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Norepinephrine Fiber Innervation Is Increased in the Amygdala of the Engrailed-2 (En2) Knock out Mouse Model of Neurodevelopmental Disorders
Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impaired social interactions and communication and repetitive/restricted behaviors and interests. Other symptoms may include abnormalities in mood and fear responses. The neural patterning transcription factor Engrailed-2 (En2) is involved in the development of the embryonic mid-hindbrain region, where monoamine neurons emerge, and has been associated with ASD. While our previous studies indicate En2 knock out (KO) mice exhibit abnormal norepinephrine (NE) systems in the forebrain associated with deficits in social interactions, fear conditioning and depression-related tasks (Brielmaier et al, 2012, 2014), nothing is known about fiber innervation of limbic systems that may contribute to the behavioral phenotype.
Objectives:
To characterize NE fiber innervation into ventral limbic systems using biochemical and anatomical approaches.
Methods:
In postnatal day 60-70 wild type (WT) and KO mice (N=4-6/genotype), we used an antibody against the norepinephrine transporter (NET) to define the levels of proteins in dissected tissue homogenates using western blotting and the numbers of NET-containing fibers in tissue sections using immunohistochemistry. Western analysis was also performed for total protein levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for NE biosynthesis.
Results:
En2 KO mice exhibited a 1.7-fold increase in total NET protein levels in the amygdala compared to WT controls (p<0.02). Furthermore, TH protein levels were increased 1.5-fold (p<0.0017), suggesting that monoamine systems were dysregulated in ventral forebrain. In parallel, NET fiber counts within the basolateral amygdala were also increased, exhibiting a 2.6-fold elevation (p<0.004), though other amygdala subnuclei displayed region-specific changes. Preliminary data also suggest increased fiber innervation in the paraventricular nucleus of the hypothalamus as well, while fiber assessments in the prefrontal cortex, nucleus accumbens, and bed nucleus of the striatum terminalis are ongoing.
Conclusions:
These observations indicate that NE fiber innervation into limbic systems may be dysregulated in this neurodevelopmental model in ways that may contribute to abnormalities in depression-related tasks (forced swim; tail suspension), fear conditioning and social interactions. These studies highlight the unexpected and complex effects of altering the levels of hindbrain gene expression on forebrain growth and development that depend on monoamine neuron projections from the brainstem. These studies may provide insight into neurobiological mechanisms that disturb long distance connectivity between brain regions that impact how the brain develops as well as maintain ongoing functional networks relevant to behavioral phenotypes. We expect that our definition of altered long distance connectivity will lay a foundation to examine the impact on functional activities in neural systems that mediate neurodevelopmental disabilities.
References: Brielmaier et al., 2012, PLoS One, 7(7):e40914. Epub 2012 Jul 19, 2012
Brielmaier et al., 2014, Genes, Brain and Behavior, 2014 13:286-298. doi:10.111/gbb.12115, 2014