Autism Mouse Model Exhibits Dysregulated Norepinephrine Innervation and Neural Activity in the Limbic System
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, fear conditioning, and depression-related tasks (forced swim, tail suspension), which implicate abnormal stress responses. Environmental stressors elicit physiological responses by increasing Hypothalamus-Pituitary-Adrenal (HPA) axis activity, which initiate 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) (Figure 1). 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.
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, in BLA, PVN, and VH following swim-stress correlates with NE innervation.
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. All perfusions occurred between 2:30 to 4:30 PM EST.
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 increased c-Fos nuclei in PVN (2.25-fold, p<0.006), suggesting an increase in neural activity. However, c-Fos nuclei were decreased in VH (50%, p<0.02) of the same animals following swim stress, suggesting decreased post-stress neural activity in VH.
Our observations indicate that En2-KO mice are hyperinnervated by NE fibers in the PVN and BLA. Additionally, neural activity in the PVN is increased >2 fold under stress conditions, changes that parallel fiber innervation. Conversely, in the hippocampus, where NE levels and fibers are reduced (Genestine et al, 2015), there is a 50% decrease in c-Fos activity. Given that the amygdala plays a stimulatory role and the VH elicits inhibition in the PVN, the changes we detect in fiber patterns and neural activity would be predicted to exaggerate stress circuit activation and responses, which in turn likely contribute to the En2-KO deficits in social interactions, fear conditioning, and depression-related tasks. More generally, these studies of the En2-KO mouse model provide insights into neurobiological mechanisms by which common genetic variants associated with neurodevelopmental disorders alter the balance of neural pathways. Consequently, these structural and functional differences may increase subject vulnerability to other genetic and environmental factors in disease causation.