Ontogeny of sex differences in amygdala and hypothalamus after neonatal trauma

Early life medical intervention, such as occurs in the neonatal intensive care unit, saves infant lives, but also is a significant risk factor for subsequent emotional, mood and sensory dysfunction, including chronic pain. Critically, there is mounting evidence of significant sex differences in NICU outcomes, with greater mortality

and gross neurological dysfunction in males and greater risk of internalizing disorders in females. However, the mechanisms underlying these sex differences are largely unknown, a key gap in the literature. Our long- term goal is to understand the neurological substrates that underlie the different effects of neonatal pain and

stress on vulnerability to negative affect disorders, including chronic pain, during the juvenile period in male and female rats. To accomplish this, we will expose neonatal rats to NICU-like treatment including a series of repetitive paw pricks which results in a latent vulnerability. Our central hypothesis is that the underlying

neuro/endocrine mechanisms differ between the sexes. For example, we have previously demonstrated that exposure to a subsequent “activating” stressor during the juvenile period unmasks alterations to negative affective behavior and pain thresholds in both sexes following neonatal pain. However, the neurobiology

underlying these changes differ. For example, Corticotropin Releasing Factor (CRF)-containing cells within the central nucleus of the amygdala (CeA) are particularly responsive during neonatal trauma and are reduced in number later in life, but only in male rats. Females, although also affected by neonatal pain, do not appear to

undergo the same changes in the amygdalar CRF system. Importantly, however, intra-amygdala CRF1 antagonists can reverse the hypersensitivity in both sexes. Thus, while CRF-expressing cells in the CeA are only involved in male rats, CRF receptors are involved in both sexes, suggesting involvement of a non-

amygdala source of CRF in females. Preliminary data point to the hypothalamus. To better understand these differences, we propose a series of innovative experiments to identify the behavioral and brain changes underlying the different effects of neonatal pain in male and female rats. Specific Aim 1 proposes hi-plex in

situ hybridization and florescent immunohistochemistry designed to assess changes in neuropeptide gene expression and cellular phenotypes in the amygdala and hypothalamus to examine differences between male and female rats exposed to NICU-like experiences. While the function of some individual genes and

biomarkers have been established, many cells co-express several neuropeptides and the various overlapping expression patterns of these neuropeptides has not been studied. Specific Aim 2 looks for changes in neuronal activation patterns in identified cell types in the amygdala and hypothalamus and correlates this with

changes in behavior, using immediate early gene signaling as a proxy for neuronal activation. Together, these experiments will fill in critical gaps in our knowledge regarding sex differences in the lasting consequences of neonatal pain.