Reelin Haploinsufficiency and Late-Adolescent Corticosterone Treatment Induce Long-Lasting and Female-Specific Molecular Changes in the Dorsal Hippocampus.

brain sciences Article Reelin Haploinsufficiency and Late-Adolescent Corticosterone Treatment Induce Long-Lasting and Female-Specific Molecular Changes in the Dorsal Hippocampus Anna Schroeder 1,2 , Maarten van den Buuse 3,4,5 and Rachel A. Hill 1,2, * 1 2 3 4 5 * The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville 3052, Australia; Department of Psychiatry, School of Clinical Sciences, Monash University, Clayton 3168, Australia School of Psychology and Public Health, La Trobe University, Bundoora 3086 Australia; Department of Pharmacology, University of Melbourne, Parkville 3052, Australia The College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville 4810, Australia Correspondence: ; Tel.: +61-3-8572-2917 Received: 21 May 2018; Accepted: 22 June 2018; Published: 25 June 2018



 Abstract: Reelin depletion and stress seem to affect similar pathways including GABAergic and glutamatergic signaling and both are implicated in psychiatric disorders in late adolescence/early adulthood. The interaction between reelin depletion and stress, however, remains unclear. To investigate this, male and female heterozygous reelin mice (HRM) and wildtype (WT) controls were treated with the stress hormone, corticosterone (CORT), during late adolescence to simulate chronic stress. Glucocorticoid receptors (GR), N-methyl-D-aspartate receptor (NMDAr) subunits, glutamic acid decarboxylase (GAD67 ) and parvalbumin (PV) were measured in the hippocampus and the prefrontal cortex (PFC) in adulthood. While no changes were seen in male mice, female HRM showed a significant reduction in GR expression in the dorsal hippocampus. In addition, CORT reduced GR levels as well as GluN2B and GluN2C subunits of NMDAr in the dorsal hippocampus in female mice only. CORT furthermore reduced GluN1 levels in the PFC of female mice. The combined effect of HRM and CORT treatment appeared to be additive in terms of GR expression in the dorsal hippocampus. Female-specific CORT-induced changes were associated with overall higher circulating CORT levels in female compared to male mice. This study shows differential effects of reelin depletion and CORT treatment on GR and NMDAr protein expression in male and female mice, suggesting that females are more susceptible to reelin haploinsufficiency as well as late-adolescent stress. These findings shed more light on female-specific vulnerability to stress and have implications for stress-associated mental illnesses with a female bias including anxiety and major depression. Keywords: reelin; corticosterone; glucocorticoid receptors; NMDA receptors; parvalbumin; GAD67 1. Introduction Reelin is an extracellular matrix protein that is secreted by Cajal-Retzius cells during embryonic brain development and mainly by cortical and hippocampal GABAergic interneurons during adulthood [1–3]. During brain development, reelin is predominantly involved in cortical layer formation [1]. Mice with homozygous loss of reelin, called reeler mice, display inversion of cortex cell layers and malposition of neurons throughout the hippocampus [4]. Heterozygous loss of reelin in mice Brain Sci. 2018, 8, 118; doi:10.3390/brainsci8070118 www.mdpi.com/journal/brainsci Brain Sci. 2018, 8, 118 2 of 15 induces a less severe phenotype showing dendritic abnormalities in the cortex and the hippocampus and only subtle abnormalities in cognitive function [5–9]. These mice are used to investigate the role of reelin in the development of psychiatric disorders as mutations in the reelin gene as well as decreased levels of mRNA are associated with autism [10], schizophrenia and bipolar disorder [2,11]. Aside from its developmental role, reelin serves a crucial function in the mature brain acting through its two major receptors, Apolipoprotein E receptor 2 (EpoER2) and very low-density lipoprotein receptor (VLDLR) [12], activating numerous downstream cascades via Dab1 phosphorylation such as Akt/mTOR or N-WASP leading to microtubule formation and actin stabilization, respectively [13]. In the mature brain, reelin signaling promotes dendritic spine maturation, synaptogenesis, synaptic transmission and plasticity, and therefore plays a critical role in the modulation of synaptic circuits [14–16]. In a comprehensive review of articles published from 1980 to 2014 on major depression a main finding was the association between both chronic stress and major depression with dendritic spine loss and aborization as well as reduced glial cells [17], thus disruption to reelin may be upstream of this broad set of functional deficits associated with major depression. Reelin signaling through ApoER2 was shown to activate NMDAR phosphorylation and calcium influx through these receptors and subunit composition [18] thus mediating synaptic plasticity and learning and memory [14,16]. NMDAr are tetramers consisting of two GluN1 subunits and two GluN2 subunits (GluN2A, GluN2B, GluN2C and GluB2D). While NMDA receptors containing GluN2C and GluN2D are mainly localized to the cerebellum and show slower kinetics compared to GluN2A and GluN2B in the adult brain [19], GluN2A and GluN2B are highly expressed in the hippocampus and the cortex and are critical mediators of synaptic function via regulating synaptic plasticity (reviewed in Sanz-Clemente, Nicoll [20]). Reelin has also been closely linked to the markers of GABAergic interneurons: glutamic acid decarboxylase (GAD67 ), the enzyme that converts glutamate to GABA, and parvalbumin (PV), as these were simultaneously decreased with reelin mRNA and protein levels in the PFC and the hippocampus in schizophrenia and bipolar post-mortem brains [21] as well as in mouse models of these neuropsychiatric disorders [22,23]. Findings on the effect of reelin deficiency on the expression of these GABAergic markers as well as NMDAr subunits remain inconsistent, possibly due to a lack of stratification for sex. Nullmeier et al. [23] for example combined male and female data when comparing GAD67 expression in reelin haploinsufficient and wild-type mice, and found a significant decrease in GAD67 and PV in multiple hippocampal regions, but did not stratify for sex in the analysis. Previously, we have shown sex differences in BDNF, TrkB and GluNR2C expression in reelin haploinsufficient mice [24,25], while others have shown that male but not female Rln +/− mice show alterations in steroid hormone levels in the cerebellum when compared to controls [26]. Due to the well-established sex differences within GABAergic development [27] as well as psychiatric disorders [28,29], it is important to discern specific differences between males and females. While several genetic variants have been associated with severe psychiatric disorders such as major depression, no single gene is likely to be causative. Rather a gene × gene or gene × environment interaction is more probable. 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