An Introduction: Quantification of the Hippocampal BDNF Content of Maternally Separated Rats Using a Western Blot Protocol

Among industrialized nations, the United States has the worst incidence of child maltreatment with 3 million cases per year (U.S. Department of Health and Human Services, 2012). Studies have shown that individuals who were maltreated when they were young are more susceptible to drug abuse such as alcohol, cocaine, and nicotine (Maddahian, Newcomb, & Bentle, 1988). Early life stress (ELS) causes hyperactivation of the Hypothalamic-Pituitary- Adrenal (HPA) Axis (Heim et al., 2000; Plotsky et al., 2005). The dysregulation of the HPA axis causes the secretion of glucocorticoid stress hormones by large amounts, which in return attenuates hippocampal Brain-derived neurotrophic factor (BDNF) (Smith, Makino, Kvetnansky, & Post, 1995). BDNF is a neurotrophin that helps the growth, maintenance, and survival of neurons and is also involved in neuronal plasticity. To mimic ELS, maternal separation is used as an animal model. Studies have shown that adult maternally separated rats have decreased hippocampal mature BDNF (Lippman et al., 2007). Exercise alters some of the effects of ELS by protecting hippocampal BDNF from the down-regulation caused by the hyperactivation of HPA axis (Maniam & Morris, 2010; Neeper et al., 1996). The purpose of this research is to investigate the effects of ELS and exercise on behaviors related to substance abuse and hippocampal BDNF content. When we attempted to measure hippocampal BDNF by the enzyme-linked immunosorbent assay (ELISA), we did not observe the previously-published down-regulation of BDNF in the hippocampi of maternally separated animals (Dold, 2013). Thus, a western blot protocol was developed for the quantification of BDNF in the same protein samples that were previously quantified using ELISA. Western blot is more specific in targeting protein due to SDS-page capability to separate protein components in accordance to their size. BDNF undergoes post-translational processing, such that both pro- BDNF (the 32 kD precursor) and mature BDNF (14 kD) are present and biologically active in rat brain tissue. Thus, western blot will allow us to distinguish between these BDNF proteins, which is important in our study since we are primarily interested in mature BDNF. We expect maternal separation (MS) to attenuate the hippocampal mature BDNF content. Furthermore, we expect that the mature BDNF content will positively correlate with the total running activity