Effects of Zinc, Mercury, or Lead on [3H]MK-801 and [3H]Fluorowillardiine Binding to Rat Synaptic Membranes.

Glutamate (Glu) is considered the most important excitatory amino acid neurotransmitter in the mammalian Central Nervous System. Zinc (Zn) is co-released with Glu during synaptic transmission and interacts with Glutamate receptors and transporters. We performed binding experiments using [3H]MK-801 (NMDA), and [3H]Fluorowillardine (AMPA) as ligands to study Zn-Glutamate interactions in rat cortical synaptic membranes. We also examined the effects of mercury and lead on NMDA or AMPA receptors. Zinc at 1 nM, significantly potentiates [3H]MK-801 binding. Lead inhibits [3H]MK-801 binding at micromolar concentrations. At millimolar concentrations, Hg also has a significant inhibitory effect. These effects are not reversed by Zn (1 nM). Zinc displaces the [3H]FW binding curve to the right. Lead (nM) and Hg (µM) inhibit [3H]FW binding. At certain concentrations, Zn reverses the effects of these metals on [3H]FW binding. These specific interactions serve to clarify the role of Zn, Hg, and Pb in physiological and pathological conditions.

Toxic effects of xylazine on endothelial cells in combination with cocaine and 6-monoacetylmorphine.

The use of xylazine as a drug of abuse has emerged worldwide in the last 7 years, including Puerto Rico. Clinical findings reported that xylazine users present greater physiological deterioration, than heroin users. The aim of this study was to assess the xylazine toxicity on endothelial cells, as this is one of the first tissues impact upon administration. Human umbilical vein endothelial cells in culture were treated with xylazine, cocaine, 6-monoacetylmorphine (heroin metabolite) and its combinations, at concentrations of 0.10-400 µM, for periods of 24, 48 and 72 h. IC50 were calculated and the Annexin V assay implemented to determine the cell death mechanism. Results indicated IC50 values at 24h as follow: xylazine 62 µM, cocaine 210 µM, 6-monoacetylmorphine 300 µM. When these drugs were combined the IC50 value was 57 µM. Annexin V results indicated cell death by an apoptosis mechanism in cells treated with xylazine or in combination. Results demonstrated that xylazine use inhibits the endothelial cell proliferation, at lower concentrations than cocaine and 6-monoacetylmorphine. These findings contribute to the understanding of the toxicity mechanisms induced by xylazine on endothelial cells.

[Intradiscal temperature variation resulting from radiofrequency thermal therapy. Cadaver study]. / Variación de la temperatura intradiscal por efecto de la terapia térmica con radiofrecuencia. Estudio en cadavers.

INTRODUCTION: Disc disease is one of the most common causes of lumbar pain. The new era of treatments for degenerative disc disease involves the use of minimally-invasive thermal technologies allowing for collagen remodeling and destruction of nociceptors in the annulus. However, a better understanding of the treatment pathophysiology is needed. The purpose of this study was to measure intradiscal temperature variation after thermodiscoplasty. MATERIAL AND METHODS: A human cadaver spine specimen was obtained and divided into blocks, each composed of two intervertebral plates and an intact disc. Radio frequency was applied at five spots with three different time intervals. Temperature was measured in each of the combinations. Units were weighed before and after treatment. Finally, the disc was exposed and the tightening achieved with each radio frequency application was measured. Data were analyzed with the SPSS software. RESULTS: The mean weight reduction obtained was 1.4 g on average (SD 0.599), with values between 0.5 and 2.6 grams. Mean temperature in the posterior rim of the annulus was 37.6 degrees C and mean temperature variation was 3.0 degrees C (SD 6.407). Mean tightening achieved in all blocks overall was 1.4 mm. DISCUSSION: The results obtained show the effectiveness of radio frequency thermodiscoplasty when performed within the safety parameters. Temperature values with radio frequency were lower than those found in comparable studies. The weight and the tightening show the effect of disc shrinking and dehydration. This report is an effective tool to define time parameters for the application of this technology.

AMPA and NMDA receptors in P2 fractions of cocaine and cocaine-prazosin-treated rats.

Cocaine sensitization results in the development of increased locomotion and stereotypy. It is accompanied by changes in glutamatergic trasmission that appear to be region-specific. The purpose of this article was to determine the effect(s) of cocaine and prazosin plus cocaine treatments on ionotropic glutamate receptors in rat cerebral cortex (CTX) and prefrontal cortex (PFC). Cocaine-sensitized rats (15 mg/kg, i.p. once for 5 days), withdrawn (7 days) and later challenged with a single cocaine dose, showed region-specific in NMDA-2A and Glu-R2 in the CTX and PFC membranes in cocaine- and prazosin-treated rats when compared to the saline controls. Co-administration of prazosin inhibits sensitization and changes in NMDA 2A and Glu-R2. Furthermore, prazosin inhibits the effect of cocaine in CTX and PFC on [(3)H]FW (AMPA agonist) binding when compared to controls. In cortex, cocaine treatment causes a marked increase in total binding, while in PFC there is a significant decrease. In both regions, cocaine-prazosin treatment attenuates the effects of cocaine. These results suggest that cocaine affects ionotropic glutamate receptors (NMDA and AMPA) and that prazosin inhibits such effects in a region-specific form in rat brain.

Veratridine, but not elevated K+, inhibits excitatory amino acid transporter activity in rat hippocampal slices.

PURPOSE: Excitatory amino acid transporter (EAAT) activity prevents Glu from reaching toxic levels, but their contribution to epileptogenesis remains controversial. We examined how the convulsant veratridine causes inhibition of EAAT activity and how it differs from the effects of another convulsant, high (50 mM) K+, that also increases Na+ conductance. METHODS: Transverse rat hippocampal slices were incubated for 1 h with 100 microM veratridine in oxygenated artificial cerebrospinal fluid (aCSF) with or without extracellular Ca2+. The medium was replaced by 50 microM[(3)H]glutamate in aCSF, and the slices incubated for 10 min at 37 degrees C. The slices were washed 3 times with cold aCSF after removal of the extracellular medium, and the radioactivity was quantified after solubilization of the slices. RESULTS: Veratridine caused a time- and dose-dependent decrease, whereas high K+ had no effect on EAAT activity. The effects of veratridine on EAAT activity were not prevented by tetrodotoxin (TTX; 10 microM). Coincubation of ouabain with veratridine resulted in further decreases of EAAT activity. Removal of extracellular Ca2+ potentiated the inhibitory effects of veratridine (and other convulsants) on EAAT activity. Chelation of intracellular Ca2+ with BAPTA also increased the inhibitory effects of veratridine on EAAT activity. CONCLUSIONS: Veratridine caused changes Ca2+ dynamics that led to inhibition of EAAT activity. Such changes in EAAT activity can contribute to the sustained epileptiform activity caused by veratridine.

Plasticity of excitatory amino acid transporters in experimental epilepsy.

PURPOSE: To examine the relationship between seizures and excitatory amino acid transporter (EAAT) activity and whether up-regulation of EAAT activity alters epileptogenicity. METHODS: In this study, we exposed rat hippocampal slices to different convulsants before measuring EAAT activity. Rats were exposed to the EAAT inhibitor pyrrolidine-2,4-dicarboxylic acid (PDC) before entorhinal cortex/hippocampal slices were obtained. These slices were exposed to low-Mg2+ buffer while electrophysiological recordings were obtained from the entorhinal cortex. mGluR III acting agents were used to study whether activation of mGluR III could regulate EAAT activity and if this regulation could overcome the effects on EAAT activity induced by the convulsants. RESULTS: Veratridine, kainic acid (KA), and pilocarpine reduced EAAT activity in rat hippocampal slices. L-2-Amino-4-phosphonobutyric acid (an mGluR III agonist) restored EAAT activity and reduced epileptiform activity to near control levels. The saturation curve for glutamate uptake in slices from KA-seized rats killed 2 hours after the first forelimb clonus was displaced to the left, suggesting a compensatory change for the enhanced excitation. On the other hand, rats injected with the EAAT inhibitor PDC (by intracerebroventricular injection) had more severe KA-induced seizures and N-methyl-D-aspartate epileptiform activity than control rats. Furthermore, hippocampal slices from KA- or KA+PDC-treated rats exposed to low Mg2+ reduced their firing rate to nearly zero once they returned to normal solution, whereas their control counterparts continued to fire, although at a lower rate. CONCLUSIONS: These results suggest a significant contribution of EAATs in some experimental epilepsy models and point to their short-term regulation by mGluR III as a possible source of their plasticity.

Efectividad del calzado ortopédico pronador en el tratamiento postoperatorio de niños con pie equino varo congénito idiopático rígido / Effectiveness of the corrective shoes in the posoperative treatment of equinovarus deformities in children

El uso de calzado ortopedico en el tratamiento postoperatorio del pie equino varo congenito idiopatico (PEVCI) es motivo de controversia. Con el fin de conocer la efectividad del uso del calzado con modificaciones especiales (botas pronadoras) en el manejo postoperatorio del paciente con PEVCI, a quienes se les efectuó una liberación posterointerna ampliada (LIPA), se realizó un estudio experimental clinico controlado ciego, que comparó dos intervenciones: al primer grupo se le formuló zapato ortopedico (botas pronadoras), al segundo se le asigno zapato normal. Para la evaluación se utilizaron los enterizos de Turco modificados. Los resultados se clasificaron como buenos regulares y malos. Se presenta un analisis interno con el objeto de detectar diferencias entre los grupos. Hasta el momento no se han encontrado diferencias por lo que se autoriza la continuación del estudio para definir con mayor precisión la efectividad de la intervención

Effects of Valeriana officinalis extracts on [3H]flunitrazepam binding, synaptosomal [3H]GABA uptake, and hippocampal [3H]GABA release.

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with gamma-aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [3H]flunitrazepam binding (EC50 4.13 x 10(-10) mg/ml). However, this increased [3H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC50 of 4.82 x 10(-1) mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [3H]flunitrazepam binding sites. Valerian extracts also potentiate K+ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [3H]GABA. Finally, inhibition of synaptosomal [3H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA(A) receptors, but can also interact at other presynaptic components of GABAergic neurons.

Proline-glutamate interactions in the CNS.

1. Crude synaptosomes (P2) and synaptosomal membranes were prepared from normal C57/B110 mouse brains and Wistar rats respectively. 2. [3H]Pro binding to mouse brain synaptic membranes was examined in the presence of competitive NMDA antagonist, MK-801, or HA-966. Conversely, the effects of l-proline on [3H]MK-801 binding were also probed. The effects of l-proline on glutamate-medicated [Ca+2]i levels were tested. 3. The authors could not detect any effect of proline on glutamate-mediated [CA+2]i levels using FURA-2 in synaptosomes or neuroblastoma cells. 4. NMDA competitive antagonists, AP-7, CPP, and CGS 19755 inhibit [3H]Pro binding to mouse brain synaptic membranes. 5. MK-801, a NMDA channel blocker, also inhibits [3H]Pro binding, but 200 mM proline is incapable of inhibiting [3H]MK-801 binding. 6. HA-966, a glycine site partial agonist inhibits [3H]Pro binding. Proline has modest effects on [3H]glycine binding.

Possible regulation of high-affinity glutamate uptake in synaptosomes of normal and epileptic mice.

Glutamate (Glu) uptake is the primary mechanism for its removal from the synapse. In genetic audiogenic seizures (AGS), Glu uptake is elevated prior to the appearance of seizures. Increased Glu uptake is also observed in synaptosomes from normal mice preincubated with lithium or nitroarginine, an NO synthase inhibitor. Pertussis and cholera toxins cause a marked reduction in Glu uptake. In contrast, neither lithium nor nitroarginine affected Glu uptake by synaptosomes from genetic epileptic mice. Arachidonic acid inhibits Glu uptake, whereas synaptosomes from epileptic mouse brain appear to be more sensitive to arachidonic acid as indicated by a shift of the inhibition curve to the left. These observations are indicative of the possible regulation of Glu uptake by second messengers and its alteration in genetic epilepsy.

Altered GABAergic and glutamatergic transmission in audiogenic seizure-susceptible mice.

The C57BL/10 SPS/sps mouse mutant are audiogenic seizure-susceptible. The enzymatic activities of glutamate decarboxylase (GAD), GABA aminotransferase (GABA-T), alanine aminotransferase (ALA-T), aspartate aminotransferase (ASP-T), and glutamate dehydrogenase (GDH) of whole brain supernatant are significantly reduced in these epileptic mice. GABA uptake is decreased in cortex, midbrain, and pons medulla. Previous studies showed the presence of two sodium-dependent GLU uptake systems in normal (SPS/SP) mice. Glutamate Umax by System 1 is significantly decreased in these mice, whereas the Umax value for System 2 is significantly increased in the epileptic mice.

High affinity [3H]glutamate uptake systems in normal and audiogenic seizure-susceptible mice.

Two high affinity sodium-dependent and PDC-sensitive glutamate (GLU) uptake systems are present in a whole brain synaptosomal preparation from adult C57BL/10 SPS/SPS normal mice. System 1 has an apparent Km of 3.65 microM while that of System 2 is 46.8 microM. Glutamate uptake in the normal mice increases gradually during development, displaying a striking peak at postnatal day 15, and decreases rapidly between PN 16 and PN 20 until it reaches adult levels. The developmental pattern of GLU uptake System 1 and System 2 in audiogenic seizure susceptible mice is similar to that described in normal mice. However, there are differences between GLU uptake system 1 and 2 during ontogenesis: (1) System 1 could not be detected until PN 15 while being markedly diminished in adulthood; and (2) GLU uptake by System 2 is increased in adults. In addition, the Umax for System 2 is significantly greater than that of normal mice at PN 2 and PN 15.

Inhibition of high-affinity [3H]L-proline binding to rat brain membranes by 2-amino-7-phosphonoheptanoic acid.

L-Glutamate and 2-amino-7-phosphonoheptanoic acid [corrected] (AP-7) (10(-9) to 10(-6) M), a competitive NMDA (N-methyl-D-aspartate) antagonist, inhibit approximately 60% of [3H]L-proline binding to rat membranes from midbrain. In hippocampal membranes, AP-7 inhibits proline binding by 80%, while in cerebellar membranes AP-7 had little effect. These results are indicative of the possible neuromodulatory role of proline in the central nervous system, possibly through the NMDA receptor(s).

GABAergic neurotransmission in the C57BL/10 sps/sps mouse mutant: a model of absence seizures.

The C57BL/10 sps/sps mouse mutant displays generalized absence seizure-like behavior. In these mice, glutamic acid decarboxylase activity is reduced in the cortex and hippocampus. Tritiated flunitrazepam binding (3H-flu) is reduced in these areas, as well as in midbrain, cerebellum, and pons-medulla. Quantitative [3H]-flunitrazepam binding autoradiography confirms these observations. GABA uptake by synaptosomes from sps/sps mice is also reduced in all the areas studied. Potassium-stimulated, Ca(2+)-dependent release of radioactivity from synaptosomes preloaded with [14C]-GABA is reduced in the hippocampus, increased in midbrain and pons-medulla, but remains unaltered in the cortex. These results suggest region-specific alterations in GABAergic neurotransmission that may be responsible for the absence-like seizures in C57BL/10 sps/sps mice.

The C57BL/10Bg sps/sps mouse: a mutant with absence-like seizures; neurochemical and behavioral correlates.

C57BL/10Bg sps/sps mice display behavioral arrest, similar to generalized absence seizures. Compared with the parent strain C57BL/10Bg SPS/SPS, the activities of glutamate decarboxylase (GAD, E. C. 2.6.1.15), GABA aminotransferase (GABA-T, E. C. 2.6.1.19), aspartate aminotransferase (ASP-T, E. C. 2.6.1.1), and glutamate dehydrogenase (GDH, E. C. 1.4.1.3) in whole brain crude supernatant were significantly reduced in the sps/sps mice. Alanine aminotransferase activity (ALA-T, E. C. 2.6.1.2), was not altered in any of the strains, and normalization of GAD, GABA-T and GDH activities by that of ALA-T, further revealed significant differences between the normal strain (SPS/SPS), the heterozygotes (SPS/sps), and behavioral arrest (sps/sps) mice. These results suggest the possible involvement of GABAergic and glutamatergic neurotransmission in the absence-like behavior displayed by sps/sps mice. Open field behavior of C57BL/10Bg sps/sps mice is characterized by periods of marked inactivity which easily distinguish affected homozygotes, from their heterozygotes littermates.

High-affinity binding of proline to mouse brain synaptic membranes.

There is evidence suggestive of the possible neuromodulatory role for L-proline in the mammalian brain. The binding of proline to whole mouse brain synaptic membranes has been partially characterized. Several binding sites for this imino acid have been identified; one in the nanomolar range and at least two in the submicromolar range. The binding of proline is inhibited by NaCl. Pipecolic acid (40 microM), ornithine, aminooxyacetic acid (AOAA), glycine, GABA, and glutamate were capable of significantly inhibiting proline binding. Although detailed pharmacological and functional studies are needed, these results are consistent with a brain-specific function for this imino acid, as well as, with the presence of specific binding site(s) for proline.

Proline binding to mouse brain synaptosomes.

L-proline has been shown to exert a variety of physiological and behavioral effects that are consistent with its possible role as a neuromodulator in the mammalian brain. The binding of l-proline to mouse brain synaptosomes has been partially characterized. Preliminary kinetic analysis shows the presence of at least two binding sites in the submicromolar range, and one in the nanomolar range. While more detailed studies are necessary as to determine the biological significance of proline binding to mouse brain synaptosomes, these results further support the possible role of proline as a neuromodulator.

Proline binding to mouse synaptosomes

La l-prolina ejerce unos efectos fisiológicos y conductúales los cuales sugieren su posible función como neuromodulador en el cerebro de los mamíferos. Se ha caracterizado parcialmente el enlazamiento de l-prolina a sinaptosomas de cerebro del ratón. Análisis preliminar de la cinética demustra la presencia de por lo menos dos sitios de enlazamiento en el rango submicromolar y de un sitio en el rango nanomolar. Aunque son necesarios estudios más detallados encaminados a esclarecer el significado biológico del enlazamiento de prolina a sinaptosomas de cerebro del ratón, estos resultados sirven de apoyo adicional a la posible función de prolina omo neuromodulador