RESUMO
In this issue of Neuron, DeVries (2001) describes experiments suggesting that acidification of the synaptic cleft can reduce Ca2+ channel activity and thereby act as a brake on tonic synaptic release of glutamate from cone cells. This work hints at a potentially important new facet to the regulation of synaptic transmission.
Assuntos
Terminações Pré-Sinápticas/fisiologia , Prótons , Transmissão Sináptica/fisiologia , AnimaisRESUMO
Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K(+) channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction.
Assuntos
Axônios/fisiologia , Moléculas de Adesão Celular Neuronais , Proteínas de Drosophila , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuroglia/fisiologia , Neuropeptídeos/fisiologia , Nervo Óptico/fisiologia , Receptores de Superfície Celular/fisiologia , Nervo Isquiático/fisiologia , Envelhecimento , Animais , Clonagem Molecular , Drosophila , Feminino , Biblioteca Genômica , Heterozigoto , Homozigoto , Humanos , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fibras Nervosas Mielinizadas/ultraestrutura , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Canais de Potássio/fisiologia , Receptores de Superfície Celular/genética , Mapeamento por RestriçãoRESUMO
Although the requirement for a strict regulation of pH in the brain is frequently emphasized, recent studies indicate that neuronal activity gives rise to significant changes in intracellular and extracellular pH. Given the sensitivity of many ion channels to hydrogen ions, this modulation of local pH might influence brain function, particularly where pH shifts are sufficiently large and rapid. Studies using pH-sensitive microelectrodes have demonstrated marked cellular and regional variability of activity-dependent pH shifts, and have begun to uncover several of their underlying mechanisms. Accumulating evidence suggests that regional and subcellular pH dynamics are governed by the respective localization of glial cells, ligand-gated ion channels, and extracellular and intracellular carbonic anhydrase.
Assuntos
Neurônios/fisiologia , Animais , Humanos , Concentração de Íons de HidrogênioRESUMO
We tested the hypothesis that extracellular membrane-bound carbonic anhydrase (CA) type IV is responsible for the regulation of interstitial pH (pH(o)) transients in brain. Rat hippocampal slices were incubated in phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the link of CA IV to the external face of plasma membranes. Then evoked alkaline pH(o) shifts were studied in a recording chamber, using pH microelectrodes. Incubation fluid was saved for later analysis. The ability to buffer a rapid alkaline load was reduced markedly in PI-PLC-treated tissue as compared with adjacent, paired control slices. The effect of benzolamide (a poorly permeant CA inhibitor) on evoked pH(o) shifts was diminished greatly in the PI-PLC-treated tissue, consistent with the washout of interstitial CA. Treatment of the incubation fluid with SDS abolished nearly all of the CA activity in fluid from controls, whereas an SDS-insensitive component remained in the fluid from PI-PLC-treated slices. These data suggested that CA type II (which is blocked by SDS) leaked from injured glial cells in both slice preparations, whereas CA type IV (which is insensitive to SDS) was liberated selectively into the fluid from PI-PLC-treated tissue. Western blot analysis was consistent with this interpretation, demonstrating a predominance of CA IV in the incubation fluid from PI-PLC-treated tissue and variable amounts of CA II in fluid from PI-PLC-treated and control slices. These results demonstrate that interstitial CA activity brain is attributable principally to membrane-bound CA IV.
Assuntos
Encéfalo/enzimologia , Anidrases Carbônicas/metabolismo , Membrana Celular/enzimologia , Espaço Extracelular/enzimologia , Animais , Benzolamida/farmacologia , Western Blotting , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Soluções Tampão , Inibidores da Anidrase Carbônica/farmacologia , Espaço Extracelular/química , Feminino , Ácido Glutâmico/administração & dosagem , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidróxidos/administração & dosagem , Técnicas In Vitro , Iontoforese , Isoenzimas/metabolismo , Masculino , Microeletrodos , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Ratos , Ratos Sprague-Dawley , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/farmacologiaRESUMO
Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown. In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient. B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.
Assuntos
Bombyx/enzimologia , Anidrases Carbônicas/metabolismo , Glândulas Exócrinas/enzimologia , Animais , Inibidores da Anidrase Carbônica/farmacologia , Glândulas Exócrinas/citologia , Concentração de Íons de Hidrogênio , Larva/enzimologia , Metazolamida/farmacologia , Força Próton-Motriz , Seda/biossínteseRESUMO
6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue, produced a chronic panencephalitis when inoculated intracerebrally into suckling ICR mice. Immunofluorescent staining revealed 6/94 viral antigen in ependyma, meninges, choroid plexus, and perivascular parenchymal sites from day 3 to 128 days after infection. Hemadsorption-neutralizing antibody was first detected between 20-25 days after infection and remained at high titers for 7 months. Using embryonated chicken eggs, virus was recovered from mouse brains for only 8 days, but could be recovered from brains grown in vitro as explants for 37 days after infection. In cell lines established from explanted brain tissue, immunofluorescence was the most sensitive indicator of virus presence, although infectious virus was not produced. Fusion of these mouse brain cells with human (W138) indicator cells was the most effective means of rescuing 6/94 virus.
Assuntos
Encefalite/microbiologia , Vírus da Parainfluenza 1 Humana , Animais , Antígenos Virais/análise , Células Cultivadas , Córtex Cerebral/microbiologia , Encefalite/imunologia , Camundongos , Esclerose Múltipla/microbiologia , Vírus da Parainfluenza 1 Humana/imunologia , Vírus da Parainfluenza 1 Humana/isolamento & purificação , Fatores de TempoRESUMO
Nearly complete brain ischemia under normoglycemic conditions results in death of only selectively vulnerable neurons. With prior elevation of brain glucose, such injury is enhanced to include pancellular necrosis (i.e., infarction), perhaps because an associated, severe lactic acidosis preferentially injures astrocytes. However, no direct physiologic measurements exist to support this hypothesis. Therefore, we used microelectrodes to measure intracellular pH and passive electrical properties of cortical astrocytes as a first approach to characterizing the physiologic behavior of these cells during hyperglycemic and complete ischemia, conditions that produce infarction in reperfused brain. Anesthesized rats (n = 26) were made extremely hyperglycemic (blood glucose, 51.4 +/- 2.8 mM) so as to create potentially the most extreme acidic conditions possible; then ischemia was induced by cardiac arrest. Two loci more acidic than the interstitial space (6.17-6.20 pH) were found. The more acidic locus [4.30 +/- 0.19 (n = 5); range: 3.82-4.89] was occasionally seen at the onset of anoxic depolarization, 3-7 min after cardiac arrest. The less acidic locus [5.30 +/- 0.07 (n = 53); range 4.46-5.93)] was seen 5-46 min after cardiac arrest. A small negative change in DC potential [8 +/- 1 mV (n = 5); range -3 to -12 mV and 7 +/- 2 mV (n = 53); range +3 to -31 mV, respectively] was always seen upon impalement of acidic loci, suggesting cellular penetration. In a separate group of five animals, electrical characteristics of these cells were specifically measured (n = 17): membrane potential was -12 +/- 0.2 mV (range -3 to -24 mV), input resistance was 114 +/- 16 M omega (range 25-250 M omega), and time constant was 4.4 +/- 0.4 ms (range 3.0-7.9 ms). Injection of horseradish peroxidase into cells from either animal group uniformly stained degenerating astrocytes. These findings establish previously unrecognized properties of ischemic astrocytes that may be prerequisites for infarction from nearly complete ischemia: the capacity to develop profound cellular acidosis and a concomitant reduction in cell membrane ion permeability.
Assuntos
Acidose/metabolismo , Astrócitos/metabolismo , Encefalopatias/etiologia , Isquemia Encefálica/complicações , Hiperglicemia/complicações , Equilíbrio Ácido-Base , Acidose/patologia , Animais , Encefalopatias/patologia , Isquemia Encefálica/metabolismo , Eletrofisiologia , Peroxidase do Rábano Silvestre , Hiperglicemia/metabolismo , Membranas Intracelulares/fisiologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
The histological organization of the filum terminale of the spinal cord in Rana catesbeiana and Rana pipiens was characterized to determine if this region possessed an organized neuropil or whether it was merely a glial remnant that persisted after absorption of the larval tail. The excised filum was maintained in vitro. Intracellular electrophysiological recording was performed with horseradish peroxidase injection. Tyrosine hydroxylase and serotonin distribution were revealed by immunocytochemical methods. Astroglia were the dominant cell type and displayed an elaborate variety of forms. The mean membrane potential was logarithmically related to the extracellular potassium concentration but displayed a sub-Nernstian slope. Oligodendroglia were also seen, as well as ependyma that extended from the central canal to the pial surface. Neuronal activity was revealed by occasional intracellular penetration of elements that displayed spontaneous excitatory postsynaptic or action potentials. The major evidence for the presence of neurons was the demonstration of tyrosine hydroxylase (TH) immunoreactivity in a large population of cerebrospinal fluid-contacting neurons that abutted the ventral half of the central canal. The axons of these cells entered a ventral bundle and ascended the cord; some fibers left this tract and apparently terminated on large arcuate neurons within the filum. Serotoninergic fibers were primarily confined to a subpial location at the dorsal midline. We conclude that the filum terminale of the frog has a sparse but functional neuropil that is organized around the central canal and supported by a profusion of elaborate glial forms.
Assuntos
Cauda Equina/citologia , Animais , Astrócitos/análise , Eletrofisiologia , Epêndima/citologia , Histocitoquímica , Peroxidase do Rábano Silvestre , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Oligodendroglia/análise , Potássio/farmacologia , Rana catesbeiana , Rana pipiens , Serotonina/análise , Tirosina 3-Mono-Oxigenase/análiseRESUMO
The effect of glutamate and aspartate iontophoresis on extracellular pH was investigated in the turtle cerebellum in vitro. Both amino acids produced a rapid alkaline transient, typically followed by a prolonged acidification. These responses could be evoked in all layers of the cerebellum. Transition from bicarbonate to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered media amplified the pH shifts. Similar alkaline-acid transients could be evoked in the molecular layer by electrical stimulation of the parallel fibers or the ipsilateral peduncle, or by superfusion of glutamate or aspartate. However, no alkaline shifts were evoked in the granular layer by either parallel fiber or peduncle stimulation. In contrast, the iontophoretically induced alkaline shifts were largest in the granular layer. Compared with the stimulus-evoked alkalinizations, the iontophoretic alkaline shifts were relatively insensitive to Mn2+ or Cd2+. These data suggest that the activity-dependent alkalinization of brain extracellular space is generated by a bicarbonate-independent mechanism related to excitatory synaptic transmission. The results are consistent with a flux of hydrogen ions through cationic channels, but do not support a direct role for voltage-dependent Ca2+ channels. In view of the sensitivity of ion channels to changes in external pH, and the magnitude of the amino acid-induced pH shifts, these results indicate that extracellular pH could play an important modulatory role in excitatory synaptic transmission.
Assuntos
Ácido Aspártico/farmacologia , Cerebelo/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Glutamatos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Animais , Bicarbonatos/farmacologia , Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Estimulação Elétrica , Espaço Extracelular/química , Feminino , Ácido Glutâmico , HEPES/farmacologia , Concentração de Íons de Hidrogênio , Canais Iônicos/efeitos dos fármacos , Iontoforese , Masculino , Manganês/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/fisiologia , Transmissão Sináptica , TartarugasRESUMO
Transient increases and decreases in extracellular potassium (delta[K+]o) were recorded from the gray matter of hemisected, neonatal rat spinal cords isolated from 3, 4, 9- and 10-day-old pups. delta[K+]o were evoked in both the ventral and dorsal regions of the gray matter by electrical stimulation. In the ventral horn, repetitive stimulation of the ventral root was required to elicit detectable delta[K+]o. By contrast, single dorsal root stimuli evoked clear delta[K+]o. In the dorsal horn, single orthodromic stimuli elicited delta[K+]o as large as 4-5 mM from a baseline of 4.5 mM. With repetitive stimulation the [K+]o reached, but never exceeded, a ceiling of 10-11 mM. Undershoots were seen only after repetitive stimulation. Spontaneous delta[K+]o were observed in the ventral horn and were well correlated with ventral root activity. Spontaneous delta[K+]o were rare in the dorsal cord, but were recorded after bath application of apamin or tetraethylammonium. The magnitude and distribution of evoked K+ transients and postsynaptic components of the evoked field potential were well correlated in both the dorsal and the ventral gray matter. delta[K+]o were reversibly blocked by 1 mM CdCl2 in the bath and diminished by 1 mM BaCl2. Bath application of mephenesin, apamin or tetraethylammonium diminished evoked delta[K+]o in a stimulus-dependent manner. In apamin and tetraethylammonium, decreases from control responses were largest with high intensity stimulation, the opposite was the case with mephenesin. These results are interpreted in terms of the spinal circuits activated by high- and low-intensity electrical stimulation. We conclude that activity-related delta[K+]o in neonatal spinal cord are large enough to modulate neuronal electrical activity and the [K+]o is well regulated compared to other immature CNS regions studied. Thus, local increases in [K+]o may, by modulating neuronal activity, play a role in neonatal spinal cord developmental processes.
Assuntos
Canais Iônicos/fisiologia , Potássio/fisiologia , Medula Espinal/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos/fisiologia , Apamina/farmacologia , Cádmio/farmacologia , Cloreto de Cádmio , Estimulação Elétrica , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Ratos , Medula Espinal/efeitos dos fármacos , Compostos de Tetraetilamônio/farmacologiaRESUMO
In a number of CNS preparations, neuronal activation has been shown to result in a rapid extracellular alkaline transient, followed by a prolonged acid shift. The isolated turtle cerebellum was used to investigate the early alkaline transient. Double-barreled ion-sensitive microelectrodes for H+, K+ and tetramethylammonium were used to measure field potentials and extracellular ion and volume shifts in response to bipolar electrical stimulation of the parallel fibers. Transition from 15 mM HEPES to 35 mM HCO3- -buffered Ringer decreased the amplitude of the alkaline shift, presumably due to a marked increase in extracellular buffering power. In HEPES-buffered Ringer, repetitive stimulation produced alkaline shifts as large as 0.3-0.4 pH. Single shocks produced an alkaline shift of 0.006 +/- 0.0002 pH with a latency as short as 70 ms. Kynurenic acid (an excitatory amino acid antagonist), or Mn2+, blocked the alkaline shift and the postsynaptic component of the field potential. The alkaline shift was not blocked by the Na-H exchange inhibitor amiloride. The relationship between pHo and extracellular volume transients was studied using tetramethylammonium as an extracellular volume indicator. In nominally HCO3-free Ringer, stimulation at 5 Hz for 10 s caused a decrease in extracellular volume of 3.0 +/- 0.2 per cent. The volume transient was unaffected by 3 mM Mn2+, while the alkaline shift was completely abolished. The data for the alkaline shift are consistent with a channel-mediated transmembrane flux of proton equivalents. The size of the pH change and the underlying perturbation it represents, indicate that acid-base shifts may be a functionally important consequence of neuronal activity.
Assuntos
Cerebelo/fisiologia , Espaço Extracelular/fisiologia , Tartarugas/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cerebelo/metabolismo , Estimulação Elétrica , Espaço Extracelular/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Tartarugas/metabolismoRESUMO
Gliotic hippocampal slices were used to study glial acid secretion in a tissue largely devoid of neural elements. Rat hippocampal slices were prepared 10-28 days after sterotaxic injection of kainate. Cresyl Violet staining and immunohistochemistry for glial fibrillary acidic protein demonstrated a loss of neurons and a proliferation of reactive astrocytes in area CA3. Extracellular pH and K+ shifts were recorded in CA3 in response to K+ iontophoresis. Elevation of K+ evoked an extracellular acid shift that was two- to three-fold larger in gliotic versus unlesioned tissue. Ba2+ caused a slow extracellular acidification, and blocked both the depolarizing responses of the glial cells and the acid shifts evoked by K+. The K(+)-evoked acid shifts were abolished in Na(+)-free media, and diminished in HEPES-buffered solutions. Inhibition of extracellular carbonic anhydrase caused a reversible enhancement of the K(+)-evoked acid shifts, an effect that could be mimicked during H+ iontophoresis in agarose gels. Gliotic acid shifts were unaffected by amiloride or its analogs, stilbenes, zero Cl- media, zero or elevated glucose, lactate transport inhibitors, zero Ca2+ or Cd2+. Smaller acid shifts could be evoked in normal slices which were also enhanced by benzolamide, and blocked by Ba2+ and zero Na+ media. It is concluded that acid secretion by reactive astrocytes is Na+ and HCO3(-)-dependent and is triggered by depolarization. The similar pharmacological and ionic sensitivity of the acid shifts in non-gliotic tissue suggest that these properties are shared by normal astrocytes. These characteristics are consistent with the operation of an electrogenic Na(+)-HCO3- co-transporter. However, the enhancement of the acid shifts by inhibitors of extracellular carbonic anhydrase suggests that CO3(2-), rather than HCO3-, is the transported acid equivalent.
Assuntos
Ácidos/metabolismo , Gliose/metabolismo , Hipocampo/metabolismo , Animais , Bário/farmacologia , Bicarbonatos/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Eletrofisiologia , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Valores de Referência , Sódio/fisiologiaRESUMO
Depolarization-induced, intracellular alkaline shifts were studied in reactive astrocytes within slices of gliotic hippocampus. Slices were prepared 10-28 days after sterotaxic injection of kainic acid into the hippocampus of anesthetized rats. Astrocytes in gliotic CA3 were impaled with double-barreled pH sensitive microelectrodes and depolarized by iontophoresis of K+ from an adjacent micropipette. Elevation of extracellular K+ produced an intracellular alkalinization that grew with increasing membrane depolarization, ranging from approximately 0.10 to 0.30 pH units. Exposure to Ba2+ depolarized the cells and produced a similar alkalinization. In the presence of Ba2+, the K(+)-induced depolarization and the associated alkaline shift were abolished. The depolarization-induced alkaline shifts were partially inhibited (40 +/- 8.9%) in Na(+)-free media and were enhanced in bicarbonate versus HEPES-buffered saline. The alkalinizations were unaffected by incubation in chloride-free media, or by the stilbene 4,4'-dinitrostilbene-2,2'-disulfonic acid. It is concluded that the depolarization-induced alkaline shift of reactive astrocytes is mediated in part by a Na+ and HCO3(-)-dependent mechanism that is insensitive to stilbenes. These characteristics correspond well with the properties of depolarization-induced acid secretion in the gliotic tissue. In addition, a separate, Na(+)-independent mechanism contributes to the depolarization-induced alkalinization. In view of the absolute Na+ dependence of acid secretion in the gliotic slices, we propose that the latter mechanism does not extrude acid across the plasma membrane.
Assuntos
Álcalis/metabolismo , Astrócitos/metabolismo , Gliose/fisiopatologia , Hipocampo/fisiopatologia , Animais , Bário/farmacologia , Bicarbonatos/metabolismo , Eletrofisiologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Sódio/fisiologiaRESUMO
Synaptic activation of central neurons has been associated with rapid extracellular alkalinization. In this report, we directly activated CA1 pyramidal cells by antidromic invasion, or by field stimulation. Antidromic activation produced no pH change, despite a robust population spike in five of 11 slices. In six slices, antidromic stimulation at 10 Hz evoked a small alkalinization in stratum pyramidale (0.04 +/- 0.01 unit pH) which grew to 0.10-0.20 unit pH at 50-100 Hz, and was blocked in 0 Ca2+ media. Simultaneous pH recordings revealed no alkalinizations in stratum radiatum, despite robust alkaline shifts in stratum pyramidale. When synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, DL-2-amino-5-phosphonovalerate and picrotoxin, the Schaffer collateral-induced alkaline shift in stratum radiatum was abolished. With adequate stimulus strength and orientation, however, alkaline shifts in stratum radiatum could still be elicited, presumably by direct activation of the CA1 population. The non-synaptic alkaline shifts ranged from 0.10-0.20 unit pH, were amplified by benzolamide, and blocked by tetrodotoxin, 0 Ca2+ saline, and 300-400 microM Cd2+. Although directly activated alkaline shifts were never observed in 0 Ca2+ saline, large stimulus evoked responses could be elicited upon addition of 5-10 mM Ba2+. The Ba(2+)-dependent responses were also amplified by benzolamide and blocked by tetrodotoxin, Cd2+ or high Mg2+. These data demonstrate that stratum pyramidale can undergo an extracellular alkaline shift independent of stratum radiatum. The ionic dependence and pharmacologic sensitivity of the alkaline shifts suggest that voltage-gated Ca2+ channels are instrumental in triggering the alkalinizing mechanism. However, the ability of Ba2+ to support the alkaline shifts indicates that Ca2+ entry is not an absolute requirement. Implications for the mechanism of these pH changes are discussed.
Assuntos
Bário/farmacologia , Canais de Cálcio/fisiologia , Cálcio/farmacologia , Hipocampo/fisiologia , Células Piramidais/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Dendritos/efeitos dos fármacos , Dendritos/fisiologia , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Espaço Extracelular , Hipocampo/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Picrotoxina/farmacologia , Células Piramidais/efeitos dos fármacos , Ratos , Transmissão Sináptica/efeitos dos fármacos , Tetrodotoxina/farmacologiaRESUMO
The role of Ca2+ in cellular injury has received particular attention in studies of acute spinal cord trauma. In this context, the spatial and temporal distribution of extracellular Ca2+ ([Ca2+]e) may have an important bearing on the development of secondary tissue injury. We therefore studied the spatial-temporal distribution of [Ca2+]e following moderate (25 g-cm) contusive injury to the rat thoracic (T9-T11) spinal cord. Double-barreled, Ca(2+)-selective microelectrodes were used to measure the magnitude and time course of [Ca2+]e at increasing depths from the dorsal spinal cord surface. After 2 h, the tissue was frozen and later analyzed for total Ca concentration using atomic absorption spectroscopy. [Ca2+]e fell at all depths, but the decrease was maximal at 250 and 500 microns from the dorsal surface, where, at 0-10 min after injury, [Ca2+]e averaged 0.09 +/- 0.03 and 0.06 +/- 0.03 mM respectively. By 2 h postinjury, [Ca2+]e recovered to nearly 1 mM across all depths. Over this time, total tissue calcium concentration ([Ca]t) was 4.54 +/- 0.16 mumol/g in injured cords vs 2.75 +/- 0.1 mumol/g in sham-operated controls. These data place emphasis on the dorsal gray matter as a principal site of ionic derangement in acute spinal cord injury. The implications of these findings are discussed with reference to secondary injury processes.
Assuntos
Cálcio/metabolismo , Contusões/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Gasometria , Pressão Sanguínea , Contusões/fisiopatologia , Espaço Extracelular/metabolismo , Feminino , Ratos , Ratos Endogâmicos , Traumatismos da Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
Glutamate receptor activation induces an extracellular alkalinization in rodent hippocampus. We studied its Ca2+ dependence and pharmacology in hippocampal slices. Glutamate-evoked alkaline shifts were blocked by 0 Ca2+ saline. Alkalinizations induced by AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and NMDA (N-methyl-D-aspartate) were abolished by 20 microM CNQX (6-cyano-7-nitro-nitroquinoxaline-2,3-dione) and 50 microM APV (DL-2-amino-5-phosphonovalerate), respectively. The AMPA- and NMDA-evoked alkaline shifts were blocked by 0 Ca2+, however, AMPA-induced [K+]o elevation was unaffected. These data suggest that the glutamate receptor-channel does not mediate H+ influx, and support a role for Ca(2+)-H+ exchange.
Assuntos
Álcalis/metabolismo , Cálcio/fisiologia , Hipocampo/metabolismo , Receptores de Glutamato/fisiologia , Animais , Espaço Extracelular/metabolismo , Ácido Glutâmico/farmacologia , Técnicas In Vitro , N-Metilaspartato/farmacologia , Potássio/metabolismo , Ratos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
The extracellular pH of the brain is subject to shifts during neural activity. To understand these pH changes, it is necessary to measure [H+], [HCO3-], [CO3(2-)] and [CO2]. In principle, this can be accomplished using CO3(2-) and pH-sensitive microelectrodes; however, interference from HCO3- and Cl-, and physiological changes in [HCO3-], complicate measurements with CO3(2-) electrodes. Calibration requires knowledge of slope response, interference constants and corrections for [HCO3-] shifts. We show that when [HCO3-] is altered at constant [CO2] in the absence of Cl-, the HCO3- interference cancels and the Nikolsky equation reduces to the Nernst equation for CO3(2-). Measurement of CO3(2-) slope response by this method yielded a value of 28.5 +/- 0.72 mV per decade change in [CO3(2-)]. In Cl(-)-containing solutions, interference coefficient for HCO3- and Cl- were determined by altering [HCO3-] at constant [CO2], changing [CO2] at constant [HCO3-], then solving the simultaneous Nikolsky equations for each transition. The mean interference constants corresponded to selectivity ratios of 245:1 and 1150:1 for CO3(2-) over HCO3- and Cl- respectively. To correct for possible changes in [HCO3-], the equilibrium relation between CO3(2-) and HCO3- was substituted into the Nikolsky equation to yield an equation in [CO3(2-)] and [H+]. By simultaneously measuring shifts in [H+] with a pH microelectrode, this equation is readily solved for [CO3(2-)]. These methods were tested by measuring [HCO3-] and [CO2] in experimental solutions, and in the extracellular fluid of rat hippocampal slices.
Assuntos
Bicarbonatos/análise , Química Encefálica/fisiologia , Dióxido de Carbono/análise , Espaço Extracelular/química , Animais , Cloretos/química , Hipocampo/química , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Eletrodos Seletivos de Íons , Microeletrodos , RatosRESUMO
The regulation of intracellular pH (pHi) was investigated in reticulospinal neurons of the lamprey using ion-selective microelectrodes. Steady-state pHi in 23 mM HCO-3-buffered Ringer was 7.44 +/- 0.03 with a membrane potential of 54 +/- 4 mV (mean +/- S.E.M., n = 6). In nominally HCO-3-free solutions, pHi recovery from acid loading was blocked by 10(-3)M amiloride. Recovery was stimulated by transition to HCO-3-containing solutions. Results suggest that pHi regulation in lamprey reticulospinal neurons is mediated by a Na+-H+ exchanger. The presence of a distinct, HCO-3-dependent pHi regulatory mechanism is postulated.
Assuntos
Tratos Extrapiramidais/metabolismo , Peixes/metabolismo , Lampreias/metabolismo , Amilorida/farmacologia , Animais , Permeabilidade da Membrana Celular , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Formação Reticular/metabolismo , Sódio/metabolismo , Medula Espinal/metabolismoRESUMO
Activity-dependent extracellular pH shifts were studied in slices of the rat dorsal lateral geniculate nucleus (dLGN) using double-barreled pH-sensitive microelectrodes. In 26 mM HCO3--buffered media, afferent activation (10 Hz, 5 s) elicited an early alkaline shift of 0.04+/-0.02 pH units associated with a later, slow acid shift of 0.05+/-0.03 pH units. Extracellular pH shifts in the ventral lateral geniculate nucleus were rare, and limited to acidifications of approximately 0.02 pH units. The alkaline shift in the dLGN increased in the presence of benzolamide (1-2 microM), an extracellular carbonic anhydrase inhibitor. The mean alkaline shift in benzolamide was 0.10+/-0.05 pH units. In 26 mM HEPES-buffered saline, the alkaline response averaged 0.09+/-0.03 pH units. The alkaline shifts persisted in 100 microM picrotoxin (PiTX) but were blocked by 25 microM CNQX/50 microM APV. If stimulation intensity was raised in the presence of CNQX/APV, a second alkalinization arose, presumably due to direct activation of dLGN neurons. The direct responses were amplified by benzolamide, and blocked by either 0 Ca2+/EGTA, Cd2+ or TTX. In 0 Ca2+, addition of 500 microM-5 mM Ba2+ restored the alkalosis. Alkaline shifts evoked with extracellular Ba2+ were larger and faster than those elicited by equimolar Ca2+. In summary, synchronous activation in the dLGN results in an extracellular H+ sink, via a Ca2+-dependent mechanism, similar to activity-dependent alkaline shifts in hippocampus.
Assuntos
Espaço Extracelular/metabolismo , Espaço Extracelular/fisiologia , Corpos Geniculados/metabolismo , Corpos Geniculados/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Benzolamida/farmacologia , Potenciais Evocados/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Feminino , Corpos Geniculados/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Microeletrodos , Picrotoxina/farmacologia , Ratos , Ratos Long-Evans , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
gamma-Aminobutyric acid (GABA) can influence conduction in a number of axonal preparations from the peripheral and central nervous system. In the spinal cord, the excitability of primary afferent terminals has long been known to be affected by GABA. Whether conduction in the long fiber tracts of the spinal cord can be similarly modulated is unknown. Since GABA causes a pronounced depression of excitability in preparations of unmyelinated axons, and myelination is incomplete in the neonatal rat, we tested whether GABA can modulate conduction in the dorsal columns of 10-17-day-old rats. Experiments were performed in vitro, on isolated dorsal column segments (n = 18). The extracellular compound action potential evoked by submaximal stimuli was recorded with a glass micropipette positioned 0.5-2.0 mm from a stimulating electrode. At concentrations of 10(-4) - 10(-3) M, GABA decreased excitability, reversibly depressing the compound action potential amplitude, and increasing the latency by 47 +/- 11% and 22 +/- 9% (mean +/- S.E.M., n = 5, 10(-3) M), respectively. These effects were blocked by picrotoxin and mimicked by isoguvacine (10(-4) M), which decreased the compound action potential amplitude by 44 +/- 10% and increased the latency by 9 +/- 4% (n = 5). Lower concentrations of these agents caused a modest increase in excitability. At 10(-5) M, GABA increased the compound action potential amplitude by 14 +/- 2% and decreased the latency by 3 +/- 2% (n = 5). Our results demonstrate that functional GABAA receptors are present in neonatal dorsal columns.(ABSTRACT TRUNCATED AT 250 WORDS)