The organotypic brain slice model is well-established

in our working group and serves as a validated tool to study toxic, degenerative and developmental changes as well as synaptic recovery, survival and cell death of neurons (Gähwiler et al., 1997, Humpel and Weis, 2002, Moser et al., 2003, Moser et al., 2006, Stoppini et al., 1991, Weis et al., 2001 and Zassler et al., 2003). In this SP600125 in vivo model cholinergic neurons are axotomized, however, the normal cytoarchitecture is retained similar to the in vivo situation and functional connections including transport and diffusion probabilities are maintained. The brain tissue is derived from postnatal day 10 brains and therefore it is not completely comparable to adult brains, which is a limitation of the present study. In further studies it would be of particular interest to investigate the effects of EtOH in adult nbM slices and thus to compare with the neuropathological changes in adult brains. In fact, culturing of adult brain slices has been reported (Bickler et al., 2010, Hassen et al., 2004 and Xiang et al., 2000), although this technique is not trivial and such slices are not easy to culture for long time. Adolescent DNA Damage inhibitor brains distinctively response to EtOH exposure compared to adult brains (Smith,

2003) and the context of ongoing plasticity in the adolescence faces the continuous production of new neurons during adult neurogenesis (Nixon et al., 2010). Indeed, adolescents are more prone to the neurotoxic effects of EtOH than adults (Crews et al., 2007). before In the present model brain slices are normally cultured for 2 weeks before staining in experiments correlating to adolescent age. In fact the basal forebrain cholinergic neurogenesis is already completed before birth (E17) (Semba and Fibiger, 1988). In the present study detection of cholinergic neurons was performed using the immunohistochemical marker for the enzyme ChAT, which is expressed in cell bodies and nerve fibers of cholinergic neurons. In our experiments control slices displayed around

120 ChAT-positive neurons, which is in line with previous work (Weis et al., 2001). ChAT serves as a marker for the functional activity of cholinergic neurons (Oda, 1999) and a decreased number directly correlates with cognitive impairment (Counts and Mufson, 2005). Indeed, a dysfunction of the cholinergic system and the loss of cholinergic neurons is in concert with low levels of acetylcholine in the cortex and resulted in cognitive impairment (Mesulam, 2010). Interestingly, an impairment of the cholinergic system (Floyd et al., 1997) and cognitive decline has also been reported after long-term EtOH treatment in vivo (Arendt et al., 1988 and Ehrlich et al., 2012). Accordingly, the activity of cholinergic neurons after EtOH exposure possibly represents a depression of the enzyme ChAT and not cell death.