than microinjection of drugs.

Fig. 6

Typical recording of systolic blood pressure and heart rate using radiotelemetry of one eGFP transfected rat under baseline conditions (left) and during exposure to air-jet stress (right). SBP: systolic blood pressure; HR: heart rate; eGFP: rats transfected with enhanced green fluorescent protein. BP and the HR of eGFP transfected rats exhibit normal values of hemodynamic and baroreflex parameters under basal conditions (Table 2). Exposure to stress induced an increase in BP and HR and a decrease in baroreflex sensitivity and re-setting of baroreflex toward higher SBP and lower PI values comparable to wild-type rats (102,103). Under basal physiological conditions, rats over-expressing V1a receptors in PVN have comparable values of BP and HR to eGFP transfected rats. However, V1a transfected rats exhibit reduced sensitivity and operating range of the baroreflex under basal physiological conditions. When exposed to air-jet stress, V1a transfected rats exhibited similar increase of SBP, HR, suppression of baroreflex sensitivity and re-setting towards higher SBP and lower PI values (Table 2). However, the operating range of the baroreflex of these rats during exposure to stress was significantly increased (Table 2).