In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450scc ) and 3/3-hydroxysteroid dehydrogenase/AS -0 4 isomerase (3/3-HSD) has been studied in the rat brain. P450 scc transforms cholesterol into pregnenolone and 3/3-HSD transforms pregnenclone into progesterone . PCR was used to amplify cDNA sequences from total RNA extracts . Classical steroidogenic tissues, like adrenal and testis, as well as the nonsteroidogenic tissue lung have been used as controls . The expression of P450 Scc and 3/3-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord . In addition, primary cultures of rat cerebellar filial cells and rat cerebellar granule cells were found to express P450 SCc and 3,8-HSD at comparable levels . Furthermore, three of the four known isoenzymes of 3,8-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products . Using RNA probes, in situ hybridization indicated that P450 scc and 3/3-HSD are expressed throughout the brain at a low level and mainly in white matter . Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450 Scc and 3,ß-HSD mRNA detected by PCR . This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes . Key Words: Neurosteroids-Side-chain cleavage enzyme-3,ß-Hydroxysteroid dehydrogenase-PCR-In situ hybridization . dehydrogenase/A S-A'' isomerase ; P450 s ,, P450 side-chain cleavage enzyme; 1 X SSC, 0.15 M NaCl/0 .015 M sodium citrate (pH 7 .6) .

In order to examine the mechanisms that underlie development of the inner ear, the normal processes were perturbed using all-trans-retinoic acid (RA). By implanting a resin exchange bead saturated with RA into stage 16 (Hamburger and Hamilton, 1951, J. Morphol. 88, 49-92) embryonic day 2.5 chick ears, it was possible to analyze its in vivo effects on inner ear development. There is a temporal window during which the developing chick inner ear is particularly susceptible to the effects of RA (stages 16-19). This RA period of sensitivity precedes evidence of gross morphologic or histologic differentiation by at least 24 h, suggesting that mechanisms controlling formation of key inner ear structures are already in progress. There is a dose dependence on RA, with increasing doses of RA generating increasingly severe phenotypic abnormalities. Data indicate that these effects are due to differential sensitivities of the various inner ear structures to RA during their formation. In general, the vestibular structures were more susceptible to RA effects than the cochlear duct. Furthermore, nonsensory structures such as semicircular canals seemed to display a greater susceptibility to RA than their associated sensory structures (i.e., cristae). Among the three semicircular canals, the superior canal was the most susceptible to RA treatment, whereas the common crus was particularly resistant, suggesting that the molecular mechanisms for each structure's formation are different. The defect in semicircular canal formation is due to problems in the initial outgrowth of the canal plate which in turn is related to a down-regulation of early otocyst cell proliferation. This perturbation model provides valuable insight into the processes involved in producing the intricate patterning of the inner ear.

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