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.

Síndrome de gorlin: reporte de un caso / Gorlin syndrome: a case report

INTRODUCCIÓN: El Síndrome de Gorlin o síndrome del nevo basocelular es una enfermedad hereditaria autosómica dominante, caracterizada por anomalías del desarrollo y predisposición a carcinomas basocelulares múltiples, que constituyen uno de los aspectos más preocupantes de esta patología. Pese a su baja prevalencia y manejo por especialistas, es importante para los médicos generales tener los conocimientos necesarios para identificar y derivar lesiones sospechosas de carcinoma basocelular. PRESENTACIÓN DEL CASO: Mujer previamente sana que a los 28 años de edad debuta con dos nevus basocelulares faciales confirmados histológicamente. En la anamnesis y el examen físico se observan prominencias frontales, implantación amplia de la nariz, pits palmares, hipertelorismo, quistes de millium y quistes odontogénicos, con lo que se llega al diagnóstico de síndrome de Gorlin. Se realiza seguimiento durante 13 años identificándose y confirmándose histopatológicamente 14 nevus basocelulares nodulares infiltrantes. DISCUSIÓN: El síndrome de Gorlin tiene una prevalencia estimada en 1/57.000 habitantes. Posee penetrancia variable por lo que no todos los pacientes tienen el antecedente familiar. Para su diagnóstico se han definido criterios mayores, de los cuales la paciente cumple cuatro: carcinomas basocelulares múltiples, quistes odontogénicos, pits palmares y calcificación de la hoz del cerebro; y criterios menores. El tratamiento de los carcinomas basocelulares múltiples essimilar al de las lesiones aisladas, sin embargo, por tratarse de una enfermedad multisistémica es necesario un manejo multidisciplinario de las alteraciones esqueléticas, odontológicas, neurológicas y genitourinarias. Asimismo, se debe enfatizar en la fotoprotección y educación del paciente para la detección de lesiones cutáneas sospechosas.

INTRODUCTION: Gorlin syndrome or nevoid basal cell carcinoma syndrome is a hereditary autosomal dominant condition characterized by developmental anomalies and predisposition to multiple basal cell carcinomas, which are the most worrying aspects of the disease. Despite its low prevalence and specialized management, it is important for general physicians to be able to identify and refer suspicious lesions of basal cell carcinoma.CASE REPORT: A 28 year-old female, without relevant medical history, presented with two facial basal cell carcinomas histologically confirmed. Physical examination evidenced frontal bossing, a broad nasal root, hypertelorism, millia and odontogeniccysts, which comprise the diagnosis of Gorlin syndrome. 13-year follow-up identified 14 infiltrating nodular basal cell carcinomas. DISCUSSION: Gorlin syndrome has an estimated prevalence of 1 per 57.000 people. It has variable penetrance, therefore not all patients have a compatible family history. Major criteria have been established for its diagnosis, from which our patient fulfills four of them: multiple basal cell carcinomas, odontogenic cysts, palmar pits and calcification of the falx cerebri; and several minor criteria. Treatment of multiple basal cell carcinomas is similar to isolated lesions, however, as a multisystemic disease requires a multidisciplinary management of skeletal disorders, dental, neurological and genitourinary manifestations. Photoprotection and patient education for detection of suspicious skin lesions should be emphasized.

Chronic fluoxetine treatment induces structural plasticity and selective changes in glutamate receptor subunits in the rat cerebral cortex.

It has been postulated that chronic administration of antidepressant drugs induces delayed structural and molecular adaptations at glutamatergic forebrain synapses that might underlie mood improvement. To gain further insight into these changes in the cerebral cortex, rats were treated with fluoxetine (flx) for 4 weeks. These animals showed decreased anxiety and learned helplessness. N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit levels (NR1, NR2A, NR2B, GluR1 and GluR2) were analysed in the forebrain by both western blot of homogenates and immunohistochemistry. Both methods demonstrated an upregulation of NR2A, GluR1 and GluR2 that was especially significant in the retrosplenial granular b cortex (RSGb). However, when analysing subunit content in postsynaptic densities and synaptic membranes, we found increases of NR2A and GluR2 but not GluR1. Instead, GluR1 was augmented in a microsomal fraction containing intracellular membranes. NR1 and GluR2 were co-immunoprecipitated from postsynaptic densities and synaptic membranes. In the immunoprecipitates, NR2A was increased while GluR1 was decreased supporting a change in receptor stoichiometry. The changes of subunit levels were associated with an upregulation of dendritic spine density and of large, mushroom-type spines. These molecular and structural adaptations might be involved in neuronal network stabilization following long-term flx treatment.

Estudio toxicológico y teratogénico del extracto metanólico de cinnamomun zeylanicum (canela) / Toxicológico and teratogénico study of the metanólico extract of cinnamomun zeylanicum (cinnamon)

Se realizó el estudio fitoquímico, toxicológico agudo y citotóxico del cinnamomum zeylanicum (canela). Para la determinación de la DL50 se utilizaron 30 ratones albinos, cuyos pesos oscilaron entre 25 y 30 gr., siguiendo el método de Probits. Igualmente se determinó la concentración letal media (CL-50) en artemia salina. La actividad citotóxica y teratogénica fue evaluada en huevos de Tetrapygus Níger (erizo de mar). Nos permite concluir, siguiendo los criterios de William, que el extracto metanólico de la canela es ligeramente tóxico y posee efecto citotóxico frente al erizo de mar, no evidenciándose efecto teratogénico, a las dosis empleadas.

We have performed a Phytochemic. Toxicologic and Cytotoxic study, of Cinamomum zeylanicum (canela) in laboratory. We have determinated the letal 50-doses (DLSO) in mice, the letal middle concentration (CL_50) in Artemia salina and the cytotoxic and teratogenic effect on Tetrapygus niger eggs (sea Hedgehog). We may conclude following the William Criterions that the metanolic extract of canela is lightly toxic and it has cytotoxic effects on sea Hedgehog at the doses studied.

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.

Effect of glutamate receptor phosphorylation by endogenous protein kinases on electrical activity of isolated postsynaptic densities of rat cortex and hippocampus.

Postsynaptic densities (PSDs) were isolated from rat brain cortex and hippocampus, purified and incorporated into giant (5-80 microns in diameter) liposomes. Gigaohm seals were obtained with a patch-clamp pipette, and a giant liposome PSD-containing membrane patch, was excised and recorded. The PSD was always oriented in an inside-out configuration. This allowed receptor agonists or antagonists to be added from the interior of the recording pipette, and also the addition of different substances, such as ATP, calcium, calmodulin and others to the 'intracellular' side of the PSD, i.e. to the bath. alpha-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor agonists such as quisqualate or AMPA induced in the PSD a complex pattern of electrical activity, that was blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by 2-aminophosphonovalerate (APV). The currents generated by 0.5-1 microM quisqualate were increased by about 100% when the PSDs were phosphorylated. Similar findings were obtained when the agonist was 0.2-2 microM kainate. These currents were also blocked by a non-N-methyl-D-aspartate (NMDA) receptor antagonist but not by APV, and were increased by about 70% by phosphorylation of the PSDs. Addition of 5-10 microM NMDA plus 1 microM glycine to the 'extracellular' side of the PSD, led to a characteristic pattern of activity, with the opening of multiple receptor ion channels. This was entirely blocked by 10 microM APV. Addition of extracellular Mg2+ (1-2 mM) induced a voltage-dependent block of the currents. Phosphorylation of the PSD led to an increase of Mg(2+)-blocked current of about 80%. The effect of phosphorylation on ion channel activity showed a markedly different requirement for calcium and for calmodulin among the AMPA, kainate and NMDA types of glutamate receptors, thus suggesting that each receptor type is coupled at the synapse with a unique complement of protein phosphokinases.

In memoriam professor Hugo Adrian.

In memoriam Professor Hugo Adrian, born 1926, deceased 1994, in Santiago, Chile. After completion of his studies in veterinary medicine, he followed a successful career in neurophysiological research. He was Research Associate (1961-1962) and Visiting Professor (1973-1976) at the Neurophysiology Department University of Wisconsin, USA. He was the first Director (1958-1960) of the Institute of Physiology at the Austral University, Valdivia, and was Professor (1963-1973; 1977-1994) and Chairman of the Department of Physiology and Biophysics, University of Chile, Santiago, Chile, where he led a group of researchers in auditory physiology, introduced the use of computer techniques to physiological studies, and developed several projects of applied neurophysiology.

In memoriam professor Hugo Adrian

In memoriam Professor Hugo Adrian, born 1926, deceased 1994, in Santiago, Chile. After completion of his studies in veterinary medicine, he followed a successful career in neurophysiological research. He was Research Associate (1961-1962) and Visiting Professor (1973-1976) at the Neurophysiology Department University of Wisconsin, USA. He was the first Director (1958-1960) of the Institute of Physiology at the Austral University, Valdivia, and was Professor (1963-1973; 1977-1994) and Chairman of the Department of Physiology and Biophysics, University of Chile, Santiago, Chile, where he led a group of researchers in auditory physiology, introduced the use of computer techniques to physiological studies, and developed several projects of applied neurophysiology

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.

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)

[Latin America: reasons for hope]. / América Latina: razones de una esperanza.

According to official figures, Latin America is almost an empty continent. The low population density, the decrease in population growth, the low use of the land and the improvement in the availability of food, health and education indexes, point to the fact that restrictive politics in population growth are not of interest to this region. On the contrary, an increase in population density is important in the development of this new civilization. In order to avoid the defects found in some industrialized countries, the anthropocetrism has to be linked to reality which is transcendent and has God as his foundation.

PIP: It is argued in this work that policies to control population growth are not needed in Latin America because of its low population density, low and declining rate of growth, and improvements in food supply, health care, and educational level over the past several years. According to the author, population density is only 13.8% of density in Asia and 27.3% of that in Europe, while only 8.6% of potentially cultivable land is cultivated. Per capita food availability increased by 11.3% between 1965 and 1989. The fresh water supply in Latin America and the Caribbean comprises 29.4% of the world total, but only 1% is utilized. The infant mortality rate declined from 94/1000 to 48/1000 live births between 1965 and 1990, while the general mortality rate fell from 11/1000 to 7/1000. To become developed, Latin America requires a much greater occupation of its agricultural potential and consequently a substantial increase in its population density. It has not been proven that deforestation in Latin America would lead to increased atmospheric carbon dioxide, and even if it did, some increase in global temperatures caused by higher carbon dioxide levels might help retard the next ice age, now relatively near. To achieve its full potential, Latin America must avoid the demographic aging and stagnation occurring in many western countries because of massive use of contraception, legal abortion, and legal euthanasia. Belief in God and the values that flow from that belief are necessary if civilization is to thrive in Latin America.

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)

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.

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.

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)

[Teaching-assistance integration: an experience of continuing education for service personnel. Colombia]. / Integración docente-asistencial: una experiencia de educación continua para el personal de servicios. Colombia.

The article describes an experience in the integration of teaching assistance involving personnel from health clinics and centers. The educational methodology was based on participation, and great progress was made in the development of health workers (professionals, technicians, auxiliaries, and promoters). However, the authors point out that the educational process in itself was not a factor in leading to better services, nor did it have a qualitative impact on the dynamics of preventive and curative assistance. The determining factor is the existing organization of the society where the program was carried out. Given this organization, it is fairly unlikely that new educational methods will be really meaningful because of the structural, social, political and economic constraints that characterize the Colombian system.

Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure.

Rat brain cortex synaptic vesicles have been isolated by 3 different procedures. The one of Hata et al. (J. Neurochem., 27 (1976) 139) gave synaptic vesicles with a high glutamate content, but also, as judged by [3H]ouabain binding and electron microscopy, with considerable contamination by plasma membrane vesicles. This did not allow a precise estimation of the glutamate content of each synaptic vesicle. The second procedure used (Life Sci., 21 (1977) 1075), in which the tissue is homogenized with an all glass homogenizer, yielded vesicles of higher purity, but with no glutamate. A slightly modified Kadota and Kadota procedure (J. Cell Biol., 58 (1973) 135) gave synaptic vesicles of a very high purity that were filtered on a Sepharose 4B column, and there, the synaptic vesicle fraction of highest purity was estimated to contain 3640 glutamate molecules in each glutamatergic vesicle. This is equivalent to an intravesicular concentration of 0.21 M, that is, at least 10 times higher than the glutamate concentration in the rat brain cortex.

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.