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

Abstract

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.

Relaxin’ the brain: a case for targeting the nucleus incertus network and relaxin‐3/RXFP3 system in neuropsychiatric disorders

“…Anxiety is a sustained state of vigilance marked by increased arousal and behavioural inhibition (Davis et al, ). Preclinical research on the mechanisms underlying anxiety places most emphasis on the role of the amygdala, bed nucleus of stria terminalis (BNST), ventral hippocampus (vHC) and prefrontal cortex (PFC) – all of which have moderate to strong connections with the NI (Goto et al, ; Olucha‐Bordonau et al, ; Santos et al, ).…”

Central amygdala relaxin‐3/relaxin family peptide receptor 3 signalling modulates alcohol seeking in rats

“…Emerging evidence suggests that CRF signalling interacts with the relaxin‐3/RXFP3 system to modulate both stress responses and alcohol seeking (Tanaka et al, ; Banerjee et al, ; Ma et al, ; Ma and Gundlach, ; Walker et al, ; Walker and Lawrence, ). Given that the CeA is a stress‐sensitive node in reward circuitry (Funk et al, ; Koob, ; Koob, ; Gilpin et al, ), receives relaxin‐3 innervation from the NI (Goto et al, ; Olucha‐Bordonau et al, ; Santos et al, ) and expresses dense RXFP3 mRNA (Ma et al, ), we tested the effects of the selective RXFP3 receptor antagonist, R3(B1‐22)R, on yohimbine‐induced reinstatement of alcohol seeking. Intra‐CeA microinjection of R3(B1‐22)R (1 μg) attenuated yohimbine‐induced reinstatement of alcohol seeking in iP rats.…”

“…One potential target for pharmacotherapeutic development to treat AUDs is the cognate GPCR for the conserved neuropeptide, relaxin‐3, known as relaxin family peptide 3 receptor (RXFP3) (Bathgate et al, ). Relaxin‐3 is predominantly expressed in GABA neurons in the hindbrain nucleus incertus (NI), which projects widely to forebrain areas, including the amygdala, bed nucleus of the stria terminalis (BNST), hippocampus and lateral hypothalamus, which also express high levels of RXFP3 (Goto et al, ; Ma et al, ; Olucha‐Bordonau et al, ; Santos et al, ). The NI relaxin‐3/RXFP3 is implicated in stress and arousal‐related behaviours (Tanaka et al, ; Banerjee et al, ; Ma and Gundlach, ; Walker and Lawrence, ).…”

“…However, since intra‐BNST RXFP3 antagonism only partially attenuated stress‐induced alcohol seeking, other brain regions are probably involved (Ryan et al, ). Given that the central nucleus of the amygdala (CeA) is consistently implicated in both addiction and fear/anxiety‐related conditions (see (Gilpin et al, ) for review), receives afferent input from relaxin‐3 neurons in the NI (Santos et al, ) and contains high levels of RXFP3 mRNA (Ma et al, ), we hypothesized that the CeA may constitute another node in which relaxin‐3/RXFP3 signalling mediates alcohol seeking.…”

Un nuevo agente en los mecanismos de la adicción al alcohol y la ingesta: el núcleo incertus y el neuropéptido relaxina-3

“…Las proyecciones subcorticales ascendentes proporcionan entradas moduladoras a la amígdala. Uno de estos aportes se origina en el NI, cuya señalización mediante RLN3 se ha implicado en la modulación de la recompensa/ motivación y la ansiedad y comportamientos depresivos en los roedores a través de acciones dentro de la amígdala [16]. Se ha demostrado la existencia de proyecciones topográficas del NI que contienen proyecciones a núcleos específicos de la amígdala, en consonancia con un papel probable para este sistema de excitación integrador putativo en la regulación de los comportamientos sociales y emocionales dependientes de la amígdala [16].…”