IMMUNOCYTOCHEMICAL AND IN-SITU HYBRIDIZATION APPROACHES TO THE OPTIMIZATION OF BRAIN SLICE PREPARATIONS

Methods are described for determining the expression of specific mRNAs and proteins in brain slices, in order to elucidate changes in gene e xpression during preparation of vibratome slices from hippocampus of a dult rats. In situ hybridization with S-35-labeled oligonucleotides wa s used to evaluate the level and distribution of c-fos and hsp72 mRNAs in 15-mu m frozen sections prepared from these slices. Commercially a vailable antibodies were used to examine the distribution of induced F os and Jun proto-oncogenes as well as expression of the neuronal cytos keletal protein, microtubule-associated protein 2 (MAP2), in 50-mu m v ibratome sections from immersion-fixed slices. These studies confirm t he induction of c-fos and hsp72 mRNAs during routine incubation, as pr eviously observed in hippocampal slices obtained with a tissue chopper and incubated under somewhat different conditions, indicating that su ch responses are likely to be common features of many slice preparatio ns. Accumulation of Fos and Jun immunoreactivities in neurons and glia was generally consistent with the distribution of c-fos mRNA inductio n observed in slices, and the neuronal component of this response was comparable to the expression of these proteins observed after transien t ischemia in vivo. MAP2 immunoreactivity detected in the dendritic pr ocesses of neurons tended to show an increase in staining intensity du ring slice incubation, although loss of dendritic staining in specific regions was occasionally observed in association with the absence of Fos and Jun expression and histological evidence of neuron damage. The se results support the use of MAP2 immunoreactivity as a sensitive ind icator of neuronal integrity in slices. Conversely, expression of Fos, Jun and other induced proteins demonstrates the activation of signal transduction pathways that may influence diverse aspects of cell funct ion, and that must be considered in relating in vitro results to those obtained in the intact brain.