Medial opening wedge high tibial osteotomy: more than ten years of experience with Puddu plate technique supports its indication.

PURPOSE: To describe the short-term and long-term results of patients who underwent a medial opening wedge high tibial osteotomy (MOW-HTO) for unicompartmental medial knee joint osteoarthritis. METHODS: A retrospective review was conducted of patients with MOW-HTO using a Puddu plate®, with more than ten year follow-up. The degree of correction, initial chondral damage, number of meniscal lesions, preoperative and 1-year postoperative functional scale scores (IKDC and Lysholm), and arthroplasty conversion rates at the ten year follow-up were registered. We assumed early indication when patients underwent the operation before they were 40 years old and delayed ≥ 40. Functional outcomes were analyzed by adjusting for pre-operative values. Fisher's exact test was used to study the association between the arthroplasty conversion rates and the timing of indication. RESULTS: Fifty-five patients were included, 37 of whom were male (67%). Twenty-nine patients had early indications for surgery (53%). All patients completed ten year follow-up. All patients improved IKDC (p < 0.01) and Lysholm (p < 0.01) scores compared to their presurgical scores at the one year post-operative evaluation. We had six minor complications, none requiring revision surgery. We had three conversions to arthroplasty, all in the late indication group, not statistically significant different. Linear regression showed that early indication was associated with a higher IKDC score when adjusting for the Outerbridge chondral damage score, the number of meniscal lesions, and sex (p < 0.01). CONCLUSION: All patients improved functional scores one year after surgery. Early indication (i.e., younger than 40 years of age) was independently associated with better functional outcomes than late indication at one year follow-up.

The chemical nature of the main central excitatory transmitter: a critical appraisal based upon release studies and synaptic vesicle localization.

The chemical nature of the central transmitter responsible for fast excitatory events and other related phenomena is analysed against the historical background that has progressively clarified the structure and function of central synapses. One of the problems posed by research in this field has been whether one or more of the numerous excitatory substances endogenous to the brain is responsible for fast excitatory synaptic transmission, or if such a substance is, or was, a previously unknown one. The second question is related to the presence in the CNS of three main receptor types related to fast excitatory transmission, the so-called alpha-amino-3-hydroxy-5-methylisoxazole propionic acid, kainate and N-methyl-D-aspartate receptors. This implies the possibility that each receptor type might have its own endogenous agonist, as has sometimes been suggested. To answer such questions, an analysis was done of how different endogenous substances, including L-glutamate, L-aspartate, L-cysteate, L-homocysteate, L-cysteine sulfinate, L-homocysteine sulfinate, N-acetyl-L-aspartyl glutamate, quinolinate, L-sulfoserine, S-sulfo-L-cysteine, as well as possible unknown compounds, were able to fulfil the more important criteria for transmitter identification, namely identity of action, induced release, and presence in synaptic vesicles. The conclusion of this analysis is that glutamate is clearly the main central excitatory transmitter, because it acts on all three of the excitatory receptors, it is released by exocytosis and, above all, it is present in synaptic vesicles in a very high concentration, comparable to the estimated number of acetylcholine molecules in a quantum, i.e. 6000 molecules. Regarding a possible transmitter role for aspartate, for which a large body of evidence has been presented, it seems, when this evidence is carefully scrutinized, that it is either inconclusive, or else negative. This suggests that aspartate is not a classical central excitatory transmitter. From this analysis, it is suggested that the terms alpha-amino-3-hydroxy-5-methylisoxazole propionic acid, kainate and N-methyl-D-aspartate receptors, should be changed to that of glutamate receptors, and, more specifically, to GLUA, GLUK and GLUN receptors, respectively. When subtypes are described, a Roman numeral may be added, as in GLUNI, GLUNII, and so on.

N-methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances.

N-Methyl-DL-aspartate, L-glutamate, kainate and DL-homocysteate were found to increase the initial rate and the maximal uptake of 45Ca into the non-inulin space of rat brain cortex slices incubated in vitro. The N-methylaspartate-stimulated calcium uptake was blocked by cadmium and cobalt ions, but not by the organic calcium channel blocker nifedipine or by tetrodotoxin, both of which stimulated the N-methylaspartate-independent calcium influx. gamma-Aminobutyrate increased the spontaneous calcium influx, and also reduced that stimulated by N-methylaspartate to the same level, as found with gamma-aminobutyrate alone. Adenosine (1-100 microM), ethanol (0.1 M), pentobarbital (10-100 microM) and morphine (0.2 mM), were unable to inhibit the N-methylaspartate-activated calcium influx. Ethanol (0.1 M), had no effect on the glutamate- or kainate-activated calcium influx. These findings suggest that the excitatory amino acids, because of their neuronal depolarizing action in brain cortex, lead to the opening of voltage-sensitive calcium channels, which may be blocked by cadmium, but not by the organic calcium channel antagonist, nifedipine. The activation of calcium channels by the excitatory amino acid N-methylaspartate, was entirely unaffected by the depressants ethanol, pentobarbital or morphine, or by the endogenous inhibitory substance, adenosine, thus suggesting that their inhibitory or depressant effects occur through interference with a neuronal mechanism unrelated to the one studied here. gamma-Aminobutyrate, on the other hand, considerably inhibited N-methylaspartate-induced calcium uptake, an effect interpreted as due to a gamma-aminobutyrate-induced increase in chloride conductance, that "clamps" the membrane potential and does not allow further depolarization by N-methylaspartate.

Electrically induced release of labelled taurine, alpha- and beta-alanine, glycine, glutamate and other amino acids from rat neocortical slices in vitro.

The electrically induced release of labelled alpha-aminoisobutyrate, L-alpha-alanine, beta-alanine, glycine, histidine, serine, glutamate, aspartate and taurine, from superfused thin slices of the rat neocortex, held on quick-transfer electrodes was studied. In no instance did the release of these substances resemble that of (3H)-labelled noradrenaline, acetylcholine or 5-hydroxytryptamine, which can be released by 0.5-3 V stimuli and whose release shows an absolute dependency on calcium ions. Small amounts of alpha-aminoisobutyrate, beta-alanine, serine, glutamate and aspartate were released with 4 V stimuli, but the release was statistically significant for the first two substances only. Following incubation with (3H)-histidine, substantial labelling of homocarnosine was found, but no electrically induced release of this dipeptide could be detected. With (14C)-taurine, however, small but significant release was found with sinewave stimuli of 1.5 V or higher. Such release was significantly increased in the absence of calcium ions. Biphasic pulses of frequencies ranging between 10 and 100 Hz. (1 V, 3 ms duration) did not evoke the release of (14C)-taurine, although this type of stimulation readily induced the release of (3H)-noradrenaline studied simultaneously. Differences in threshold, calcium dependency and shape of the taurine efflux peak, relative to that seen with (3H)-noradrenaline and other transmitters, suggest that taurine release occurs by mechanisms unrelated to those that mediate transmitter secretion. The release of all the above amino acids can readily be elicited, however, if stimuli that are too intense, prolonged or damaging are utilized. The occurrence of these artifacts in the present and in previous work is discussed.

A study in rat brain cortex synaptic vesicles of endogenous ligands for N-methyl-D-aspartate receptors.

The presence of endogenous ligands for the N-methyl-D-aspartate receptor was looked for in highly purified rat brain cortex synaptic vesicles, the contents of which were extracted and fractionated by gel filtration on Sephadex G-10, or by three different high-voltage electrophoresis procedures. The presence of endogenous ligands was detected by their ability to compete with 50 nM L-[3H]glutamate for binding to whole rat brain N-methyl-D-aspartate receptors. The receptor preparations used were those present in purified postsynaptic densities, in which the quisqualate receptors were blocked by 10 microM quisqualate. Synaptic vesicles had a high content of N-methyl-D-aspartate receptor ligands, which on fractionation always coincided with glutamate or aspartate. A variable and very small amount of a highly acidic endogenous ligand was also found. The latter substance did not coincide in the electrophoresis with homocysteic, cysteic, quinolinic, cysteine sulphinic or homocysteine sulphinic acids, or with N-acetyl-aspartyl-glutamic acid, S-sulphocysteine or sulphoserine. We also found that a single centrifugation, in 0.25 M sucrose, 25 mM Tris-citrate, pH 7.1, of purified synaptic vesicles, at 135,000 gmax for 45 min, led to a 51% loss of endogenous glutamate, but did not change their aspartate content. Thus, in uncentrifuged vesicles the glutamate/aspartate ratio was 9.4, while in centrifuged ones the ratio was 3.9 ATP markedly enhanced L-[3H]glutamate uptake into synaptic vesicles, but did not change the binding of L-[3H]aspartate. Differences in labelled aspartate and glutamate efflux from the vesicles were also found.(ABSTRACT TRUNCATED AT 250 WORDS)

Kainate-induced seizures alter protein composition and N-methyl-D-aspartate receptor function of rat forebrain postsynaptic densities.

The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1alpha, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, beta-actin, alpha-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl-D-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations. From these data, we suggest that intense synaptic activity associated with seizures modifies the composition of postsynaptic densities and has profound consequences on the function of the N-methyl-D-aspartate receptors present in them. This rearrangement may accompany impairment of synaptic plasticity.

Differential effects of ouabain and 2,4-dinitrophenol on contractile tension of and on sodium and calcium efflux from frog heart ventricular strips.

1 The efflux, from heart ventricular strips of Rana pipiens, of sodium ((22)Na) and calcium ((45)Ca) was measured simultaneously.2(22)Na efflux could be resolved into two first order kinetic components: k(I) = 0.105 min(-1), thought to represent efflux from the extracellular space, and k(II) = 0.0182 min(-1) representing efflux from the cells.3(45)Ca efflux was also resolved into an extracellular component, k(I) = 0.1216 min(-1); and an intracellular one, k(II) = 0.0102 min(-1). (45)Ca k(II) was greatly increased by 2,4-dinitrophenol (DNP), but unchanged by caffeine. This suggests that it represents a mitochondrial calcium compartment.4(22)Na k(II) was not changed by DNP. This indicates that, at the time of DNP addition, (22)Na was passively bound to undefined intracellular components.5 Ouabain (10(-6) M) decreased (45)Ca efflux (k(II)) initially but at later periods slightly increased it. The former effect is thought to be due to an action at the plasma membrane level, while the latter probably represents an increased exchangeability of mitochondrial calcium. The same effects were always found when ouabain was applied at different times of strip superfusion.6 Ouabain (0.25 to 4 muM) did not decrease the k(II) of (22)Na efflux. Kinetic reasons are presented which indicate that, in this preparation, the activity of the sodium pump may be too fast to be measured by means of (22)Na efflux, therefore these findings do not necessarily mean that ouabain does not inhibit active sodium transport.7 The time course of the inotropic effect of ouabain was also studied in ventricular strips of Rana pipiens heart that were stimulated at 0.2 Hz with biphasic, 2 ms pulses of supramaximal intensity, and incubated in Ringer solution containing 1.1 mM calcium, or in ;calcium-free' Ringer (residual calcium: 5.2 muM), or in ;calcium-free' Ringer with 0.1 mM of the calcium chelator ethyleneglycol bis (beta-aminoethylether) N,N'-tetraacetic acid (EGTA).8 In Ringer, the inotropic effect of ouabain was already observed at 5-10 s after steroid addition, even with the lowest concentration tested (0.25 muM), while signs of toxicity appeared only after 15 min in 4 muM ouabain, the highest concentration used.9 When the strips were incubated in ;calcium-free' Ringer solution, force of contraction decayed to 1-2% of that in 1.1 mM calcium. Addition of 4 muM ouabain to these hypodynamic strips led to a progressive increase in contractile force of up to 300%, that started after a 50 s latency period. No signs of toxicity were observed.10 Incubation of the strips in EGTA-Ringer also reduced contractile force to about 2% of that in Ringer, and 4 muM ouabain also increased force of contraction by approximately the same amount as seen in ;calcium-free' Ringer, but the effect began after a 10 min latency period. The concentration of calcium ion (Ca(2+)) in the extracellular space of strips incubated in EGTA-Ringer, was approximately 800 fold lower than in Ringer, and 60 fold lower than in ;calcium-free' Ringer solution.11 Caffeine (20 mM) induced, in strips previously incubated for 1 h in 4.4 mM calcium Ringer solution plus 10(-6) M ouabain, a marked initial contracture, that relaxed spontaneously, and was followed by slow waves of contracture. This was not observed if the strips were incubated, prior to caffeine, in 4.4 mM calcium Ringer without ouabain, or in 1.1 mM calcium Ringer solution that contained 10(-6) M ouabain.12 Based on these findings, a hypothesis that can explain the inotropic effect of cardioactive steroids is presented.

Effects of guanethidine on electron transport and proton movements in rat heart, brain and liver mitochondria.

Guanethidine at 5-25 mM concentrations was found to induce up to 79% inhibition of ADP-stimulated (state III) oxygen consumption in isolated rat heart, brain or liver mitochondria, when the added substrate was glutamate or succinate, but the inhibition was considerably lower (24% or less) when respiration was supported by ascorbate plus tetramethylphenylenediamine (TMPD). Comparable results were seen regarding ADP-stimulated proton uptake, where even greater inhibition (up to 94% with glutamate or succinate, but not ascorbate plus TMPD) was found. Similar but somewhat less marked effects were also seen in resting (state IV) respiration and on the acceptor control ratio (state III/state IV respiration). 2,4-Dinitrophenol was unable to relieve guanethidine-induced inhibition of electron transport. These results indicate that guanethidine inhibits primarily mitochondrial electron transport itself, and that the site where such inhibition is more marked is located in the span between ubiquinone and cytochrome c of the respiratory chain. It is, therefore, suggested that active guanethidine uptake by noradrenergic neurons can lead to a high drug concentration in their cytoplasm and hence to mitochondrial alterations that can contribute to the pharmacological effect of this drug. Our results demonstrate the interaction between guanethidine and the electron transport chain of mitochondria derived from different tissues and, therefore, support this hypothesis.

A search for endogenous modulators of the GABA receptor in different regions of the rat CNS.

The possible existence of endogenous substances other than ?-aminobutyric acid (GABA), that can also bind to rat brain GABA receptors, has been investigated in synaptic membranes derived from whole rat brain, or from cerebral cortex; as well as in isolated synaptic vesicles obtained from cerebral cortex, striatum, hypothalamus, cerebellum and spinal cord and in the superfusion fluid of electrically stimulated brain cortex slices, where a GABA-like substance is released by a calcium-dependent process. The detector used to study the presence of such presumed non-GABA endogenous ligands, were frozen and thawed rat brain synaptic membranes, that had been treated with 0.05% Triton X-100 and thoroughly washed. With this highly sensitive preparation, at least 5 pmol of GABA/ml could be detected. The extracts of the different preparations where these hypothetical ligands were looked for, were analyzed by means of gel filtration on Sephadez G-10, paper chromatography and high voltage electrophoresis. In a very great number of experiments performed, the only endogenous ligand detected was GABA itself. The possible influence of a number of peptides on binding of GABA to its receptor, was also looked for. No significant effect was found for substance P, neurotensin, cholecystokinin octapeptide sulfated, somatostatin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, methionine enkephalin (all 10(?5) M), angiotensin II (10(?4) M), ACTH (3 x 10(?7)M), poly-l-lysine (30 ?g/ml) or poly-l-glutamate (30 ?g/ml).

Differential effects of veratridine and calcium on the release of [(3)H]noradrenaline and [(14)C]?-aminoisobutyrate from rat brain cortex slices.

The efflux of [(3)H]noradrenaline (NA) and of the non transmitter, non metabolizable, amino acid [(14)C]?-aminoisobutyrate (AIB), was followed simultaneously from superfused rat brain cortex thin slices, that had been preloaded with those substances. Short (2 min) "pulses" of increasing veratridine concentrations were applied at 10 min intervals. When calcium in the superfusion fluid was 1 mM, [(3)H]NA efflux increased progressively with pulses of 1, 3, 10 and 30 ?M veratridine, but further increase to 100 ?M resulted in a decrease of the induced (3)H-efflux. Veratridine-enhanced [(3)H]NA efflux decreased considerably in 0.1 mM calcium and was virtually suppressed when no calcium was added to the superfusion fluid. In 1 mM calcium, the efflux of [(14)C] AIB was increased progressively by pulses of 10, 30 and 100 ?M veratridine, but no increase in efflux was seen with 1 or 3 ?M drug. In 0.1 mM, or without added calcium, the induced efflux of [(14)C]AIB was markedly increased. Similar findings were seen when a long (10 min) pulse of 10 ?M veratridine was given. After such long pulses there was a rapid return of AIB efflux to pre-veratridine levels if calcium was 1 mM, but in the absence of added calcium, the return to baseline levels of both [(3)H]NA and, especially, that of [(14)C]AIB efflux, was greatly impaired. The veratridine enhanced efflux of both NA and AIB was entirely blocked by 1 ?M tetrodotoxin.

Recordings of glutamate receptor channels in isolated postsynaptic densities.

We have isolated highly purified rat brain postsynaptic densities (PSDs), that are known to contain glutamate receptors of the AMPA and NMDA types. These PSDs were incorporated into liposomes, and grown, by a cycle of partial de- and rehydration in 5% ethylene glycol, into giant (5-100 microns in diameter) liposomes. These giant liposomes were then made to form Gigaohm (10-20 G omega) seals with conventional patch-clamp electrodes, which, when withdrawn, retain an excised patch in an inside-out configuration. When 5-10 microM L-glutamate (or 10 microM NMDA) plus 1 microM glycine were present inside the patch pipette, but not in the external fluid, a highly complex pattern of currents was seen in about 55% of the cases. This was characterized by very fast kinetics, conductances as high as 460 pS and multiple lower levels of 45, 80, 120, 230 and 340 pS. These currents, when evoked by NMDA plus glycine, were entirely suppressed by the NMDA antagonist 2-amino-5-phosphonovalerate, APV. However, those activated by L-glutamate plus glycine still appeared in the presence of APV in about 18% of the cases, but with lower conductance levels. Current kinetics similar to the latter ones were also induced by the AMPA receptor agonist quisqualate (10 microM) in 16% of the cases. This indicated that both NMDA and AMPA receptors were present, in a functionally well preserved state, in isolated postsynaptic densities. Indirect evidence also suggested that in our experiments, in which 212 seals were studied, only a single postsynaptic density was present in the patches in which channel activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous ligands for the quisqualate receptor: presence in rat brain cortex synaptic vesicles.

The presence in highly purified rat brain cortex synaptic vesicles of endogenous ligands for rat brain quisqualate receptors was investigated. The vesicles were extracted, and their contents fractionated by high voltage electrophoresis. Endogenous ligands were detected by a radioreceptor assay in which such ligands competed with 50 nM L-[3H]glutamate for binding to quisqualate receptors present in rat brain postsynaptic densities (PSDs). Binding of L-[3H]glutamate to N-methyl-D-aspartate (NMDA) receptors, also present in PSDs, was blocked by 100 microM NMDA. We found that the endogenous ligands present in brain cortex synaptic vesicles for quisqualate receptors, were glutamate and aspartate, in a molar ratio of about two to one. The quisqualate receptor had an affinity 130-fold higher for glutamate (Kd 0.3 microM) than for aspartate, and the latter amino acid also showed a marked negative cooperative for binding (Hill number 0.29, against 0.67 for glutamate). These findings suggest that glutamate is the natural transmitter that activates quisqualate receptors at some central excitatory synapses, and also that aspartate may be a classical transmitter, the receptor for which still remains to be shown.

Interaction of glycine with brain-cortex membrane fragments: kinetics, ionic requirements and amino acid specificity.

The binding of [14]glycine to rat brain-cortex membrane fragments, incubated in artificial cerebrospinal fluid, was studied in vitro by means of a nitrocellulose filter assay. The membranes were obtained from the large granule fraction (P2) of a brain-cortex homogenate, which was osmotically shocked and the larger membrane fractions isolated by centrifugation. Initial binding velocity lasts for about 2 min and equilibrium is reached in 10 min. The binding reaction is reversible, and [14C]glycine can de displaced by an excess of [12C]glycine or by dilution. Binding is strongly dependent on temperature and on sodium ions. The latter activate the binding process in a cooperative manner. Two binding components may be discerned: one with high affinity for glycine (Km = 40 +/- 8 muM) and one with lower affinity. Lowering the sodium concentration to 60 mM increases the Km of the high-affinity component to 59 muM, with no change in Vmax. The bound product is, after incubating the membranes at 37 degrees C for 10 min, 85% glycine. A large fraction of it may released by hypo-osmotic media.

A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations.

The possible excitatory effect of N-acetyl-alpha- aspartylglutamate ( NAAG ) was studied in 3 different systems. First on the increase in 45Ca2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM L-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of L-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 micrograms) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.

A search in rat brain cortex synaptic vesicles for endogenous ligands for kainic acid receptors.

Conditions were found for stabilizing rat brain cortex kainic acid (KA) receptors. Such receptors had the same Hill number (about 0.6) for KA and for glutamate. The receptors were then used as detectors for endogenous ligands present in brain cortex synaptic vesicle (SV) soluble extracts. When these SV extracts were fractionated by gel filtration on Sephadex G-10, by thin-layer chromatography, or by high voltage electrophoresis, a single endogenous component, that in all cases comigrated with glutamic acid, was found.

Continuous measurement of net potassium movements in rat brain cortex suspensions. Effects of glutamate, veratridine, creatine and other substances.

Net K fluxes in in vitro suspensions of sliced rat brain cortex were studied by means of a K-sensitive electrode. When incubation was in 3 mM K, a net K efflux occurred. It could be resolved into two first-order rate constants: k1 = 0.486 min-1, and k2 = 0.0102 min-1, that originated from compartments that contained 18% and 82% of tissue K, respectively. k1 Was suppressed by tetrodotoxin (TTX), and k2 was increased 38-fold by veratridine. The latter effect was blocked by TTX, methylphenidate (1 mM), creatine (25 mM), apamin (50 nM), quinine (100 microM), verapamil (22 microM) or D-600 (38 microM). Net K loss was greatly increased by 1 mM ouabain, and enhanced by sodium azide plus iodoacetamide, but not by 0.1 M ethanol. Glutamate (5 mM) induced a considerable and rapid net uptake of K, while aspartate or N-methylaspartate increased K efflux.

Electrically induced release of [3H]dopamine from slices obtained from different rat brain cortex regions. Evidence for a widespread dopaminergic innervation of the neocortex.

Slices obtained from the deeper layers of the rat dorsal frontal, parietal and occipital brain cortex were incubated in vitro with 6.25 X 10-7 M [3H]dopamine (DA), and subsequently superfused and electrically stimulated, while held on quick transfer electrodes, and changes in the efflux of 3H and of the individual amines measured. The separation of the amines, with quantitative recoveries, was performed by chromatography on cation-exchange resins eluted sequentially with water, 1 N HCL AND 6 M urea i 1 N HCl. When no drugs were used, the prestimulation efflux was entirely formed by deaminated metabolites, while following stimulation there was an increase in the efflux of deaminated metabolites, and considerable amounts of [3H]-noradrenaline (NA) now appeared. No DA was present in the pre- or poststimulation medium. Similar results were obtained in all the regions studied. When the slices were incubated with 10-5 M desmethylimipramine (DMI), 10-4 Mnialamide and 10-4 M tropolone, before and during incubation with [3H]DA, it was observed that, prior to stimulation, the efflux was composed of deaminated metabolites, DA and 3-methoxytyramine (MTA), and following the electrical stimulus there was an increased release of DA, NA and deaminated compounds (in order of decreasing release), while no change in that of MTA was evident. The stimulus-induced release of DA was greatest from frontal slices, intermediate from parietal, and lowest from occipital ones. DMI-resistant uptake of [3H]DA also diminished when passing from frontal to occipital. These findings are interpreted as due to the presence of dopaminergic axon terminals in all the regions studied, but with a density that diminishes in a rostrocaudal direction.

Veratridine-induced release of endogenous glutamate from rat brain cortex slices: a reappraisal of the role of calcium.

The efflux of endogenous glutamate from thin slices of rat brain cortex superfused in vitro with artificial cerebrospinal fluid (ACSF) was studied. Initially, glutamate efflux was very high (2.5 nmol/mg protein/min), possibly because of the cutting procedure, but declined sharply, and at 30 min of superfusion was 25 pmol/mg protein/min. In ACSF without added calcium, spontaneous glutamate efflux was always higher than that in calcium-containing medium, e.g. at 30 min it was 75 pmol/mg protein/min. Addition of 10 microM veratridine for 2 min, between 30 and 32 min of superfusion, led, in ACSF with calcium, to an increase in glutamate efflux of 288%, when the maximum efflux following veratridine is compared to the glutamate efflux that immediately preceded the application of this drug (from 25 to 97 pmol/mg protein/min), while in ACSF without added calcium, veratridine induced an increase of only 117% (from 75 to 163 pmol/mg protein/min). These results are interpreted as due to the dual effect of veratridine. In calcium-containing ACSF, veratridine increases sodium influx which depolarizes the neurons and opens voltage-sensitive calcium channels. The increased intraneuronal calcium induces glutamate release from synaptic vesicles, while increased intracellular sodium enhances the release of soluble cytoplasmic glutamate by the reverse operation of the plasma membrane, sodium-dependent glutamate carrier. In ACSF without calcium, the release of vesicular glutamate is suppressed, while the sodium-dependent mechanism remains. This appears as if veratridine-induced glutamate efflux were only partially calcium-dependent.

Neuropathological changes in the rat brain cortex in vitro: effects of kainic acid and of ion substitutions.

The ionic mechanisms that may contribute to the neurotoxicity of kainic acid, were studied in a system of rat thin neocortical slices superfused in vitro. Slices superfused for 3 h under control conditions showed an essentially normal aspect when studied by light microscopy. Presence of 30 microM kainate in the superfusion fluid induced neuronal swelling, nuclear condensation and signs of necrosis in some cells, while other neurons, especially in deeper layers, appeared dark and condensed, with microvacuolation. The neuropil presented numerous profiles of swollen dendrites. When the slices were superfused with chloride-free medium, a large number of pyknotic neurons was seen. This was further enhanced by 30 microM kainate, which produced no swelling in this medium. These effects of Cl-free medium were almost entirely prevented in Cl-free medium without calcium and with 0.1 mM of EGTA. Sodium-free medium induced a marked neuronal swelling that was not much changed by kainate. When calcium in an otherwise normal superfusion fluid was reduced to 0.1 mM, a large number of pyknotic neurons, some with incrustations, were seen. Kainate (30 microM) in this low calcium medium led to a very large swelling and destruction of neurons, and to a spongy neuropil. These effects of kainate were greatly intensified in calcium-free-EGTA (0.1 mM) medium. Ca-free-EGTA medium by itself induced considerable neuronal and neuropil swelling. It is concluded that kainate induces neuronal swelling by a sodium- and chloride-dependent mechanism, and the enhancement of swelling in low calcium is due to an increased sodium uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular distribution of rat brain cortex high-affinity, sodium-dependent, glycine transport sites.

The subcellular distribution of the membrane components, present in rat brain cortex homogenates, that interact with glycine in the presence of sodium ions was studied. The distribution in the primary fractions, as per cent of total binding in the homogenate, was: P1 ('nuclear'), 58%; P2 (large granule), 39%; P3 (microsomal), 2%9 Of the subfractions obtained by centrifuging P1 in a linear 0.32--1.5 M sucrose gradient, only the lighter fraction (P1-III) formed by large myelin fragments was enriched in specific binding activity with respect to P1. The pellet formed by purified nuclei had negligible binding, and fractions of intermediate density had a lower activity than P1. Transient exposure of P1-III to 1.5 M sucrose did not diminish its binding ability. Similarly, in the subfractions obtained by centrifuging P1 in a discontinuous sucrose gradient, only the least dense one, P1-A, that is formed exclusively by large myelin fragments, was enriched with respect to P1. The electron microscopy of these fractions is presented. The P2 subfractions, obtained in a linear 2--18% Ficoll gradient, had the following sodium-dependent activity (counts/min/mg protein, fractions being in the order of decreasing density): pellet, 0; P2-I, O; P2-II, 450; P2-III, 1770; P2-IV, 4130; unfractionated P2, 880; P2-IV, the least dense fraction being composed mainly of myelin. With P2 subfractions obtained in a discontinuous sucrose gradient (0.32, 0.8 and 1.2 M sucrose layers), it was also found that sodium-dependent glycine binding was only enriched, with respect to P2, in the myelin fraction P2-A. Glycine binding to purified brain cortex myelin was also found to be very high, while binding to non-myelin membranes, obtained during the purification procedure, was only 0--7% of that seen with myelin. These results suggest that high-affinity glycine binding is located in myelin proper, and possibly also in some other glial plasma membranes, but not in nuclei, mitochondria, endoplasmic reticulum or synaptosomes. The relevance of these findings for interpreting previous reports on high-affinity glycine transport in the central nervous system is analyzed.