SUMMARY A quick‐freeze, freeze‐substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a ‘double‐fixation’ protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick‐frozen in Freon 22 and freeze‐substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon‐Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze‐substitution fixative. However, if glutaraldehyde is used alone as a freeze‐substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.

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We examined the effects of maternal hyperphenylalaninemia on the morphological development of the fetal and neonatal rat brain. High concentrations of phenylalanine were induced in pregnant rats from embryonic days 14 through 21 by subcutaneous injections of alpha-methylphenylalanine (mPhe) (to inhibit maternal phenylalanine hydroxylase) at a dosage of 30 mg/100 g body weight plus phenylalanine (Phe) supplementation (to raise fetal plasma phenylalanine) at a dosage of 60 mg/100 g body weight two times daily at 12-h intervals. This treatment resulted in a significant hyperphenylalaninemia compared with vehicle-injected, pair-fed control rats. At embryonic day 21, mPhe/Phe-treated embryos displayed a reduced thickness of the cortical plate and marginal zone, a decrease in the size of postmitotic neurons, and an increase in the packing density of cells in the cortical plate. There was an increase in the number of pyknotic cells (cell death) and in the reactive microglia in the mPhe/Phe-treated group. During the postnatal period the differences between mPhe/Phe-treated and control rats became fewer and by postnatal day 7 the morphology of the mPhe/Phe-exposed brains was similar to the controls. These morphologic data, in conjunction with our previous biochemical finding of reduced cerebral DNA, RNA, and protein contents of mPhe/Phe-treated rats, indicate that the induced hyperphenylalaninemia caused a significant delay in the development of the cerebral cortex which was able to undergo recovery during the postnatal period.