Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA299

Poster Communications

Functional connection of prolactin-releasing peptide neurones from dorsomedial to paraventricular hypothalamic nucleus

C. H. Feetham1, N. Nunn1, S. M. Luckman1

1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

Prolactin-releasing peptide (PrRP) is expressed in three neuronal populations of the brain; the dorsomedial hypothalamus (DMH), the nucleus tractus solitarii and the ventrolateral medulla1. We have shown that the thermogenic action of leptin is dependent upon PrRP neurones in the DMH2. Others have shown PrRP fibres are present within the paraventricular nucleus of the hypothalamus (PVN), in apposition with oxytocin- and corticotrophin-releasing hormone (CRH)-expressing neurones1,3. Furthermore, intracerebroventricular PrRP activates PVN oxytocin and CRH neurones of wild-type but not PrRP receptor knock-out mice4. It is unknown which PrRP population is responsible for these effects, demonstrating the need to decipher the neuronal circuitry involved in mediating the effects of PrRP. Tracing studies were performed using PrRP-cre::EYFP reporter mice injected with cre-dependent anterograde tracer AAV-hSynaptophysin-mCherry into the DMH (DMH-PrRPhsyn)5. During surgery mice were initially anaesthetised with 3% gaseous isoflurane, and maintained with 1-2%. Mice were left for two weeks before further anaesthesia and perfusion with 4% paraformaldehyde. Projections were confirmed with retrograde tracer injections into the following areas in PrRP-cre::EYFP mice: medial preoptic (MPO), median preoptic (MnPO), PVN and raphe pallidus (RPa). PrRP-cre::EYFP mice were also injected intra-DMH with a cre-dependent stimulatory DREADD hM3Dq-mCherry (DMH-PrRPhM3Dq) and administered saline or clozapine N-oxide (CNO intraperitoneal, 1 mg/kg). Immunohistochemistry using DsRed and GFP antibodies verified spread of tracers and colocalisation with the EYFP reporter. PVN projections were investigated using DsRed and oxtyocin antibodies in DMH-PrRPhsyn mice. Neuronal activation of DMH-PrRPhM3Dq mice was verified by immunohistochemistry for DsRed and c-Fos. All results are mean±SEM. DMH-PrRPhsyn injections (n=3) confirmed projections from DMH PrRP neurones to the PVN, but also to the MnPO, MPO, as well as local projections in the DMH. No projections were seen to the RPa. Investigation of PVN projections confirmed proximity of PrRP terminals with oxytocin neurones in the PVN (ipsilateral side); 34±6% anterior PVN (n=4), 26±4% medial PVN (n=3) and 49±9% posterior PVN (n=3). Projections to the PVN (n=4), MnPO (n=5) and MPO (n=4), but not in the RPa (n=3) were confirmed also by colocalisation of retrograde tracer in PrRP DMH neurones. Furthermore, DMH-PrRPhM3Dq mice injected with CNO showed increased c-Fos in the PVN compared with saline-treated mice; 104±17 cells (n=5) vs 28±5 cells (n=6), respectively (p<0.01, unpaired t-test). Our results show DMH PrRP neurones project to brain areas involved in thermogenesis. In addition, we have shown a functional connection to neurones within the PVN, an important area for modulation of sympathetic activity.

Where applicable, experiments conform with Society ethical requirements