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Tetrodotoxin-sensitive uptake of ions and water byslices of rat brain in vitro.

ABSTRACT: 1. Under certain conditions the processes that bring about increased Na(+) influx and water uptake into rat brain slices have a tetrodotoxin-sensitive component. 2. Incubation of slices in the absence of glucose, or in the presence of ouabain (0.1mm), protoveratrine (10mum) or electrical stimuli, leads to increased uptakes of water and Na(+) that are partially suppressed by tetrodotoxin (3mum). 3. The increased water uptake and Na(+) influx due to the presence of 30mum-2,4-dinitrophenol or of 100mm-potassium chloride are unaffected by tetrodotoxin. 4. The additional presence of 27mm-potassium chloride diminishes, or abolishes, the inhibitory effects of tetrodotoxin on the increased uptakes of water and Na(+) found on incubation in the absence of glucose or in the presence of protoveratrine. 5. Ouabain increases Na(+) influx into caudate nuclei from rat brain and this process is suppressed by tetrodotoxin. Ouabain does not increase water uptake in the caudate nuclei. 6. Diminution of Na(+) influx in slices due to tetrodotoxin is not accompanied by an equal efflux of K(+). 7. It is concluded that glial-cell membranes are permeable to K(+) and Cl(-) and, as a consequence, when K(+) is released from neurons in association with action potentials it, together with water, moves into the glial cells. The major consequence of K(+) (and Cl(-)) permeability of the glial cell is therefore a buffering of the extracellular K(+) concentration. 8. l-Glutamate presents an anomalous feature that may indicate that its effects are not restricted to neurons. 9. Calculations are made of the increase of K(+) flux into the glia in the presence of ouabain or of protoveratrine or in the absence of glucose or on application of electrical impulses.

SUBMITTER: Okamoto K

PROVIDER: S-EPMC1179566 | biostudies-other | 1970 Nov

REPOSITORIES: biostudies-other

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Project description:1. Amino acids, particularly glutamate, gamma-aminobutyrate, aspartate and glycine, were released from rat brain slices on incubation with protoveratrine (especially in a Ca(2+)-deficient medium) or with ouabain or in the absence of glucose. Release was partially or wholly suppressed by tetrodotoxin. 2. Tetrodotoxin did not affect the release of glutamine under various incubation conditions, nor did protoveratrine accelerate it. 3. Protoveratrine caused an increased rate of formation of glutamine in incubated brain slices. 4. Increased K(+) in the incubation medium caused release of gamma-aminobutyrate, the process being partly suppressed by tetrodotoxin. 5. Incubation of brain slices in a glucose-free medium led to increased production of aspartate and to diminished tissue contents of glutamates, glutamine and glycine. 6. Use of tetrodotoxin to suppress the release of amino acids from neurons in slices caused by the joint action of protoveratrine and ouabain (the latter being added to diminish reuptake of amino acids), it was shown that the major pools of glutamate, aspartate, glycine, serine and probably gamma-aminobutyrate are in the neurons. 7. The major pool of glutamine lies not in the neurons but in the glia. 8. The tricarboxylic cycle inhibitors, fluoroacetate and malonate, exerted different effects on amino acid contents in, and on amino acid release from, brain slices incubated in the presence of protoveratrine. Fluoroacetate (3mm) diminished the content of glutamine, increased that of glutamate and gamma-aminobutyrate and did not affect respiration. Malonate (2mm) diminished aspartate and gamma-aminobutyrate content, suppressed respiration and did not affect glutamine content. It is suggested that malonate acts mainly on the neurons, and that fluoroacetate acts mainly on the glia, at the concentrations quoted. 9. Glutamine was more effective than glutamate as a precursor of gamma-aminobutyrate. 10. It is suggested that glutamate released from neurons is partly taken up by glia and converted there into glutamine. This is returned to the neurons where it is hydrolysed and converted into glutamate and gamma-aminobutyrate.

Project description:Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

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Tetrodotoxin-sensitive uptake of ions and water byslices of rat brain in vitro.

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