Functional Neuroanatomy of the Rat Nucleus Incertus–Medial Septum Tract: Implications for the Cell-Specific Control of the Septohippocampal Pathway

Abstract

The medial septum (MS) is critically involved in theta rhythmogenesis and control of the hippocampal network, with which it is reciprocally connected. MS activity is influenced by brainstem structures, including the stress-sensitive, nucleus incertus (NI), the main source of the neuropeptide relaxin-3 (RLN3). In the current study, we conducted a comprehensive neurochemical and electrophysiological characterization of NI neurons innervating the MS in the rat, by employing classical and viral-based neural tract-tracing and electrophysiological approaches, and multiplex fluorescent in situ hybridization. We confirmed earlier reports that the MS is innervated by RLN3 NI neurons and documented putative glutamatergic (vGlut2 mRNA-expressing) neurons as a relevant NI neuronal population within the NI–MS tract. Moreover, we observed that NI neurons innervating MS can display a dual phenotype for GABAergic and glutamatergic neurotransmission, and that 40% of MS-projecting NI neurons express the corticotropin-releasing hormone-1 receptor. We demonstrated that an identified cholecystokinin (CCK)-positive NI neuronal population is part of the NI–MS tract, and that RLN3 and CCK NI neurons belong to a neuronal pool expressing the calcium-binding proteins, calbindin and calretinin. Finally, our electrophysiological studies revealed that MS is innervated by A-type potassium current-expressing, type I NI neurons, and that type I and II NI neurons differ markedly in their neurophysiological properties. Together these findings indicate that the MS is controlled by a discrete NI neuronal network with specific electrophysiological and neurochemical features; and these data are of particular importance for understanding neuronal mechanisms underlying the control of the septohippocampal system and related behaviors.

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

“…RLN3 is co-expressed with GAD-and GABA-IR in GABAergic projection neurons in rats [14,33,51]. In humans, we also observed GAD65/67-IR co-localized with RLN3-IR and mRNA encoding the vGAT1 co-localized with RLN3 mRNA.…”

“…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

“…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 …”

“…214,217,220,265 In turn, the RXFP3 distribution in brain is consistent with proposed roles for relaxin-3/RXFP3 signaling in the modulation of stress responses, feeding and metabolism, motivation and reward, exploratory navigation, emotion (anxiety) and social recognition, memory and cognition, and sleep and circadian rhythm. 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.…”