Septal GABA and Glutamate Neurons Express RXFP3 mRNA and Depletion of Septal RXFP3 Impaired Spatial Search Strategy and Long-Term Reference Memory in Adult Mice.

Mouna Haidar,Mohsen Nategh,Cary Zhang,Jake Rogers,João Covita,Andrew L Gundlach,Kimberly Tin,Alexander D Wykes

doi:10.3389/fnana.2019.00030

Abstract

Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3loxP/loxP mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV(1/2)-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.

Highlights

In order to confirm that the Associated Viral (AAV)(1/2)-Cre-internal ribosome entry site (IRES)-enhanced green fluorescent protein (eGFP) vector drove Cre-recombinase expression in vivo and transduced MS and DB neurons, sections from adult floxed-Rxfp3 mice injected in the MS with AAV(1/2)-Cre-IRES-eGFP were processed for Cre-immunoreactivity (IR) and eGFP fluorescence
AAV(1/2)-Cre-IRES-eGFP viral transduction was observed in areas of the MS and DB that contain high levels of Rxfp3 mRNA (Ma et al, 2007; Smith et al, 2010), with viral spread along the rostrocaudal extent of the medial septum/diagonal band of Broca (MS/DB) region spanning +1.18–0.38 mm relative to bregma
An analysis of search strategies indicated that both AAV(1/2)-Cre-IRES-eGFP and AAV(1/2)-eGFP injected mice adopted spatial learning over acquisition days, as the adjusted odds ratio (OR) of adopting an allocentric search strategy increased by ∼30% per acquisition day (learning effect, day: OR: 1.28, 95% CI (1.22; 1.35), p < 0.001, n = 8–11 per group, Figure 2A)

Summary

Introduction

The medial septum/diagonal band of Broca (MS/DB) sends strong GABAergic and cholinergic efferent projections to the hippocampal formation (Freund and Antal, 1988; Dutar et al, 1995; Vertes and Kocsis, 1997), which together with a number of other subcortical structures [e.g., supramammillary nucleus (Vertes and Kocsis, 1997) and median raphe (MR; Vertes et al, 1999)] constitute the ‘‘septohippocampal system (SHS)’’ (Vertes and Kocsis, 1997). NI afferent fibers were observed in close apposition to septal cholinergic and parvalbumin (PV) GABA neurons that project to the hippocampus (Olucha-Bordonau et al, 2012), suggesting the NI relaxin3/RXFP3 system plays a functional role in modulating cognitive related behaviors via modulation of the SHS Consistent with this hypothesis, injection of a RXFP3-selective agonist peptide [R3/I5 (Liu et al, 2005)] into the MS increased hippocampal theta rhythm in urethane-anesthetized rats, which was significantly attenuated by prior injection of a selective RXFP3 antagonist [R3(B∆23–27)R/I5 (Kuei et al, 2007)]. Similar anatomical and functional studies have not been conducted in the mouse

Methods

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Discussion

Conclusion