Samples of rat and human cerebral cortex were frozen, stored, and thawed under a variety of conditions to define further the optimal procedure for storing human brain samples for subsequent metabolic and functional studies that use incubated synaptosomes. Tissue samples were best preserved by immersing them in isotonic sucrose prior to slow freezing, but there was no advantage in first chopping up the material. High concentrations of sucrose, rather than exerting a cryoprotective effect, were detrimental to subsequent synaptosomal performance (oxygen uptake, K+ accumulation, stimulus-induced release of amino acid neurotransmitters). However, good activity was observed in preparations from rat brain frozen in the absence of fluid. This result was confirmed by studies on the uptake of gamma-aminobutyrate (GABA) into an osmotically sensitive compartment in synaptosomes prepared from frozen human autopsy material transshipped from the brain tissue collection ("brain bank") at Harvard Medical School, MA, USA, to Sydney, Australia. Although activity was slowly lost over a 3-mo period in rat tissue samples stored at -20 degrees C, there was little or no such loss at -70 degrees C.

Quinolone antibiotics, which can be proconvulsant in susceptible patients, were found to inhibit the specific binding of the adenosine receptor ligands L-3H-N6-phenylisopropyladenosine (L-3H-PIA) and 3H-N-ethylcarboxamidoadenosine (3H-NECA) to rat brain synaptic membranes. The inhibitions were concentration dependent, and for both ligands the order of potency was rosoxacin greater than nalidixic acid greater than oxolinic acid greater than or equal to ciprofloxacin greater than norfloxacin greater than enoxacin: IC20 values (concentrations causing a 20% inhibition of specific binding) ranged from 30-35 microM to 1-3 mM. Hill coefficients were approximately 0.5, suggesting that the compounds are probably antagonists at these sites. Most of the compounds did not alter 3H-diazepam binding directly, although rosoxacin showed relatively strong, and enoxacin weak, concentration-dependent inhibition. At 50 microM the compounds enhanced the maximal gamma-aminobutyric acid (GABA) activation of 3H-diazepam binding to varying degrees, without altering the EC50 of activation, whereas at 200 microM they tended to reduce GABA activation. Most noteworthy was the large increase in GABA-stimulated 3H-diazepam binding caused by 50 microM nalidixic acid. The compounds did not alter the Ca2+/Cl- -dependent binding of 3H-glutamate, nor of the binding of the glutamate site-selective ligands 3H-kainate and alpha-3H-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA); the uptake of the non-metabolized glutamate analogue D-3H-aspartate by cortical homogenates was also unaffected. The CNS side effects of these antibiotics may result, in part, from interaction with sites which mediate the inhibitory neurotransmission of adenosine and, possibly, GABA.

Inherited myoclonus of Poll Hereford calves is characterized by hyperesthesia and myoclonic jerks of the skeletal musculature, which occur spontaneously and in response to sensory stimuli. The disease shows autosomal recessive inheritance, and significant proportions of the Poll Hereford herds in many countries are thought to be carriers of the mutant gene. Studies revealed a specific and marked (90 to 95 percent) deficit in [3H]strychnine binding sites in spinal cord membranes from myoclonic animals compared to controls, reflecting a loss of, or defect in, glycine/strychnine receptors. Spinal cord synaptosomes prepared from affected animals showed a significantly increased ability to accumulate [3H]glycine, indicating an increased capacity of the high-affinity neuronal uptake system for glycine. In contrast, stimulus-induced glycine release and spinal cord glycine concentrations were unaltered.

Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.

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