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

Fulltext embargoed for: 12 months post date of publicationThe 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

GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3) mRNA

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3 mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChAT mRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm

Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala

The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or "hubs") within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin (PV), calretinin (CR) and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus. In contrast, sparse anterogradely-labeled and relaxin-3-immunoreactive fibers were observed in other amygdala nuclei, including the lateral, central and basal nuclei, while the nucleus accumbens lacked any innervation. Using synaptophysin as a synaptic marker, we identified relaxin-3 positive synaptic terminals in the medial amygdala, BST and endopiriform nucleus of amygdala. Our findings demonstrate the existence of topographic NI and relaxin-3-containing projections to specific nuclei of the extended amygdala, consistent with a likely role for this putative integrative arousal system in the regulation of amygdala-dependent social and emotional behaviors