Characterization and stress induced modulations of RXFP3+ neurons in the mouse BNST

Abstract

Stress induced relapse and stressful emotional responses occurring during drug withdrawal are common problems for drug addicts. The relaxin-3/relaxin family peptide receptor 3 (RXFP3) system modulates stress responses and reward seeking behaviour. RXFP3 is expressed within the bed nucleus of the stria terminalis (BNST), and bilateral intra-BNST injections of an RXFP3 antagonist decreased self-administration and stress induced reinstatement of alcohol-seeking in rats. The electrophysiological properties of RXFP3+ neurons in the BNST and whether they are responsive to stress are presently not known. Similarly, the possibility of convergent signalling with other neuropeptides such as corticotropin releasing factor (CRF) in the BNST is not clear either.

Therefore, whole cell recordings were conducted on defined RXFP3+ neurons in brain slices taken from adult RXFP3-Cre x tdTomato mice (n=38 mice for naive group). To study any alterations in RXFP3+ dorsal BNST neurons induced by chronic stress, a group of mice (n=13) were subjected to swim stress daily for 5 days. Mice were anaesthetised (5% isoflurane), decapitated and 250 µm coronal slices through the BNST were obtained.

Based on Hammack’s characterization, the majority of recorded RXFP3+ neurons (~75%) were classified as Type II neurons and a new type of neuron, Type IV neurons were found within the dorsal BNST. Surprisingly, the electrophysiological profile of RXFP3+ neurons was not fixed; five days of swim stress induced plasticity, shifting a population of Type II neurons to Type III or Type IV neurons. Notably, stress induced plasticity of BNST RXFP3+ neurons was not only found in terms of neuron types, but also in their responsiveness to afferent inputs. The decay time of both spontaneous inhibitory postsynaptic currents (P=0.0005, Mann-Whitney test) and spontaneous excitatory postsynaptic currents (P=0.0028, Mann-Whitney test) were significantly increased after stress (naive, n=70 cells from 38 mice; stress, n=32 cells from 13 mice).

Most BNST RXFP3+ neurons from the naive group (6 out of 9) were hyperpolarised following bath application of a selective RXFP3 agonist (A2, 100 nM), in line with an inhibitory role of this neuropeptide. Bath application of CRF (100 or 300 nM) onto brain slices during gap free voltage clamp recordings were conducted (n=14) to examine potential interactions between these peptide systems. CRF induced an increase of GABA release onto RXFP3+ neurons (~40% of the RXFP3+ neurons recorded), but decreased GABA release for ~35% of RXFP3+ neurons. In addition, CRF also caused direct postsynaptic effects on RXFP3+ neurons.

Overall, these experiments have helped to elucidate the electrophysiological properties of RXFP3+ BNST cells, their responsiveness to stress, their interaction with a prototypic stress neuropeptide and their potential role in stress induced reward-seeking.