RESUMO
High-frequency stimulation (HFS) applied to stratum radiatum of a rat hippocampal slice in the presence of ionotropic glutamate receptor antagonists evokes a biphasic GABA(A) receptor-dependent response in CA1 pyramidal neurons, with a brief hyperpolarizing IPSP (hIPSP) followed by a long-lasting depolarization. We show now that it is possible to pharmacologically separate the hIPSP and late depolarization from one another. In neurons intracellularly perfused for 1-2 hr with F(-) as the major anion and no ATP, the hIPSP (and the corresponding current, hIPSC) evoked by HFS was blocked, whereas neither the late depolarization nor its underlying current was attenuated. In contrast, internal perfusion with a high concentration (5 mM) of the impermeant lidocaine derivative QX-314 selectively abolished the depolarizing component of the biphasic response and also strongly reduced depolarizations evoked by extracellular microinjection of K(+). Bath application of quinine (0. 2-0.5 mM) or quinidine (0.1 mM) resulted in a pronounced inhibition of the HFS-induced extracellular K(+) concentration ([K(+)](o)) transient but not of the bicarbonate-dependent alkaline shift in extracellular pH. The attenuation of the [K(+)](o) transient was closely paralleled by a suppression of the HFS-evoked depolarization but not of the hIPSP. Quini(di)ne did not affect depolarizations induced by exogenous K(+) either. These data provide direct pharmacological evidence for the view that the HFS-induced biphasic response of the pyramidal neuron is composed of mechanistically distinct components: a direct GABA(A) receptor-mediated phase, which is followed by a slow, nonsynaptic [K(+)](o)-mediated depolarization. The bicarbonate-dependent, activity-induced [K(+)](o) transient can be blocked by quini(di)ne, whereas its depolarizing action in the pyramidal neuron is inhibited by internal QX-314. The presence of fundamentally distinct components in GABA(A) receptor-mediated actions evoked by HFS calls for further investigations of their functional role(s) in standard experimental maneuvers, such as those used in studies of synaptic plasticity and induction of gamma oscillations.
Assuntos
Potenciais Evocados/fisiologia , Antagonistas GABAérgicos/farmacologia , Hipocampo/fisiologia , Células Piramidais/fisiologia , Receptores de GABA-A/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Trifosfato de Adenosina/farmacologia , Anestésicos Locais/farmacologia , Animais , Potenciais Evocados/efeitos dos fármacos , Fluoretos/farmacologia , Técnicas In Vitro , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Masculino , Modelos Neurológicos , Neurônios/fisiologia , Compostos Organofosforados/farmacologia , Ácidos Fosfínicos/farmacologia , Propanolaminas/farmacologia , Células Piramidais/efeitos dos fármacos , Quinina/farmacologia , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
We investigated the pathophysiological mechanisms of glutamate-induced delayed neuronal damage in rat hippocampal slice cultures [Stoppini et al. (1991) J. Neurosci. Methods 37, 173-182], with propidium iodide as a marker of cell death. Exposure of the cultures to growth medium containing 10 mM glutamate for 30 min resulted in a slowly developing degeneration of hippocampal principal cells, starting from the medial end of the CA1 region and reaching the dentate gyrus by 48 h. By 24 h, most pyramidal cells in CA1 were damaged. An acute phase of degeneration preceded the delayed damage at 2-6 h, affecting cells in a spatially diffuse manner. When tetrodotoxin (0.5 microM) was present during the glutamate insult, a marked protection (mean 57%, P<0.001) of the CA1 damage was observed. Rather strikingly, when tetrodotoxin was applied immediately following or even with a delay of 30 min after the insult, a similar amount of protection was achieved. In field recordings carried out after the insult, the glutamate-treated slices exhibited spontaneously occurring negative shifts with a duration of 1-10 s and an amplitude of up to 400 microV in the CA3 region, whereas the control slices were always quiescent. Taken together, the results suggest that post-insult neuronal network activity, rather than the direct action of exogenous glutamate, is a major cause of delayed CA1 pyramidal cell death in the organotypic slices. These observations may have implications in the design of neuroprotective strategies for the treatment of brain traumas which are accompanied by delayed and/or distal neuronal damage.
Assuntos
Lesões Encefálicas/metabolismo , Morte Celular/fisiologia , Ácido Glutâmico/toxicidade , Hipocampo/efeitos dos fármacos , Degeneração Neural/metabolismo , Neurotoxinas/toxicidade , Células Piramidais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Morte Celular/efeitos dos fármacos , Epilepsia/metabolismo , Epilepsia/patologia , Epilepsia/fisiopatologia , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/metabolismo , Rede Nervosa/fisiopatologia , Neurotoxinas/metabolismo , Técnicas de Cultura de Órgãos , Células Piramidais/metabolismo , Ratos , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
Extracellular pH transients were evoked in rat hippocampal brain slices by activation of a monosynaptic inhibitory pathway following pharmacological blockade of glutaminergic transmission. Repetitive stimulation in stratum radiatum near the recording site in stratum pyramidale evoked an immediate alkaline shift which was potentiated by pentobarbital and blocked by picrotoxin but not by 2-hydroxy-saclofen. Benzolamide, a poorly permeant inhibitor of carbonic anhydrase (CA), and prontosil-dextran 5000, a macromolecular CA inhibitor, abolished the alkaline transients evoked by stimulation and by exogenous GABA. Thus an extracellular CA is involved in regulating interstitial pH in brain, and the stimulation-induced alkaline transients are caused by net influx of CO2 into CA1 neurons in response to efflux of bicarbonate across postsynaptic GABAA receptor channels.
Assuntos
Hipocampo/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Reflexo Monosináptico/efeitos dos fármacos , Animais , Inibidores da Anidrase Carbônica/farmacologia , Estimulação Elétrica , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Transmissão Sináptica/efeitos dos fármacosRESUMO
The effect of dexamethasone on exercise-induced adrenocorticotropin (ACTH) secretion and dental analgesia was studied in healthy human subjects. Different levels of exercise (100-200 W) were produced by a cycle ergometer. Dental pain thresholds were tested with a constant current stimulator. Dental pain thresholds were elevated with increasing work loads, and the elevation was still significant 30 min after the end of the exercise. Dexamethasone produced a significant reversal of exercise-induced pain threshold elevations concomitantly with the suppression of exercise-induced ACTH release. The results suggest that the corticotropin releasing factor-ACTH axis is involved in the exercise-induced analgesia.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Analgesia , Dexametasona/farmacologia , Dor/fisiopatologia , Esforço Físico , Dente/inervação , Hormônio Adrenocorticotrópico/sangue , Estimulação Elétrica , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Limiar Sensorial/efeitos dos fármacosRESUMO
1. The role of voltage-gated calcium channels in the generation of activity-induced alkaline shifts in extracellular pH (pHo) was studied in rat hippocampal slices (area CAI) by means of Ca(2+)-and H(+)-selective microlectrodes inserted into the stratum pyramidale and/or stratum radiatum. 2. After complete pharmacological blockade of ionotropic glutamate receptors and gamma-aminobutyric acid-A (GABAA) receptors, trains (5-10 Hz, 5-10s) of antidromic spikes in pyramidal neurons were associated with a fast alkaline transient of up to 0.17 pH units and a fall in the extracellular Ca2+ concentration ([Ca2+]o). The alkaline shift was strongly enhanced upon inhibition of extracellular carbonic anhydrase. 3. Application of 100 microM Ni2+ plus 100 microM Cd2+ inhibited both the fall in [Ca2+]o and the alkaline transient triggered by antidromic spikes. The alkaline shift was abolished in the absence of extracellular Ca2+. 4. In the absence of postsynaptic receptor antagonists, alkaline transients linked to a given level of synaptic excitation in s. radiatum were strongly suppressed after blockade of somatic (and, consequently, of dendritic "backpropagating") spikes by microdrop application of tetrodotoxin to the cell-body layer. 5. We have previously shown that activity-induced alkaline transients in the CAI region are due to an influx of Ca2+ into neurons, which triggers an influx of H+ ions probably caused by activation of a plasmalemmal Ca2+/H+ ATPase. The present results indicate that much (in s. pyramidale perhaps all) of the pH-changing influx of Ca2+ is mediated by voltage-gated Ca2+ channels.
Assuntos
Canais de Cálcio/fisiologia , Hipocampo/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Benzolamida/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacologia , Dendritos/efeitos dos fármacos , Dendritos/fisiologia , Estimulação Elétrica , Eletrofisiologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Tetrodotoxina/farmacologiaRESUMO
The effect of dynamic cycle ergometer exercise and isometric leg exercise on skin sensitivity was studied in man. Exercise was performed at different loads. Cutaneous sensitivity to innocuous and noxious thermal stimuli was tested using a contact thermostimulator and sensitivity to tactile stimuli was tested using electrical stimuli. During isometric exercise a segmental (the exercising limb), but not a multisegmental, phasic decrease of cutaneous thermal sensitivity to innocuous stimuli was found. At the isometric forces used the effect on tactile and heat pain sensitivity was not significant. During dynamic exercise a multisegmental, load-dependent decrease of sensitivity in all tested sensory modalities was found and this attenuation disappeared gradually after the end of exercise. In contrast to isometric exercise, the decrease of sensitivity produced by dynamic exercise was most evident in tactile sensitivity. The size of the stimulus area (7.9 vs 11.8 cm2) did not have a significant effect on the magnitude of the exercise-induced decrease of cutaneous thermal sensitivity to innocuous stimuli. It was concluded that underlying the modulation of skin sensitivity by dynamic and isometric exercise were mechanisms that were different, at least to a small extent. Isometric exercise produced a segmental modulation of skin sensitivity due to central neuronal mechanisms, independent of exercise-induced stress. Exercise-induced stress could have caused the modulation of skin sensitivity by dynamic exercise.
Assuntos
Exercício Físico , Sensação , Fenômenos Fisiológicos da Pele , Adulto , Temperatura Baixa , Temperatura Alta , Humanos , Perna (Membro) , Masculino , Fenômenos Fisiológicos do Sistema Nervoso , Tato/fisiologiaRESUMO
1. The ionic mechanism of extracellular alkaline transients mediated by alpha-amino-3-hydroxy-5-methylisoxazolate-4-propionic acid (AMPA) receptor channels was studied in the rat hippocampal slice (area CA1) by means of H(+)-, Ca(2+)- and Na(+)-selective microelectrodes. 2. Alkaline transients mediated by selective synaptic activation of AMPA receptors were coupled to a transient fall in extracellular Ca2+ ([Ca2+]o). 3. Removal of [Ca2+]o blocked the alkaline transient evoked by microinjection of AMPA, but had little effect on the simultaneous tetrodotoxin-resistant fall in extracellular Na+ ([Na+]o). 4. Alkaline transients evoked by microinjection of gamma-aminobutyric acid (GABA) were not affected by removal of [Ca2+]o. GABA had no effect on [Na+]o. 5. The present results indicate that activity-induced glutamatergic alkaline transients in the hippocampal slice are due to an influx of Ca2+, and not to a conductive movement of H+ ions across glutamate-gated cation channels.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Espaço Extracelular/fisiologia , Hipocampo/fisiologia , Canais Iônicos/fisiologia , Receptores de Glutamato/fisiologia , Animais , Cálcio/metabolismo , Técnicas de Cultura , Potenciais da Membrana/fisiologia , Ratos , Receptores de AMPA/fisiologia , Sódio/metabolismo , Ácido gama-Aminobutírico/fisiologiaRESUMO
1. Inhibitors of extracellular carbonic anhydrase (CAo) offer much promise as diagnostic tools in the study of the synaptic basis of activity-induced alkaline transients in the brain. However, most of the present information related to the effects of CAo blockers in nervous tissue comes from experiments that involve simultaneous synaptic activation of various types of postsynaptic receptor channels. In the present work, double-barreled H(+)-selective microelectrodes were used to study alkaline shifts in extracellular pH (pHo) evoked by selective synaptic and pharmacological activation of glutamate and gamma-aminobutyric acid (GABA) receptors in the CA1 cell body layer in rat hippocampal slices. Inhibition of CAo was achieved with the use of the poorly permeant carbonic anhydrase inhibitor, benzolamide (10 microM; applied in the bath solution) or the impermeant macromolecular inhibitor, prontosil-dextran 5000 (PD 5000; applied in microdrops). 2. Alkaline transients that were exclusively attributable to synaptic activation of glutamate receptors were induced by stimulation of Schaffer collaterals in the presence of picrotoxin (PiTX, 100 microM). An enhancement by the CAo inhibitors of these alkaline transients took place at all stimulus frequencies (5-200 Hz) and stimulus train durations (0.5-20 s) examined. 3. Inhibition of CAo enhanced the alkaline transients evoked by selective synaptic activation of alpha-amino-3-hydroxy-5-methyli-oxazolate- 4-propionic acid (AMPA)/kainate receptors in experiments involving stimulation of Schaffer collaterals in the simultaneous presence of PiTX and D-2-amino-5-phosphonopentoate (AP5, 40-80 microM). 4. Alkaline shifts evoked by selective synaptic activation of N-methyl-D-aspartate (NMDA) receptors were enhanced after inhibition of CAo as seen in experiments where Schaffer collaterals were stimulated in the simultaneous presence of PiTX and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX, 20-40 microM) in an Mg(2+)-free solution. 5. Benzolamide and PD 5000 also enhanced the alkaline shifts seen upon pressure injection of glutamate, AMPA, or NMDA. The glutamate-induced alkaline shifts were inhibited by AP5+CNQX, suggesting that uptake of glutamate did not significantly contribute to their generation. 6. Stimuli applied at 5-10 Hz in stratum radiatum close (within 0.5 mm) to the recording site evoked alkaline shifts that were blocked by CNQX plus AP5. In the continuous presence of the two glutamate antagonists, PiTX-sensitive alkaline transients were observed in response to brief high-frequency (20-100 Hz) trains consisting of 100 stimuli. Upon application of pentobarbital sodium (100 microM), these apparently monosynaptically evoked GABAA receptor-mediated alkaline transients were evident also at low stimulation frequencies (5-10 Hz).(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Potenciais Evocados/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Receptores de AMPA/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Benzolamida/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
1. The neuronal basis of alkaline shifts in extracellular pH (pHo) evoked by stimulation of Schaffer collaterals was studied by means of double-barreled H(+)-selective microelectrodes in the area CA1 of rat hippocampal slices. 2. Alkaline transients in stratum pyramidale evoked by stimulation at a low frequency (5-10 Hz) were enhanced by pentobarbital sodium (100 microM). In the absence of the drug, inhibition of extracellular carbonic anhydrase (CAo) by benzolamide or by prontosildextran 5000 (PD 5000) resulted in an increase in the alkaline shifts. In contrast to this, alkaloses evoked by low-frequency stimulation in the presence of pentobarbital were attenuated by a subsequent inhibition of CAo. 3. Blockade of gamma-aminobutyric acid-A (GABAA) receptors with picrotoxin (PiTX; 100 microM) resulted in an enhancement of alkaline transients in s. pyramidale evoked by low-frequency stimulation (10 Hz) but suppressed alkaline shifts evoked by brief high-frequency (1 s, 100 Hz) trains of stimuli. 4. Application of trains of stimuli consisting of a constant number of pulses (50 or 100) revealed a striking dependence of the effect of benzolamide on stimulation frequency (10-200 Hz) in s. pyramidale: the enhancement of the alkaloses seen upon inhibition of CAo became progressively smaller with an increase in frequency, and at 100-200 Hz benzolamide produced a suppression or a complete block of the alkaline transients. However, alkaline transients evoked with the use of a constant train duration (5 s) were enhanced by benzolamide at all stimulation frequencies examined (5-200 Hz).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Álcalis/farmacologia , Potenciais Evocados/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Benzolamida/farmacologia , Concentração de Íons de Hidrogênio , Picrotoxina/farmacologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
1. The ionic mechanism underlying the fast, GABAA receptor-mediated inhibitory postsynaptic potential (IPSPA) was examined in rat neocortical neurones using intracellular recording techniques. Synaptic responses were evoked by orthodromic stimulation applied to the subcortical white matter or to the pial surface. All experiments were carried out at a constant extracellular Cl- concentration. 2. The resting membrane potential was -76.2 +/- 1.0 mV (mean +/- S.E.M., n = 32) and in most cells IPSPA was depolarizing. The reversal potential of IPSPA (EIPSP-A) was -70.2 +/- 0.9 mV (n = 32) and that of a more slowly developing hyperpolarizing response (IPSPB) was -91.4 +/- 1.3 mV (n = 28). 3. An examination of the temporal relationships between excitatory postsynaptic potentials (EPSPs) and IPSPAs in different cells suggested that, despite partial overlap of these responses, EPSPs had little influence on the measured values of EIPSP-A. 4. Application of 20 mM trimethylamine (TriMA), a membrane-permeant weak base which is expected to produce a rise in pHi (and hence in intracellular HCO3-), induced a reversible positive shift in EIPSP-A of up to +9.0 mV (mean + 4.2 mV) at an extracellular pH (pHo) of 7.4. In some experiments, the shift in reversal potential was associated with a change in the polarity of IPSPA from hyperpolarizing to depolarizing. 5. Application of 20 mM lactate (a membrane-permeant weak acid which is expected to produce a fall in pHi and hence in intracellular HCO3-) at pHo 7.0 produced a hyperpolarizing shift in EIPS-A of up to -7.5 mV (mean -5.6 mV). In some experiments, exposure to lactate changed the polarity of IPSPA from depolarizing to hyperpolarizing. 6. Changes in pHo from 7.4 to 7.0 reduced the effect of TriMA and augmented that of lactate on EIPSP-A, as could be expected on the basis of the pHo-dependent change in the fraction of membrane permeable non-charged weak base or acid. 7. Under control conditions, a change in pHo from 7.4 to 7.0 produced a slight positive shift (< +2 mV) in EIPSP-A. In the presence of TriMA, a similar change in pHo gave rise to a negative shift (-1.8 to -2.7 mV). 8. The results obtained indicate that HCO3- ions contribute significantly to the IPSPA, thereby making EIPSP-A more positive than the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 400 WORDS)