tetrodotoxin and Hypercapnia

The parafacial respiratory group (pFRG) in the rostral ventrolateral medulla of the newborn rat is predominantly composed of pre-inspiratory (Pre-I) neurons and is involved in respiratory rhythm generation. The subgroup located close to the ventral surface (at least partially overlapping the retrotrapezoid nucleus, RTN) expresses the Phox2b transcription factor and responds to hypercapnic stimulation with strong depolarization, which suggests it has a role in central chemoreception. Although a CO(2) response of pFRG/RTN neurons has been confirmed in the presence of tetrodotoxin (TTX), it is unknown whether the depolarization involved in this response is induced by a direct postsynaptic response of pFRG/RTN neurons or by any presynaptic components mediated by Ca(2+)-dependent mechanisms. In this study, we examined the effects of ATP or substance P receptor antagonists on hypercapnic responses of rostral pFRG/RTN neurons. We tested effects of Cd(2+) and low Ca(2+)-high Mg(2+) in the presence of TTX. The experiments were performed in in vitro brainstem–spinal cord preparations from newborn rats in which Pre-I neurons reflect the discharge pattern of the pFRG. We found that ATP receptor and substance P receptor antagonists do not block membrane potential responses to hypercapnic stimulation (2%→8%) of pFRG/RTN neurons in the rostral parafacial region.Moreover, rostral pFRG/RTN neurons were depolarized by hypercapnia under conditions where the contribution of presynaptic components was inhibited in the presence of TTX and Cd(2+) or in a low Ca(2+)-high Mg(2+) solution containing TTX and Cd(2+). All cases (except some cases in a low Ca(2+)-high Mg(2+) solution) of membrane depolarization by hypercapnic stimulation were accompanied with an increase in input resistance. These neurons were predominantly Phox2b immunoreactive. Our findings suggest that the response of pFRG/RTN neurons to hypercapnia is induced by direct action on the postsynaptic membrane via closing of K(+) channels.

Topics: Adenosine Diphosphate; Animals; Animals, Newborn; Cadmium; Calcium; Calcium Channel Blockers; Carbon Dioxide; Homeodomain Proteins; Hypercapnia; In Vitro Techniques; Magnesium; Medulla Oblongata; Neurokinin-1 Receptor Antagonists; Neurons; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinuclidines; Rats; Rats, Wistar; Receptors, Neurokinin-1; Receptors, Purinergic P2; Respiratory Center; Substance P; Tetrodotoxin; Transcription Factors

We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells in AM slices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74 +/- 0.19 pC/min at 5% CO2 to 6.36 +/- 0.77 pC/min at 10% CO2. This response to CO2 was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2+ influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2 microm), a membrane-permeant carbonic anhydrase (CA) inhibitor. We detected the presence of two CA isoforms (CAI and CAII) in neonatal AM slices by in situ hybridization and real-time PCR. The expression of these enzymes decreased in adult AM together with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2+ influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K+ current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors.

Topics: Adrenal Medulla; Animals; Animals, Newborn; Calcium; Carbon Dioxide; Carbonic Anhydrases; Catecholamines; Chromaffin Cells; Dose-Response Relationship, Drug; Enzyme Induction; Extracellular Fluid; Hydrogen-Ion Concentration; Hypercapnia; Intracellular Fluid; Ion Transport; Isoenzymes; Methazolamide; Patch-Clamp Techniques; Polymerase Chain Reaction; Potassium; Rats; Rats, Wistar; Sodium; Tetrodotoxin

We studied chemosensitive signaling in locus coeruleus (LC) neurons using both perforated and whole cell patch techniques. Upon inhibition of fast Na(+) spikes by tetrodotoxin (TTX), hypercapnic acidosis [HA; 15% CO(2), extracellular pH (pH(o)) 6.8] induced small, slow spikes. These spikes were inhibited by Co(2+) or nifedipine and were attributed to activation of L-type Ca(2+) channels by HA. Upon inhibition of both Na(+) and Ca(2+) spikes, HA resulted in a membrane depolarization of 3.52 +/- 0.61 mV (n = 17) that was reduced by tetraethylammonium (TEA) (1.49 +/- 0.70 mV, n = 7; P < 0.05) and absent (-0.97 +/- 0.73 mV, n = 7; P < 0.001) upon exposure to isohydric hypercapnia (IH; 15% CO(2), 77 mM HCO(3)(-), pH(o) 7.45). Either HA or IH, but not 50 mM Na-propionate, activated Ca(2+) channels. Inhibition of L-type Ca(2+) channels by nifedipine reduced HA-induced increased firing rate and eliminated IH-induced increased firing rate. We conclude that chemosensitive signals (e.g., HA or IH) have multiple targets in LC neurons, including TEA-sensitive K(+) channels and TWIK-related acid-sensitive K(+) (TASK) channels. Furthermore, HA and IH activate L-type Ca(2+) channels, and this activation is part of chemosensitive signaling in LC neurons.

Topics: Action Potentials; Animals; Animals, Newborn; Calcium Channels; Chemoreceptor Cells; Hypercapnia; In Vitro Techniques; Locus Coeruleus; Neurons; Potassium Channels; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetrodotoxin

The anatomical structure of central respiratory chemoreceptors in the superficial ventral medulla of rats was studied by using hypercapnia-induced c-fos labeling to identify cells directly stimulated by extracellular pH or PCO(2). The distribution of c-fos-positive cells was found to be predominantly perivascular to surface vessels. In the superficial ventral medullary midline, parapyramidal, and ventrolateral regions where c-fos-positive cells were concentrated, we found a common, characteristic, anatomical architecture. The medullary surface showed an indentation covered by a surface vessel, and the marginal glial layer was thickened. We classified c-fos-positive cells into two types. One (type I cell) was small, was located inside the marginal glial layer and close to the medullary surface, and surrounded fine vessels. The other (type II cell) was large and located dorsal to the marginal glial layer. c-fos Expression under synaptic blockade suggested that type I cells are intrinsically chemosensitive. The chemosensitivity of surface cells (possible type I cells) surrounding vessels was confirmed electrophysiologically in slice preparations. We suggest that this characteristic anatomical structure may be the central chemoreceptor.

Topics: Anesthetics, Local; Animals; Calcium; Carbon Dioxide; Chemoreceptor Cells; Hypercapnia; Magnesium; Medulla Oblongata; Membrane Potentials; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Respiratory Center; Stimulation, Chemical; Tetrodotoxin

The chemosensitive response of locus coeruleus (LC) neurones to changes in intracellular pH (pH(i)), extracellular pH (pH(o)) and molecular CO(2) were investigated using neonatal rat brainstem slices. A new technique was developed that involves the use of perforated patch recordings in combination with fluorescence imaging microscopy to simultaneously measure pH(i) and membrane potential (V(m)). Hypercapnic acidosis (15 % CO(2), pH(o) 6.8) resulted in a maintained fall in pH(i) of 0.31 pH units and a 93 % increase in the firing rate of LC neurones. On the other hand, isohydric hypercapnia (15 % CO(2), 77 mM HCO(3)(-), pH(o) 7.45) resulted in a smaller and transient fall in pH(i) of about 0.17 pH units and an increase in firing rate of 76 %. Acidified Hepes (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid)-buffered medium (pH(o) 6.8) resulted in a progressive fall in pH(i) of over 0.43 pH units and an increase in firing rate of 126 %. Isosmotic addition of 50 mM propionate to the standard HCO(3)(-)-buffered medium (5 % CO(2), 26 mM HCO(3)(-), pH(o) 7.45) resulted in a transient fall in pH(i) of 0.18 pH units but little increase in firing rate. Isocapnic acidosis (5 % CO(2), 7 mM HCO(3)(-), pH(o) 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH(i) preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH(o). In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH(i), indicating that a decrease in pH(i) is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.

Topics: Acidosis; Animals; Animals, Newborn; Carbon Dioxide; Electrophysiology; Extracellular Space; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; Kinetics; Locus Coeruleus; Membrane Potentials; Microscopy, Fluorescence; Neurons; Patch-Clamp Techniques; Pons; Propionates; Rats; Rats, Sprague-Dawley; Tetrodotoxin

7-Nitroindazole, an inhibitor of neuronal nitric oxide synthase, reportedly inhibits hypercapnic dilation, but tetrodotoxin, an inhibitor of neuronal transmission, reportedly does not. Thus, evidence does not uniformly support the hypothesis of a neurogenic link to the hypercapnic response. Others suggest the hypercapnic response is mediated by a K(ATP) ion channel. In the following studies, we observed that topically administered tetrodotoxin inhibited dilations produced by hypercapnia. In addition, topical tetrodotoxin and either topical or intraperitoneal 7-nitroindazole, inhibited dilations produced by the K(ATP) channel openers, cromakalim and pinacidil. Inhibition of hypercapnic dilation and inhibition of dilation by the openers of the K(ATP) channel was immediately reversed by either L-lysine or L-arginine, amino acids previously shown to facilitate opening of the channel. The data strongly supports the previous conclusion that there is a K(ATP) ion channel link in the response of pial arterioles to hypercapnia. The location of the channel is not established by these data, nor is it known whether the action of tetrodotoxin on the channel was direct or indirect.

Topics: Animals; Arginine; Arterioles; Carbon Dioxide; Cromakalim; Hypercapnia; Indazoles; Ion Channel Gating; Lysine; Male; Nitric Oxide Synthase; Nitroprusside; Pinacidil; Potassium Channels; Rats; Rats, Sprague-Dawley; Respiration; Telencephalon; Tetrodotoxin; Vasodilation; Vasodilator Agents

The response to hypercapnic acidosis (2-8% CO2, bath pH 7.8-7.2) was examined in whole cell recordings from neonatal (P1 to P5) rat Locus coeruleus (LC) neurones in the in vitro brainstem-spinal cord preparation exposed to low Ca2+ (0.2 mM)-high Mg2+ (5 mM). This medium suppressed chemical synaptic transmission and resulted in a pattern of subthreshold oscillations of membrane potential and rhythmic burst discharge which was synchronized throughout the network. The oscillation was suppressed, and the discharge of individual neurones desynchronized, by the gap junction uncoupler, carbenoxolone, indicating that in low Ca2+-high Mg2+ LC neurones form an electrically coupled network. Switching from 2 to 8% CO2 decreased the oscillation amplitude and increased its frequency. The oscillation was suppressed by external Cd2+ and by TTX. but persisted during injection into the cell soma of QX-314. We conclude that in LC neurones acidosis increases the frequency of a Ca2+- and Na+-dependent dendritic oscillator which is synchronized by gap junction coupling throughout the network. This coupling is retained during acidosis.

Topics: Acidosis; Animals; Cadmium; Calcium; Cells, Cultured; Culture Media; Hydrogen-Ion Concentration; Hypercapnia; Lidocaine; Locus Coeruleus; Magnesium; Membrane Potentials; Neurons; Rats; Rats, Sprague-Dawley; Tetrodotoxin

The purpose of the present study was to quantify the inhibitory effect of inhaled halothane and isoflurane on acute hypercapnia-induced responses of capacitance-regulating veins and related cardiovascular variables in response to sequential 40-s periods of 5%, 10%, 15%, and 20% inspired CO2 (FICO2). Measurements were made in normoxic alpha-chloralose-anesthetized rabbits before, during, and after either 0.75 minimum alveolar anesthetic concentration inhaled halothane or isoflurane. The graded hypercapnia caused graded venoconstriction and bradycardia but minimal pressor responses. Hypercapnia-induced venoconstriction was blocked by prior local superfusion of the exposed veins with 3 x 10(-6) M tetrodotoxin. Both the hypercapnia-induced venoconstriction and bradycardia responses were significantly attenuated by halothane or isoflurane and did not fully recover after removal of the anesthetics from the circulation. Both anesthetics produced a significant baseline (i.e., prehypercapnia) hypotension and a tendency toward a resultant tachycardia. The baseline hypotension did not recover completely after elimination of the anesthetic. Neither anesthetic altered baseline vein diameter. These results agree with previous studies demonstrating that hypercapnic acidosis produces mesenteric venoconstriction by elevating excitatory sympathetic efferent neural input via activation of peripheral and central chemoreceptors and that bradycardia results from activation of compensatory baroreflexes. The neural components of these reflexes are possible primary sites for attenuation of these cardiovascular responses by halothane and isoflurane.

Topics: Acute Disease; Anesthetics, Inhalation; Animals; Blood Pressure; Bradycardia; Halothane; Heart Rate; Hypercapnia; Isoflurane; Mesenteric Veins; Rabbits; Tetrodotoxin; Vasoconstriction

1. Using the isolated medulla and spinal cord of the neonatal rat, the response to CO2-induced changes in superfusate pH was examined in whole cell and perforated patch recordings from ventral medullary neurones which were identified by injection of Lucifer Yellow. The respiratory response to changing the CO2 concentration (from 2 to 8%) consisted of an increase in phrenic burst frequency, which could be accompanied by an increase, decrease or no change in burst amplitude. 2. Five classes of neurone - inspiratory, post-inspiratory, expiratory, respiration-modulated and ionic - were distinguished on the basis of their membrane potential and discharge patterns. Almost all (112 of 123) responded rapidly to 8% CO2 with a sustained change in membrane potential. Depolarizing responses (3-18 mV) occurred in inspiratory, respiration-modulated and 45% of tonic neurones. Hyperpolarizing responses (2-19 mV) occurred in expiratory and post-inspiratory neurones. The remaining tonic neurones were inhibited or showed no response. 3. In representatives of each class of neurone, membrane potential responses to 8% CO2 were retained when tested in the presence of tetrodotoxin (n = 7), low (0.2 mM) Ca(2+)-high (5 mM) Mg2+ (n = 23) or Cd2+ (0.2 mM) (n = 3)-containing superfusate, implying that they are mediated by intrinsic membrane or cellular mechanisms. 4. Neurones were distributed between 1200 microns rostral and 400 microns caudal to obex, and their cell bodies were located between 50 and 700 microns below the ventral surface (n = 104). Almost all responsive neurones (n = 78) showed dendritic projections to within 50 microns of the surface. 6. These experiments indicate that significant numbers of ventral medullary neurones, including respiratory neurones, are intrinsically chemosensitive. The consistency with which these neurones show surface dendritic projections suggests that this sensitivity may arise in part at this level.

Topics: Animals; Animals, Newborn; Brain Stem; Calcium; Carbon Dioxide; Cell Size; Chemoreceptor Cells; Dendrites; Hypercapnia; In Vitro Techniques; Magnesium; Medulla Oblongata; Membrane Potentials; Neurons; Patch-Clamp Techniques; Phrenic Nerve; Rats; Respiration; Spinal Cord; Tetrodotoxin

The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-L-arginine (L-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this relaxation by 54% and 70%, respectively. The effect of L-NOARG was completely reversed by L-arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO2 elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response to hypercapnia. L-NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 M HCl resulted in relaxation identical to that obtained by lowering the pH with 15% CO2. In vessel segments in which the endothelium had been removed beforehand 15% CO2 induced relaxation that was not different from that seen in vessels with intact endothelium. L-NOARG had no affect in endothelium denuded vessels. The results suggest that high CO2 elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by L-NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Basilar Artery; Cyclic GMP; Hydrogen-Ion Concentration; Hypercapnia; Male; Methylene Blue; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Wistar; Tetrodotoxin; Vasodilation

We examined the effect of nitric oxide synthase (NOS) inhibition and tetrodotoxin (TTX) on the increase of cerebral blood flow (CBF) in parietal (CoBF) and cerebellar cortex (CeBF) in response to hypercapnia. Rats were anesthetized with halothane and artificially ventilated. Hypercapnia was induced by adding 5% CO2 to the inhalation mixture. CoBF and CeBF were measured by laser-Doppler flowmetry. NOS inhibition was achieved by intravenous (30 mg/kg) and/or topical application (1 mM) of NG-nitro-L-arginine (L-NNA). Activity in perivascular nerves around pial and cortical vessels was inhibited by topical application of TTX (20 microM). Under control conditions, hypercapnia (66 +/- 1 mmHg) increased CoBF by 70 +/- 4% and CeBF by 96 +/- 5%. Systemic L-NNA decreased the baseline level of CoBF and CeBF by 11 +/- 3%, but topical L-NNA did not affect baseline flow. Intravenous L-NNA attenuated the hypercapnic increase of CoBF by 77 +/- 5% and CeBF by 63 +/- 4% within 10-20 min. Topical L-NNA attenuated the hypercapnic increase of CoBF by 52 +/- 6% and CeBF by 29 +/- 5% after 45-min exposure. Both CoBF and CeBF decreased rapidly when L-NNA was infused during sustained hypercapnia, but not when L-NNA was applied topically. Effect of intravenous L-NNA was partially prevented by pretreatment with intravenous L-arginine. Intravenous or topical L-NNA enhanced the rise of CBF elicited by cortical spreading depression, adenosine (1 mM), or sodium nitroprusside (300 microM), except in the cerebellum where topical L-NNA attenuated the rise of CBF elicited by adenosine by 53%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arginine; Cerebellar Cortex; Cerebrovascular Circulation; Electrocardiography; Hypercapnia; Male; Nitric Oxide; Nitroarginine; Parietal Lobe; Rats; Tetrodotoxin