Relaxin-3 Innervation From the Nucleus Incertus to the Parahippocampal Cortex of the Rat

Abstract

Spatial learning and memory processes depend on anatomical and functional interactions between the hippocampus and the entorhinal cortex. A key neurophysiological component of these processes is hippocampal theta rhythm, which can be driven from subcortical areas including the pontine nucleus incertus (NI). The NI contains the largest population of neurons that produce and presumably release the neuropeptide, relaxin-3, which acts via the Gi/o-protein-coupled receptor, relaxin-family peptide 3 receptor (RXFP3). NI activation induces general arousal including hippocampal theta, and inactivation induces impairment of spatial memory acquisition or retrieval. The primary aim of this study was to map the NI/relaxin-3 innervation of the parahippocampal cortex (PHC), including the medial and lateral entorhinal cortex, endopiriform cortex, perirhinal, postrhinal, and ectorhinal cortex, the amygdalohippocampal transition area and posteromedial cortical amygdala. Retrograde tracer injections were placed in different parts of the medial and lateral entorhinal cortex, which produced prominent retrograde labeling in the ipsilateral NI and some labeling in the contralateral NI. Anterograde tracer injections into the NI and immunostaining for relaxin-3 produced fiber labeling in deep layers of all parahippocampal areas and some dispersed fibers in superficial layers. Double-labeling studies revealed that both hippocampal projecting and calcium-binding protein-positive (presumed GABAergic) neurons received a relaxin-3 NI innervation. Some of these fibers also displayed synaptophysin (Syn) immunoreactivity, consistent with the presence of the peptide at synapses; and relaxin-3-positive fibers containing Syn bouton-like staining were frequently observed in contact with hippocampal-projecting or calcium-binding protein-positive neuronal somata and more distal elements. Finally, in situ hybridization studies revealed that entorhinal neurons in the superficial layers, and to a lesser extent in deep layers, contain RXFP3 mRNA. Together, our data support functional actions of the NI/relaxin-3-parahippocampal innervation on processes related to memory, spatial navigation and contextual analysis.

Unveiling a Novel Memory Center in Humans: Neurochemical Identification of theNucleus Incertus, a Key Pontine Locus Implicated in Stress and Neuropathology

“…Numerous earlier anatomical studies have characterized the connectivity and neurophysiological activity of the NI in rat and mouse brain, including the strong projections to the SHS system, and to multiple cortical and limbic areas involved in emotional cognition [14,17,18, 21], while others have characterized the nature of the transmitters, neuropeptides, and receptors expressed by different populations of NI neurons in these species [14][15][16][17][18][19][20][21][22], which provide an insight to the likely functional role of the NI in the human brain.…”

“…Thereafter, many decades later, a large majority of neurons in the rat NI or nucleus O [12] was shown to express the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), as reflected by staining against the GABA-synthesizing enzyme, GAD, and in situ hybridization (ISH) for vGAT1 mRNA [13,14]. Since then, the neuroanatomy of the NI and its connections throughout the brain has been systematically mapped using various histological methods in rats [14][15][16][17][18][19], mice [20][21][22][23][24], zebrafish [25], and in non-human primate [26], but not in humans.…”

History of key regulatory peptide systems and perspectives for future research

“…206,214,220,[266][267][268][269][270][271][272][273][274][275][276][277] In earlier studies, and more recently, comprehensive investigations have characterized receptors for various transmitters/peptides that are present on relaxin-3 neurons within the nucleus incertus [278][279][280][281] and further details of the anatomical networks utilizing relaxin-3/RXFP3 signaling in rat and mouse, 282 including those in the medial septum, 283 hippocampus, and piriform cortex. 284,285 A further aspect of relaxin-3/RXFP3 signaling recently reported was an association with the signaling of cellular aging. Using in vivo co-regulation analyses Maudsley and colleagues 286,287 observed that It should be noted that the structural similarities of relaxin-3 and relaxin, allow relaxin-3 to be tested pharmacologically as a likely RXFP1 agonist in various models, 288 and several reports describe its actions under these conditions.…”

“…In earlier studies, and more recently, comprehensive investigations have characterized receptors for various transmitters/peptides that are present on relaxin‐3 neurons within the nucleus incertus 278–281 and further details of the anatomical networks utilizing relaxin‐3/RXFP3 signaling in rat and mouse, 282 including those in the medial septum, 283 hippocampus, and piriform cortex 284,285 …”

The Relaxin-3 Receptor, RXFP3, Is a Modulator of Aging-Related Disease

“…Anxiety, along with associated disorders such as post-traumatic stress disorder (PTSD), have in recent years been strongly associated with premature aging conditions [150][151][152][153]. With respect to the impact of RXFP3 upon anxiety-related disorders, it has been shown that specific central stimulation generates an anxiolytic effect in model organisms [154].…”