Search

The Na+-dependency of γ-aminobutyric acid (GABA) binding to particulate fractions of mouse brain was studied, by increasing the concentration of Na+ in the homogenizing fluid the retention of both [3H] GABA and [14C] sucrose was increased in the crude nuclear fractions. But, at Na+ concentrations greater than 10 meq/l, the retention of both substances was decreased in the synaptosomal-mitochondrial fractions. Although the total [3H] GABA bound to all fractions was increased by increasing the Na+ concentration of the homogenizing medium, the total [14C] sucrose entrapped in these fractions remained rather constant. The changes in GABA binding caused by Na+ thus could not be accounted for by the presence of entrapped homogenizing fluid in pellets. The decreased binding of GABA to synaptosome fractions found in the presence of 40 to 100 meq/l Na+ was due to “clumping” effects caused by the excess salt. With high salt concentrations many of the particles which bind GABA and which are normally associated with the synaptosome fraction were sedimented in the crude nuclear fraction. In sum, these results indicated that the binding of GABA occurs maximally at a Na+ concentration of about 40 meq/l.

After the subcutaneous injection of U-14C- d -glucose, a greater amount of 14C was incorporated into the brains of aggregated mice than into those of their isolated littermates. The increase in radioactivity occurred rather uniformly throughout the several brain structures studied. The percentage difference in cerebral radioactivity between the groups was smaller at 0930–1200 hr and 1800–2000 hr than at 1000–1500 hr. This difference was least at 1800–2000 hr, which is the time that coincides with the peak of general motor activity.

In unanesthetized rhesus monkeys equipped with intracerebral chemitrodes (push-pull cannulae plus multiple contacts), injections of lithium chloride into the amygdala-hippocampus region produced high-voltage, 1/sec sharp waves, and episodes of electrical afterdischarges without motor manifestations. The effect, which began after a delay of a few minutes, was strictly localized to the injection site and lasted for minutes or hours. No change in motor thresholds was observed, but afterdischarge thresholds increased considerably. Spontaneous restlessness and aggressiveness of the animals were diminished. After pretreatment with lithium, when the local electrical disturbance had subsided, residual effects were demonstrated by injection of ( a ) l -glutamate, which produced a reappearance of typical lithium waves; or ( b ) acetylcholine, which produced a mixture of lithium and acetylcholine waves. After pre-treatment with l -glutamate, when its characteristic effects had subsided, injection of lithium re-activated the mechanism for producing afterdischarges resembling those of glutamate. We suggest that the well-known excitatory properties of l -glutamate and acetylcholine on CNS neurons may be related to a selective increase in membrane permeability to sodium or lithium if present. Therapeutic effects of lithium may be related to its specific effect on the limbic system.

Using subcellular fractionation and a double-isotope method, it was shown that differential housing of male Swiss albino mice for 6 to 7 weeks caused pronounced neurochemical changes in a region of discontinuous sucrose gradient fractions which contains synaptosomes. The “binding” of the possible inhibitory neurotransmitters, [14C]GABA (2.4 × 10−5 m ) and [3H] glycine (9 × 10−8 m ), and the total protein content of these fractions were significantly lower for the brains of aggressive “isolated” mice than for those of their “aggregated” counterparts. These differences in protein content and in amino acid binding (when expressed in mole amino acid/fraction) occurred in particles that were localized just above the 1.2:1.6 m sucrose interface of the gradients. However, when the “binding” data were expressed in mole amino acid/mg protein, no differences existed between gradient subfractions of the brains of isolated and aggregated mice. Because the protein content and total amino acid binding capacities of synaptosomal fractions were lower for the brains of isolated mice than for those of their aggregated counterparts, while no differences existed in amino acid binding on a protein basis, it seems apparent than an environmentally-induced morphological change had been produced in the brain—the brains of isolated mice contained a lesser number of subcellular binding sites for GABA and glycine. These environmentally-sensitive subcellular structures may reside on “heavy” synaptosomes, as they were localized just above the 1.2:1.6 m sucrose (“mitochondrial”) interface. This demonstration of environmentally-sensitive constituents of cerebral nerve-ending particles further validates the view that the brain exhibits structural plasticity when an animal is adapting to conditions which produce dramatic changes in behavior.

The “binding” affinities and capacities of [ 3 H]-γ-aminobutyric acid ( 3 H-GABA) and [ 14 C]-glycine to synaptosome-enriched fractions of rat cerebral cortex and spinal cord have been compared over a 10 −8 to 10 −3 m concentration range in isosmotic sucrose solutions containing 32 m m NaCl. At 0 C, the binding of both GABA and glycine to particles of the cerebral cortex appeared to take place by “triple-affinity” processes. The respective binding affinities (reciprocals of K B values) of GABA for particles of the cortex were generally greater than those for glycine, whereas maximal binding capacities ( B max values) for GABA were about twice as great as those for glycine. In fractions of spinal cord, at 0 C, the binding of both GABA and glycine occurred by “double-affinity” processes, and though the binding affinities of GABA were about twice those of glycine, B max values for GABA were smaller than those for glycine. Striking changes occurred when particles were allowed to stand in contact with [ 3 H]-GABA plus [ 14 C]-glycine at 23 C for 45 min. Although double-affinity mechanisms persisted at the higher temperature for the binding of both amino acids to particles of spinal cord (GABA still being bound with greater affinities than glycine), the binding of both amino acids to particles of cerebral cortex was best resolved as “single-affinity” processes. The differences between B max values for GABA and glycine in particles of cerebral cortex and spinal cord appeared to reflect their different endogenous concentrations.

The interaction of N -methyl-bicuculline and γ-aminobutyric acid (GABA) on a synaptosomal fraction of rat cerebral cortex was examined at 0 C in a balanced bicarbonate-buffered medium using differential centrifugation and double-isotope scintillation spectrometry. The drug, at 10 −4 m , produced a significant decrease in the binding of [ 3 H]GABA (10 −8 , 10 −7 m ) to the particles. Further experiments revealed that N -methyl-bicuculline, at concentrations of 10 −7 –10 −3 m , significantly decreased the “binding” of GABA (4.8 × 10 −8 m ) to the particles. This bicuculline-induced reduction in GABA binding exhibited linear characteristics over a 10 −10 –10 −3 m concentration range of the drug, and when present at 10 −3 m , the drug decreased GABA binding by about 23%. This demonstration of a bicuculline-sensitive component of GABA binding to synaptosomal particles adds support to neurophysiological studies which have indicated that bicuculline may serve as an antagonist to the GABA receptor in vivo .

Synaptosomal fractions, prepared from the brains of mice which had been differentially-housed (isolated vs. aggregated) for 8–10 weeks, differed in their capacities to bind the inhibitory neurotransmitter suspects, glycine and γ-aminobutyric acid. These environmentally-induced changes were shown at 0 C in buffered physiological medium. The binding of both glycine and GABA (1.2 × 10 −5 m ) to these particles was decreased in the presence of 10 −8 and 10 −4 m strychnine-SO 4 . These results provided evidence that changes in cerebral synaptic morphology caused by environmental modification can be demonstrated by measuring the binding of possible inhibitory neuro-transmitters. Hence, the binding of these substances appears to occur by adaptable, plastic processes.

Desheathed frog sciatic nerves, previously loaded with labeled l -glutamate, released greater amounts of radioactivity when stimulated electrically, at intensities ranging from juxtathreshold to twice that needed to evoke maximal α, β -action potentials. Incubations of control and stimulated companion nerves were carried out simultaneously. When an increase in the efflux of glutamate was not evident from the general shapes of the efflux curves, it became clear by determining the percentage of total radioactivity released in 120 min. It seems that there exists a discrete compartment of glutamate in peripheral nerve which is a part of the total exchangeable compartment and which can be released by electrical stimulation. The extra glutamate release accounts for about one-tenth of the total glutamate efflux and may be related to the increase in membrane permeability which occurs during conduction.