25550
An Autism Mouse Model Exhibits Limbic System Alterations That Enhance Susceptibility to Stress
Autism Spectrum Disorder (ASD) is characterized by abnormalities in social interaction and restricted/repetitive behaviors. Engrailed-2 (En2), a gene associated with ASD, is a neural patterning transcription factor involved in the development of the embryonic mid-hindbrain region, where norepinephrine (NE) producing neurons emerge. Previous studies indicate that En2 knockout (En2-KO) mice display ASD-like deficits in social interactions, and show depressive phenotypes behaviorally (forced swim) and physiologically (decreased cell number in the dentate gyrus and decreased neurons produced in adult neurogenesis), which implicate abnormal stress responses. Environmental stressors elicit physiological responses by increasing Hypothalamic-Pituitary-Adrenal (HPA) axis activity, which initiates in the paraventricular nucleus of the hypothalamus (PVN). HPA axis activation, specifically PVN activity, is modulated by projections from limbic structures, including the amygdala (stimulatory) and the ventral hippocampus (VH) (inhibitory). While dorsal forebrain structures in the En2-KO exhibit reduced NE levels and fiber innervation, innervation patterns into the ventral limbic system, including amygdala and PVN, are undefined.
Objectives:
Characterize NE fiber innervation into the basolateral amygdala (BLA) and PVN using biochemical and anatomical approaches, and determine whether neural activity, indicated by c-Fos immunohistochemistry, PVN, and VH following swim-stress correlates with NE innervation.
Methods:
In postnatal day 60-70 wild type (WT) and KO mice (N=4-6/genotype), western blot analysis was performed to determine protein levels of norepinephrine transporter (NET) and tyrosine hydroxylase (TH). NET-containing fibers in BLA and PVN were assessed on tissue sections using immunohistochemistry. To measure stress response, animals were given 10 minutes of swim stress followed by PFA fixation via cardiac perfusion at 120 to 140 minutes. Brain sections were cut at 40 µm, and stained for c-fos using DAB. All forced swim was performed between 12:00 to 2:00 PM EST.
Results:
En2-KO mice exhibited increased NET (1.7-fold, p<0.02) and TH (1.5-fold, p<0.002) protein levels in the amygdala. NET fiber counts were also increased in BLA (2.3-fold, p<0.0007) and PVN (1.7-fold, p<0.016). Following swim stress, En2-KO mice exhibited a 6-fold increase in c-fos nuclei in the locus coeruleus (the origin of NE fibers) compared to unstressed KO mice, while WT exhibited a 2-fold increase compared to unstressed WT. Further, the KO stress group also had a ~2-fold increase in c-fos signal in the PVN compared to the WT stress. Conversely, the En2-KO ventral hippocampus exhibited a blunted response to stress, where the increase in c-fos levels were approximately a quarter of those exhibited in the WT.
Conclusions:
Our observations indicate that NE fiber innervation in the En2-KO mice is markedly increased in the PVN and BLA, whereas it is reduced in the hippocampus. In turn, this pattern of enhanced excitatory signaling accompanied by reduced inhibitory activity will likely lead to excessive activation of the HPA axis, an outcome under current investigation.