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Notes: Abstract— (1) Mouse cerebrum slices swell in tris-buffered Krebs-Ringer medium. Swelling is rapid at first, then slows to a more or less constant rate. Even after 3 hr incubation, water content/g of tissue dry wt. shows no sign of an asymptotic limit. Swelling is the same at 37° and at 0°.(2) Tissue water measured by incubation with tritiated water is equal to total tissue water measured by drying slices. Equilibration between tritiated water and tissue water is complete within 2 min.(3) Tissue liquid can be divided into three phenomenologically distinguishable compartments: first inulin space, which is the compartment permeable to inulin at both 0° and 37°; second inulin space, which is the compartment permeable to inulin at 37° but not at 0°; and 37°non-inulin space, which is the compartment impermeable to inulin at both 0° and 37°. The evidence for this is:(a) Penetration of inulin into tissue is greater at 37° than at 0°. After the first 20 min the rate of penetration at 0° is approximately equal to the rate of penetration at 37°, and only slightly less than the rate of increase of total tissue water. Therefore the smaller inulin space observed at 0° cannot be due to slower entry of inulin.(b) The inulin content of slices incubated in inulin-containing medium at 37° and cooled to 0° in the same medium is the same as the inulin content of tissue incubated at 37° without subsequent cooling. In contrast, the inulin content of tissues preincubated in inulin-free medium at 37° and then incubated in inulin-containing medium at 0° is the same as the inulin content of tissues incubated in inulin-containing medium at 0° without preincubation at 37°. Therefore the smaller inulin space at 0° than at 37°can be due neither to a reversible temperature-dependent change in the size of one single inulin space nor to an irreversible, greater swelling of a single inulin space at the higher temperature, but is due to some portion of the 37° inulin space becoming impermeable to inulin at 0°.(c) Some inulin is retained by tissue incubated with inulin at 37°, then transferred to inulin-free medium at 0°; the amount of retained inulin is equal to the difference between inulin content of tissue incubated with inulin at 37° and tissue incubated with inulin at 0°. This confirms 3b above and in addition shows that inulin which has entered the second inulin space at 37° is trapped there when this space becomes impermeable to inulin at 0°.(4) The penetration of the amino acids, L-lysine and D-glutamate at 0° is equal to the penetration of inulin at 37°. This confirms the real existence of the 37° inulin space at 0°, and shows that the barrier at 0° between the first and second inulin spaces does not exist for these substances.(5) The amino acids L-leucine and glycine penetrate total tissue water at 0°. L-leucine is actively transported at this temperature.(6) The amino acids α-aminoisobutyric acid, L-leucine, and L-lysine at 2 mm have no effect at 37° on either the inulin space or the non-inulin space.(7) The inulin space is insensitive at 37° to physiologically significant changes in the medium. In contrast, the non-inulin space is quite sensitive to these changes. Addition of D-glutamate greatly increases the non-inulin space; addition of ouabain or cyanide, or omission of glucose, increases the non-inulin space slightly; and replacement of Na+ ion by choline+ ion greatly decreases this space. These changes are independent and roughly additive.

Notes: (1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices.(2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used.(3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals.(4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals.(5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices.(6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.

Notes: Abstract— (1) Incubation in physiological saline at 37º caused brain slices from chick embryos to lose water (shrink), whereas slices from young chicks gained water (swell). Shrinkage decreased with age in embryonic brain, and swelling increased with age in chick's brain.(2) Incubation at 0º diminished shrinkage in 8-day old embryos, caused an increasing degree of swelling in older embryos, and enhanced swelling in young chick's brain slices.(3) Inulin equilibrated more rapidly with tissue water in embryonic than in young adult brains. Measured ‘inulin space’ values at 37º changed little with age, whereas 0º‘inulin space’ values significantly decreased with age. Calculated zero time ‘inulin space’ values also decreased with age.(4) The measured ‘inulin space’ at 0º was larger than that measured at 37º (but not of the calculated zero time ‘inulin space’) in young embryos, then became progressively smaller, so that in young chicks the situation was reversed.(5) The calculated extracellular space ranges from about 37 per cent (8-day old embryos) to about 14 per cent (15-day old chicks).(6) Incubation in the absence of glucose had more effect on brain slices from young chicks than from embryos. The opposite was true when a hypotonic medium or a medium containing d-glutamate was used.

Notes: Abstract— Synaptosomal release and exchange of [3H]GABA were studied by a superfusion technique which minimizes reuptake. The release of [3H]GABA was increased by depolarizing concentrations of KCl and showed calcium-dependence. Superfusion with 1-1000 μm unlabelled GABA caused a dose dependent, saturable increase in the release of radioactivity by homoexchange. The exchange process showed high substrate specificity: among the various amino acids and putative neurotransmitters tested, only γ-amino-β-hydroxybutyric acid was a good stimulator of [3H]GABA release. Superfusion with sodium-free medium (NaCl replaced by sucrose) virtually abolished homoexchange. Ouabain also increased the release of [3H]GABA, and its action was additive to that of unlabelled GABA.The presence of exchange at concentrations that are in the range of the high affinity uptake system, the apparent similarity between calculated rates of exchange and initial uptake rates, the non-detectability of exchange in a condition (Na+ deprivation) which inhibits high affinity uptake, and the lack of decrease of actual GABA concentration in incubation media used for uptake experiments, all suggest that homoexchange accounts for a substantial part of the synaptosomal accumulation of [3H]GABA generally interpreted as high affinity uptake.

Notes: Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE2 and 8-epi-PGF2α following infusion of N-methyl-d-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE2 and 8-epi-PGF2α basal levels. These effects were largely prevented by the specific cytosolic phospholipase A2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.