Bruce K. Schrier

Representative of Marshall Nirenberg's early work as part of the Biochemical Genetics group at NIH, and specifically Nirenberg's experiments involving gene expression in neurons, this article assesses the activities of marker enzymes in surface cultures of new born mouse brain cells, and in glial and nonbrain cell lines.

Abstract The synthesis, uptake, and efflux of some putative neurotransmitters by cultured rat glial tumor cells were studied. Three glial cell clones were capable of pyridoxal-dependent synthesis of γ-aminobutyrate both in cell-free homogenates and in intact cells in monolayers. Substantial catabolism of γ-aminobutyrate was not found. Synthesis of taurine and isethionic acid, but not β-alanine, by one of the glioma lines was also shown. Glioma lines were found to take up glutamate and exhibited Na+-dependent uptake of γ-aminobutyrate. The uptake of γ-aminobutyrate consisted of a slow saturable component (Kt ∼ 13 to 30 µm) and a rapid nonsaturable component. Both of these were antagonized by some structural analogs of γ-aminobutyrate as well as by taurine, β-alanine, bicuculline, and low temperature. Similar kinetic parameters were found for three different glioma lines. Fibroblast-like cells obtained from rat brain cell cultures had only the nonsaturable component of γ-aminobutyrate uptake. Taurine uptake also consisted of two Na+-dependent and temperature-dependent components: a rapid saturable component (Kt ∼ 10 to 17 µm) and a nonsaturable component which varied in magnitude between cell lines. These uptakes were antagonized by β-alanine but not by γ-aminobutyrate. The glioma lines also excreted the concentrated γ-aminobutyrate into the extracellular milieu, but could maintain cell to medium concentration ratios of g80-fold. In taurine efflux experiments, cell to medium concentration ratios in excess of 1500-fold could be maintained by one of the gliomas. The data are consistent with a possible role of central nervous system glial cells in the modulation of neuronal excitability via control of the levels of neuroactive substances in the extracellular milieu of neurons.

The activity of choline acetyltransferase was more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord cells alone. This increase was associated with the formation of functional neuromuscular junctions in culture. Counts of silver-stained cells and determinations of other enzyme activities indicated that the increased choline acetyltransferase activity was not due to nonspecific neuronal survival but reflected greater activity in the surviving neurons. Hence, muscle had a marked, highly specific trophic effect on the cholinergic neurons that innervated it.

Abstract— The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 10 8 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 10 8 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 10 9 copies per brain (18 copies per cell) to 2.4 x 10 5 copies per brain (1.5 x 10 −3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 10 4 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 10 8 copies per brain (5.5 copies per cell) to 3.2 x 10 5 copies per brain (2 x 10 −3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level. Brain and liver RNA diversities were measured under double‐blind experimental conditions in three experiments with rats raised in experientially enriched (EC) or impoverished (IC) environments. Liver RNA diversity of EC animals was not different from that of IC animals. Brain total RNA of EC animals, at equivalent R 0 ts of 184,000‐212,000, hybridized to 10.6% of rat unique DNA (mean of 11 separate groups of rats). The average hybridization of brain RNA from 11 groups of IC animals in the same range of equivalent Rot was 8.2% of the unique DNA. The difference was statistically significant at P < 0.02. Of 10 groups of 3 littermate pairs (paired across EC and IC groups) brain RNA diversity was greater in EC animals in 8 cases. A least squares fit of the kinetics of hybridization to a pseudo first order reaction showed that, at saturation, the RNA from brains of EC animals was complementary to 16.4% of the unique DNA while that from IC animals was complementary to 9.1%. This difference was found in the least abundant class of rat brain RNA. These changes in sequence diversity reflected either an increase in the number of diverse RNA species present or an increase in the number of copies of certain RNA species in the rats raised in an enriched environment. A change in brain RNA populations of this magnitude may reflect a significant difference in brain function between EC and IC animals.