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— The calcium-dependent incorporation of L-[3-14C]serine and [1,2−14C]ethanolamine into the phospholipid of isolated neuronal and glial cells from rabbit brain was studied, and the distribution of the enzymic system among the correspondent subfractions was examined. The neuronal cell-enriched fraction was found to possess a much higher rate of exchange of both bases than the glial cell-enriched fraction. Among the sub-fractions isolated from the neuronal and glial cells, those corresponding to neuronal plasma membranes and microsomes showed a noticeably higher exchange of serine and ethanolamine compared to the corresponding subfractions from glia. Neuronal/glial ratios of about 6–8 were found for the exchange activity in both plasma membrane-enriched fraction and in microsomes. Synaptosomes and synaptosomal subfractions contained low activities. It is concluded that the calcium-dependent enzymic system for the exchange of serine, ethanolamine and other nitrogenous bases with endogenous phospholipid is concentrated mostly in the neuronal perikaryal membranes, and could be used as a neuronal marker.

— Soluble proteins were studied in preparations from rabbit brain cortex enriched in neuronal or glial cells and in subcellular cortical fractions. Analytical polyacrylamide gels were used for acidic (pH 9-5) and basic (pH 4-3) proteins and qualitative and quantitative differences are described. The isozymes of lactic dehydrogenase, brain specific proteins and radioactive labelling patterns were used to characterize some soluble proteins.

— The distribution of protein-bound radioactivity among subcellular organelles of cerebral cortex was followed after intravenous administration of [3H]leucine and after incubation of brain slices in the presence of [3H]leucine. Neuronal and glial cell-enriched fractions were prepared by discontinuous sucroseFicol1 gradient centrifugation of cerebral cortex cell suspensions. Subcellular fractions were obtained from each of the cell prepara- tions and the protein-bound radioactivity determined after in uiuo and in vitro incorporation of [3H]leucine. The unfractionated neuronal material had a considerably higher level of protein-bound radioactivity than the glial material. The most marked neuronal-glial dif- ferences were observed in microsomes and soluble proteins, while the radioactive labelling of the nuclear and mitochondria1 fractions was similar for the two cell types.

— A modification of the available methods was used for preparation of nerve and glia cell-enriched fractions from rabbit brain and spinal cord. The rate of incorporation of tritiated leucine and the turnover rates of protein during 10 days was studied in the bulkprepared cell fractions. The rate of incorporation into the nerve cell fraction was approximately three times greater than in the glia fraction. The nerve cells had one rapid and one slow phase in decline of radioactivity, while the glial cells were characterized by a more uniform decline. The soluble radioactivity was followed in whole tissue of brain and spinal cord and certain differences between the two were observed.

The low-molecular weight beta-trace protein constitutes about seven per cent of the total protein content in human cerebrospinal fluid. Within the central nervous system the protein is found predominantly in white matter and fractions enriched in glial cells. Immunofluorescence studies on sections of monkey brains show that beta-trace protein is particularly localized to cells such as astrocytes and oligodendrocytes. Significantly higher amounts of beta-trace protein are found in brain tumours derived from glial cells than in other tumours.

Brain proteins in one cerebral hemisphere were labeled by means of intracarotid perfusion with 3H-leucine by a 40 s pulse. Recirculating precursor contributed little to the protein bound radioactivity. This radioactivity reached a peak level 30 min after perfusion. Fractions enriched in neuronal perikarya and glial cells showed a similar time course of labeling, but the neuronal fraction demonstrated the highest level of protein bound radioactivity. Subcellular fractions from whole brain were studied by the same system. The highest protein bound radioactivity was observed in the nuclear and microsomal fractions. The brain entry of the precursor by means of a controlled intracarotid pulse of short duration offers particular advantages in short-term experiments since the systemic metabolism of the labeled precursor is largely avoided. The easily achieved high labeling of proteins facilitates assay of the radio-activities in different cellular and subcellular fractions and also allows analyses of relative turnover rates in electrophoretically separated proteins.

Tritiated methylmercuty hydroxide (MeHgOH) with a specific activity of 306 curies/mol was synthesized by a Grignard reagent from tritiated methyl iodide with a yield of 70%. Tritiated MeHg was given to squirrel monkeys by stomach tube in weekly doses of 0.8 mg/kg. The animals were killed after one dose or six doses. The distribution of the substance in the brain was then studied by microautoradiography and by fractionation of brain tissue into cellular and subcellular fractions. Part of the MeHg in the brain is in a water-soluble form. Methylmercuty was found autoradiographically mainly in the neuropile of the cortex. At subtoxic doses (one dose), more protein-bound radioactivity was found in the glial fraction than in the neuronal fraction. At toxic dose (six doses), the protein-bound fraction of MeHg increased significantly (P less than .02) in the neurons relative to nontoxic dose (one dose). Methylmercury was demonstrated autoradiographically in damaged neurons but not in undamaged neurons.

Amino acid incorporation in neuronal and glial cells has been investigated in several laboratories employing bulk-separation techniques to obtain cell-enriched fractions. The relative rates of incorporation into the proteins of both cell types vary substantially with the method of isotope administration. Through the use of single-pulse perfusions with a duration of 30-40 sec the early time course of labeling has been studied. The difference between neuronal and glial cells with respect to indicating cell interactions will be discussed. The in vitro amino acid incorporation has been measured in neurons and glia after slice incubation. In material from animals developing experimental allergic encephalitis the rate of 3H-leucine incorporation more than doubles in the unfractionated brain. Glial cells increased their rate of incorporation by approximately 400% under the same conditions. The involvement of specific proteins in the cells and organelles has been studied by gel electrophoresis. The high uptake capacity of glial cells for certain amino acids with possible transmitter function has been further characterized. The release of these substances is measured in a superfusion system where beds of cells, preloaded with the labeled substance, are used. High potassium pulses stimulate release of, for example, GABA in both neuronal and glial cells.

— Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.