Heterogeneous responses of nucleus incertus neurons to corticotrophin‐releasing factor and coherent activity with hippocampal theta rhythm in the rat

Abstract

Key points• The nucleus incertus (NI) is a stress and arousal responsive, hindbrain region involved in ascending control of septohippocampal theta rhythm.• NI neurons express high levels of the neuropeptide relaxin-3 and corticotrophin-releasing factor (CRF) receptor-1 (CRF-R1).• We report the first in-depth characterization of NI neurons, using in vivo and in vitro electrophysiological techniques, which reveal a population of relaxin-3-containing NI neurons activated by CRF via postsynaptic CRF-R1 and a non-relaxin-3 neuron population inhibited or unaffected by CRF.• Relaxin-3 NI neurons exhibit strong phase-locked firing with the ascending phase of hippocampal theta oscillations.• These findings suggest the NI is a heterogeneous neuronal population and key site of CRF action with the capacity to modulate cognition in response to stress.Abstract The nucleus incertus (NI) of the rat hindbrain is a putative node in the ascending control of the septohippocampal system and hippocampal theta rhythm and is stress and arousal responsive. NI contains GABA neurons that express multiple neuropeptides, including relaxin-3 (RLN3) and neuropeptide receptors, including corticotrophin-releasing factor receptor-1 (CRF-R1), but the precise anatomical and physiological characteristics of NI neurons are unclear. Therefore, we examined the firing properties of NI neurons and their responses to CRF, the correlation of these responses with occurrence of relaxin-3, and NI neuron morphology in the rat. Most NI neurons excited by intracerebroventricular CRF infusion were RLN3-positive (9 of 10), whereas all inhibited cells were RLN3-negative (8 of 8). The spontaneous firing of RLN3 (n = 6) but not non-RLN3 neurons (n = 6) was strongly modulated and phase-locked with the initial ascending phase of hippocampal theta oscillations. In brain slices, the majority of recorded NI neurons (15 of 19) displayed excitatory responses to CRF, which uniformly increased action potential frequency and membrane potential depolarization in the presence of tetrodotoxin, indicating a direct, postsynaptic action of CRF on NI neurons. This excitation was associated with reduction in the slow component of afterhyperpolarization and a strong depolarization. Quantitative analysis in naïve rats of validated CRF-R1, RLN3 and neuronal nuclear antigen (NeuN) immunoreactivity revealed 52% of NI neurons as CRF-R1 positive, of which 53% were RLN3 positive, while 48% of NI neurons lacked CRF-R1 and RLN3. All RLN3 neurons expressed CRF-R1. CRF neurons that projected to the NI were identified in lateral preoptic hypothalamus, but not in paraventricular hypothalamus, bed nucleus of stria terminalis or central amygdala. Our findings suggest NI is an important site for CRF modulation of hippocampal theta rhythm via effects on GABA/RLN3 transmission.

Central relaxin-3 receptor (RXFP3) activation reduces elevated, but not basal, anxiety-like behaviour in C57BL/6J mice

“…The neuroanatomy of the relaxin-3/RXFP3 system suggests a broad role as an ascending neuromodulatory network [20,21], akin to the monoamine systems including serotonin, and noradrenaline [22][23][24][25]. Anatomical and functional data [15][16][17][18] suggest that relaxin-3/RXFP3 systems may interact directly with monoamine [19,26] and other peptide systems [27][28][29], and/or act at shared downstream limbic and hypothalamic target areas to modulate 'anxiety' and other stress-related responses [30][31][32][33][34].…”

Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro

“…Rxfp3-KO and wild-type littermate mice display similar serum corticosterone concentrations at baseline and after acute restraint stress As our acute RXFP3-A2 injection studies may have been somewhat confounded by the stress stimulus associated with the protocol, and in light of earlier evidence suggesting links between relaxin-3 signalling and stress-responses and CRH related signalling Lenglos et al 2013;Ma et al 2013;Lenglos et al 2015), we used a characterised strain of whole-of-life (null) Rxfp3 KO mice (Hosken et al 2015) to investigate whether a global deletion of RXFP3 from the brain altered basal corticosterone levels in serum, and whether the HPA axis associated increase in corticosterone levels in response to acute stress would be altered in these mice.…”

“…Sections from Crh-IRES-Cre;Ai14 were incubated with a characterised monoclonal relaxin-3 antibody (a 1:5 dilution of cell culture media) raised against an N-terminal region of the mature relaxin-3 peptide in mouse (Kizawa et al 2003;Ma et al 2013), whereas sections from colchicine-treated C57BL/ 6J mice were incubated with anti-relaxin-3 (1:5) and a 1:500 dilution of a specific rabbit anti-CRH antibody (kindly provided by the Wylie Vale Laboratory, Salk Institute, San Diego, USA). Antibodies were diluted in a 2% NHS and 0.1% Triton X-100 in PB solution, and sections were incubated for 48 h at 4°C.…”

CRF and the nucleus incertus: a node for integration of stress signals

“…CRF infusion into, or electrical stimulation of, the NI impairs long-term potentiation (LTP) of hippocampal-medial prefrontal cortical synapses 11 , whereas intra-NI infusion of the CRFR1 antagonist antalarmin reversed stress-induced suppression of LTP in this pathway 12 . Intra-NI infusion of the CRFR1 antagonist CP-376395, but not the CRFR2 antagonist astressin 2B, considerably reduced the reinstatement of alcohol seeking in rats that was induced by administration of the pharmacological stressor yohimbine 13 -an effect that is probably mediated by CRFR1 activation of relaxin-3-positive NI neurons 6,14 . The NI is therefore a stressresponsive nucleus and, through CRFR1, contributes to memory and learning, stressinduced reward seeking, impairments in neuronal plasticity, and arousal behaviours [9][10][11][12][13] .…”

“…This highly conserved structure consists mainly of GABAergic projection neurons, innervates many forebrain regions 3 and has been implicated in various behaviours including arousal and responses to stress 2,4 . NI neurons express CRF receptor type 1 (CRFR1) protein and mRNA in abundance 5 , and electrophysiological characterization in vitro and in vivo has revealed that CRF depolarizes NI cells via postsynaptic CRFR1 in a long-lasting and non-desensitizing manner 6 . Substantial evidence confirms the importance of CRF signalling in the NI in relation to various stress-related disorders, such as anxiety (reviewed in REFS 2,7).…”