Characterization of cardiovascular reflexes evoked by airway stimulation with allylisothiocyanate, capsaicin, and ATP in Sprague-Dawley rats.

Acute inhalation of airborne pollutants alters cardiovascular function and evidence suggests that pollutant-induced activation of airway sensory nerves via the gating of ion channels is critical to these systemic responses. Here, we have investigated the effect of capsaicin [transient receptor potential (TRP) vanilloid 1 (TRPV1) agonist], AITC [TRP ankyrin 1 (TRPA1) agonist], and ATP (P2X2/3 agonist) on bronchopulmonary sensory activity and cardiovascular responses of conscious Sprague-Dawley (SD) rats. Single fiber recordings show that allyl isothiocyanate (AITC) and capsaicin selectively activate C fibers, whereas subpopulations of both A and C fibers are activated by stimulation of P2X2/3 receptors. Inhalation of the agonists by conscious rats caused significant bradycardia, atrioventricular (AV) block, and prolonged PR intervals, although ATP-induced responses were lesser than those evoked by AITC or capsaicin. Responses to AITC were inhibited by the TRP channel blocker ruthenium red and the muscarinic antagonist atropine. AITC inhalation also caused a biphasic blood pressure response: a brief hypertensive phase followed by a hypotensive phase. Atropine accentuated the hypertensive phase, while preventing the hypotension. AITC-evoked bradycardia was not abolished by terazosin, the α1-adrenoceptor inhibitor, which prevented the hypertensive response. Anesthetics had profound effects on AITC-evoked bradycardia and AV block, which was abolished by urethane, ketamine, and isoflurane. Nevertheless, AITC inhalation caused bradycardia and AV block in paralyzed and ventilated rats following precollicular decerebration. In conclusion, we provide evidence that activation of ion channels expressed on nociceptive airway sensory nerves causes significant cardiovascular effects in conscious SD rats via reflex modulation of the autonomic nervous system.

Peripheral chemoreceptors tune inspiratory drive via tonic expiratory neuron hubs in the medullary ventral respiratory column network.

Models of brain stem ventral respiratory column (VRC) circuits typically emphasize populations of neurons, each active during a particular phase of the respiratory cycle. We have proposed that "tonic" pericolumnar expiratory (t-E) neurons tune breathing during baroreceptor-evoked reductions and central chemoreceptor-evoked enhancements of inspiratory (I) drive. The aims of this study were to further characterize the coordinated activity of t-E neurons and test the hypothesis that peripheral chemoreceptors also modulate drive via inhibition of t-E neurons and disinhibition of their inspiratory neuron targets. Spike trains of 828 VRC neurons were acquired by multielectrode arrays along with phrenic nerve signals from 22 decerebrate, vagotomized, neuromuscularly blocked, artificially ventilated adult cats. Forty-eight of 191 t-E neurons fired synchronously with another t-E neuron as indicated by cross-correlogram central peaks; 32 of the 39 synchronous pairs were elements of groups with mutual pairwise correlations. Gravitational clustering identified fluctuations in t-E neuron synchrony. A network model supported the prediction that inhibitory populations with spike synchrony reduce target neuron firing probabilities, resulting in offset or central correlogram troughs. In five animals, stimulation of carotid chemoreceptors evoked changes in the firing rates of 179 of 240 neurons. Thirty-two neuron pairs had correlogram troughs consistent with convergent and divergent t-E inhibition of I cells and disinhibitory enhancement of drive. Four of 10 t-E neurons that responded to sequential stimulation of peripheral and central chemoreceptors triggered 25 cross-correlograms with offset features. The results support the hypothesis that multiple afferent systems dynamically tune inspiratory drive in part via coordinated t-E neurons.

Normobaric hyperoxia (95% O2) stimulates CO2-sensitive and CO2-insensitive neurons in the caudal solitary complex of rat medullary tissue slices maintained in 40% O2.

We tested the hypothesis that decreasing the control level of O2 from 95% to 40% reduces tissue partial pressure of oxygen (pO2), decreases extracellular nitric oxide (NO) and decreases intracellular superoxide (O2(-)) while maintaining viability in caudal solitary complex (cSC) neurons in slices (∼300-400 µm; neonatal rat P2-22; 34-37°C). We also tested the hypothesis that normobaric hyperoxia is a general stimulant of cSC neurons, including CO2-excited neurons. Whole-cell recordings of cSC neurons maintained in 40% O2 were comparable to recordings made in 95% O2 in duration and quality. In 40% O2, cSC neurons had a significantly lower spontaneous firing rate but similar membrane potentials and input resistances as cSC neurons maintained in 95% O2. Tissue pO2 was threefold lower in 40% O2 versus 95% O2. Likewise, extracellular NO and intracellular O2(-) were lower in 40% versus 95% O2. 67% of neurons maintained in 40% O2 control were stimulated by hyperoxia (95% O2) compared to 81% of neurons maintained in 95% O2 that were stimulated during hyperoxic reoxygenation following acute exposure to 0-40% O2. cSC slices maintained in 40% O2 exhibited CO2-chemosensitive neurons, including CO2-excited (31.5%) and a higher incidence of CO2-inhibited (31.5%) neurons than previously reported. Likewise, a higher incidence of CO2-inhibited and lower incidence of CO2-excited neurons were observed in 85-95% O2. 82% of O2-excited neurons were also CO2-chemosensitive; CO2-excited (86%) and CO2-inhibited neurons (84%) were equally stimulated by hyperoxia. Our findings demonstrate that chronic (hours) and acute (minutes) exposure to hyperoxia stimulates firing rate in the majority of cSC neurons, most of which are also CO2 chemosensitive. Our findings support the hypothesis that recurring exposures to acute hyperoxia and hyperoxic reoxygenation-a repeating surge in tissue pO2-activate redox and nitrosative signaling mechanisms in CO2-chemosensitive neurons that alter expression of CO2 chemosensitivity (e.g., increased expression of CO2-inhibition) compared to sustained hyperoxia (85-95% O2).

High doses of pseudoephedrine hydrochloride accelerate onset of CNS oxygen toxicity seizures in unanesthetized rats.

Pseudoephedrine (PSE) salts (hydrochloride and sulfate) are commonly used as nasal and paranasal decongestants by scuba divers. Anecdotal reports from the Divers Alert Network suggest that taking PSE prior to diving while breathing pure O2 increases the risk for CNS oxygen toxicity (CNS-OT), which manifests as seizures. We hypothesized that high doses of PSE reduce the latency time to seizure (LS) in unanesthetized rats breathing 5 atmospheres absolute (ATA) of hyperbaric oxygen. Sixty-three male rats were implanted with radio-transmitters that recorded electroencephalogram activity and body temperature. After ≥7-day recovery, and 2 h before "diving", each rat was administered either saline solution (control) or PSE hydrochloride intragastrically at the following doses (mg PSE/kg): 0, 40, 80, 100, 120, 160, and 320. Rats breathed pure O2 and were dived to 5ATA until the onset of behavioral seizures coincident with neurological seizures. LS was the time elapsed between reaching 5ATA and exhibiting seizures. We observed a significant dose-dependent decrease in the LS at doses of 100-320 mg/kg, whereas no significant differences in LS from control value were observed at doses ≤80 mg/kg. Our findings showed that high doses of PSE accelerate the onset of CNS-OT seizures in unanesthetized rats breathing 5ATA of poikilocapnic hyperoxia. Extrapolating our findings to humans, we conclude that the recommended daily dose of PSE should not be abused prior to diving with oxygen-enriched gas mixes or pure O2.

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications.

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.

Acute hyperoxia increases lipid peroxidation and induces plasma membrane blebbing in human U87 glioblastoma cells.

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H(2)O(2)) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O(2), normobaric hyperoxia (0.95 ATA O(2)) or hyperbaric hyperoxia (HBO(2), 3.25 ATA O(2)) for 60 min. H(2)O(2) (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H(2)O(2). Average membrane roughness (R(a)) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The R(a) of the plasma membrane was 34+/-3, 57+/-3 and 63+/-5 nm in 0.20 ATA O(2), 0.95 ATA O(2) and HBO(2), respectively. R(a) was 56+/-7 and 138+/-14 nm in 0.2 and 2 mM H(2)O(2). Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H(2)O(2) and correlated with O(2)-induced membrane blebbing (r(2)=0.93). Coapplication of antioxidant, Trolox-C (150 microM), significantly reduced membrane R(a) and MDA levels during hyperoxia. Hyperoxia-induced H(2)O(2) production increased 189%+/-5% (0.95 ATA O(2)) and 236%+/-5% (4 ATA O(2)) above control (0.20 ATA O(2)). We conclude that MLP and membrane blebbing increase with increasing O(2) concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage.

Role of intracellular and extracellular pH in the chemosensitive response of rat locus coeruleus neurones.

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.

Cell-cell coupling in CO(2)/H(+)-excited neurons in brainstem slices.

The indirect and direct electrical and anatomical evidence for the hypothesis that central chemoreceptor neurons in the dorsal brainstem (solitary complex, SC; locus coeruleus, LC) are coupled by gap junctions, as reported primarily in rat brainstem slices, and the methods used to study gap junctions in brain slices, are critiqued and reviewed. Gap junctions allow intercellular communication that could be important in either electrical coupling (intercellular flow of ionic current), metabolic coupling (intercellular flow of signaling molecules), or both, ultimately influencing excitability within the SC and LC during respiratory acidosis. Gap junctions may also provide a mechanism for modulating neuronal activity in the network under conditions that lead to increased or decreased central respiratory chemosensitivity. Indirect measures of electrical coupling suggest that junctional conductance between chemosensitive neurons is relatively insensitive to a broad range of intracellular pH (pH(i)), ranging from pH(i) approximately 7.49 to approximately 6.71 at 35-37 degrees C. In contrast, further reductions in pH(i), down through pH(i) approximately 6.67, abolish indirect measures of electrical coupling.

Development of in vivo ventilatory and single chemosensitive neuron responses to hypercapnia in rats.

We used pressure plethysmography to study breathing patterns of neonatal and adult rats acutely exposed to elevated levels of CO2. Ventilation (VE) increased progressively with increasing inspired CO2. The rise in VE was associated with an increase in tidal volume, but not respiratory rate. In all animals studied, the CO2 sensitivity (determined from the slope of the VE vs. inspired % CO2 curve) was variable on a day to day basis. Chemosensitivity was high in neonates 1 day after birth (P1) and fell throughout the first week to a minimum at about P8. Chemosensitivity rose again to somewhat higher values in P10 through adult rats. The developmental pattern of these in vivo ventilatory responses was different than individual locus coeruleus (LC) neuron responses to increased CO2. The membrane potential (V(m)) of LC neurons was measured using perforated patch (amphotericin B) techniques in brain slices. At all ages studied, LC neurons increased their firing rate by approximately 44% in response to hypercapnic acidosis (10% CO2, pH 7.0). Thus the in vivo ventilatory response to hypercapnia was not correlated with the V(m) response of individual LC neurons to hypercapnic acidosis in neonatal rats. These data suggest that CO2 sensitivity of ventilation in rats may exist in two forms, a high-sensitivity neonatal (or fetal) form and a lower-sensitivity adult form, with a critical window of very low sensitivity during the period of transition between the two (approximately P8).

Oxygen measurements in brain stem slices exposed to normobaric hyperoxia and hyperbaric oxygen.

We previously reported (J Appl Physiol 89: 807-822, 2000) that < or =10 min of hyperbaric oxygen (HBO(2); < or = 2,468 Torr) stimulates solitary complex neurons. To better define the hyperoxic stimulus, we measured PO(2) in the solitary complex of 300-microm-thick rat medullary slices, using polarographic carbon fiber microelectrodes, during perfusion with media having PO(2) values ranging from 156 to 2,468 Torr. Under control conditions, slices equilibrated with 95% O(2) at barometric pressure of 1 atmospheres absolute had minimum PO(2) values at their centers (291 +/- 20 Torr) that were approximately 10-fold greater than PO(2) values measured in the intact central nervous system (10-34 Torr). During HBO(2), PO(2) increased at the center of the slice from 616 +/- 16 to 1,517 +/- 15 Torr. Tissue oxygen consumption tended to decrease at medium PO(2) or = 1,675 Torr to levels not different from values measured at PO(2) found in all media in metabolically poisoned slices (2-deoxy-D-glucose and antimycin A). We conclude that control medium used in most brain slice studies is hyperoxic at normobaric pressure. During HBO(2), slice PO(2) increases to levels that appear to reduce metabolism.

Continuous intracellular recording from mammalian neurons exposed to hyperbaric helium, oxygen, or air.

We developed a hyperbaric chamber for intracellular recording in rat brain stem slices during continuous compression and decompression of the tissue bath with the inert gas helium. Air, rather than helium, was also used as the compression medium in some cases to increase tissue nitrogen levels. An important feature is the chamber door, which opens or closes rapidly at 1 atmosphere absolute (ATA) for increased accessibility of the microelectrode. The door also closes and seals smoothly without disrupting the intracellular recording. Hyperbaric oxygen was administered during helium compression using a separate pressure cylinder filled with perfusate equilibrated with 2. 3-3.3 ATA oxygen. Measurements of tissue/bath PO(2) and pH confirmed that the effects of compression using helium or air could be differentiated from those due to increased PO(2). One hundred and thirteen neurons were studied during 375 compression cycles ranging from 1 to 20 ATA (mode 3.0 ATA). We conclude that it is technically feasible to record intracellularly from the same mammalian neuron while changing ambient pressure over a physiologically important range. These techniques will be useful for studying how various hyperbaric environments affect neurophysiological mechanisms.

Hyperbaric oxygen depolarizes solitary complex neurons in tissue slices of rat medulla oblongata.

Hyperbaric oxygen (HBO2) at approximately 3 atmospheres absolute (ATA) pressure is toxic to the mammalian CNS due to excessive O2 free radical production. No study has ever determined the effects of < or = 3 ATA of O2 on the membrane potential and firing rate of neurons in the mammalian brainstem. Likewise, no study has ever determined the effects of < or = 3 ATA pressure per se on brainstem neurons. Accordingly, we initiated intracellular recordings at 1 ATA in solitary complex neurons in slices (300 microns) of rat caudal medulla oblongata that were maintained inside a 72 liter hyperbaric chamber. Helium, which is inert and without narcotic effect at moderate levels of hyperbaria, was used to hydrostatically compress the submerged brain slice to determine the effects of pressure per se. Tissue oxygen tension and extracellular pH were also measured during exposure to hyperbaric gases. Six of 19 neurons were affected by hyperbaric helium; 5 cells were depolarized and 1 cell was hyperpolarized. Input resistance (Rin) either increased (n = 1) or decreased (n = 3). When control perfusate (0.95 ATA O2) was switched to perfusate saturated with 98% O2 (balance CO2, pH = 7.3-7.4, pO2 = 2.5-3.4 ATA; 2-18 minutes of exposure) in a separate pressure vessel, 8 of 13 neurons were depolarized and 5 neurons were insensitive. In the 8 O2-responsive neurons, Rin either increased (n = 5), decreased (n = 2) or was unchanged (n = 1). Three of 8 neurons depolarized by HBO2 were also depolarized by hyperbaric helium, usually with an additional change in Rin. We conclude that hydrostatic (helium) pressure and HBO2 independently increase excitability in certain solitary complex neurons. We hypothesize that these responses contribute, in part, to neural events that either precede or occur during CNS O2 toxicity.

Response of intracellular pH to acute anoxia in individual neurons from chemosensitive and nonchemosensitive regions of the medulla.

The effect of acute (10 minutes) exposure to anoxia on intracellular pH (pHi) in individual brainstem neurons, in slices from neonatal (P7 to P11) rats, was studied using a fluorescence microscopy imaging technique. Neurons from 4 regions of the medulla were studied, two of which contained chemosensitive neurons (nucleus tractus solitarius, NTS, and ventrolateral medulla, VLM) and two regions which did not contain chemosensitive neurons (hypoglossal, Hyp, and inferior olivary, IO). Acute anoxia caused a rapid and maintained acidification of 0.1-0.3 pH unit that was not different in neurons from chemosensitive vs. nonchemosensitive regions. Blocking the contribution of Na+/H+ exchange (NHE) to pHi regulation by exposing neurons to acute anoxia in the presence of the exchange inhibitor amiloride (1 mM) did not affect the degree of acidification seen in neurons from the NTS and VLM region, but significantly increased acidification (to about 0.35 pH unit) in Hyp and IO neurons. In summary, anoxia-induced intracellular acidification is not different between neurons from chemosensitive and nonchemosensitive regions, but NHE activity blunts acidification in neurons from the latter regions. These data suggest that neurons from chemosensitive areas might have a smaller acid load in response to anoxia than neurons from nonchemosensitive regions of the brainstem.

Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive areas of the medulla.

Intracellular pH (pHi) regulation was studied in neurons from two chemosensitive [nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM)] and two nonchemosensitive [hypoglossal (Hyp) and inferior olive (IO)] areas of the medulla oblongata. Intrinsic buffering power (betaint) was the same in neurons from all regions (46 mM/pH U). Na+/H+ exchange mediated recovery from acidification in all neurons [Ritucci, N. A., J. B. Dean, and R. W. Putnam. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R433-R441, 1997]. Cl-/HCO-3 exchange mediated recovery from alkalinization in VLM, Hyp, and IO neurons but was absent from most NTS neurons. The Na+/H+ exchanger from NTS and VLM neurons was fully inhibited when extracellular pH (pHo) <7.0, whereas the exchanger from Hyp and IO neurons was fully inhibited only when pHo <6.7. The Cl-/HCO-3 exchanger from VLM, but not Hyp and IO neurons, was inhibited by pHo of 7.9. These pH regulatory properties resulted in steeper pHi-pHo relationships in neurons from chemosensitive regions compared with those from nonchemosensitive regions. These differences are consistent with a role for changes of pHi as the proximate signal in central chemoreception and changes of pHo in modulating pHi changes.

Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts.

Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 microm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential-like activity, which had peak-to-peak amplitudes of < or = 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade; and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip < or = 3 microm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, wits averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording which typically lasted for 1-3 h. Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (<1 min) steady-state perforation at 22-25 degrees C. This adaptation of conventional perforated patch recording was termed "rapid perforated patch recording". The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].

Cell-cell coupling between CO2-excited neurons in the dorsal medulla oblongata.

Anatomically coupled neurons (17 of 137) and non-coupled neurons (120 of 137), in and near the nucleus tractus solitarius and dorsal motor nucleus (i.e. solitary complex), were studied by rapid perforated patch recording in slices (rat, 150-350 microm thick, postnatal day 0-21) before, during and after exposure to hypercapnic acidosis. Anatomical coupling refers to the intercellular transfer of Lucifer Yellow and Biocytin into adjoining neurons, presumably via gap junctions [see Dean et al. (1997) Neuroscience 80, 21-40]. Eighty-six per cent of the anatomically coupled neurons (12 of 14) were depolarized by hypercapnic acidosis, a response referred to as CO2 excitation or CO2 chemosensitivity. In all, 28% (12 of 43) of the CO2-excited neurons were anatomically coupled to at least one other neuron. None (0 of 39) of the CO2-inhibited neurons were anatomically coupled, and only 4% (two of 46) of the CO2-insensitive neurons were anatomically coupled. Increasing the fractional concentration of CO2 from five to 10 and 15% in constant bicarbonate (26 mM) decreased intracellular pH (control 7.3 7.4, 22-25 degrees C) by approximately 1.0 and 1.5 pH units, respectively, as measured using the pH-sensitive fluorescent dye, 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein. Nine of the anatomically coupled neurons (six CO2-excited, one CO2-insensitive and two unidentified) exhibited spontaneous electrotonic postsynaptic potential-like activity, suggesting that they were also electrotonically coupled. During hypercapnic acidosis, the amplitudes of electrotonic postsynaptic potentials were unchanged, concomitant with small changes in input resistance. The frequency of electrotonic postsynaptic potentials increased during hypercapnic acidosis in many anatomically coupled neurons (eight of nine), indicating that both neurons of the coupled pair were stimulated. Cell-cell coupling occurred preferentially in and between CO2-excited neurons of the solitary complex. Further, CO2-excited neurons were not electrotonically uncoupled during intracellular acidosis, in contrast to the effect that decreased intracellular pH has on many other types of coupled cells. It was not determined whether anatomical coupling was affected by hypercapnic acidosis since dye mixture was always administered under normocapnic conditions. The high correlation between anatomical coupling, electrotonic coupling activity and CO2-induced depolarization suggests that cell-cell coupling is an important electroanatomical feature in CO2-excited neurons of the solitary complex. CO2-excited neurons have been hypothesized to function in central chemoreception for the cardiorespiratory control systems, suggesting that cell cell coupling may contribute in part to central chemoreception of CO2 and H+.

Intracellular pH response to hypercapnia in neurons from chemosensitive areas of the medulla.

We investigated whether neurons in two chemosensitive areas of the medulla oblongata [nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM)] respond to hypercapnia differently than neurons in two nonchemosensitive areas of the medulla oblongata [inferior olive (IO) and hypoglossal nucleus (Hyp)]. Medullary brain slices from preweanling Sprague-Dawley rats were loaded with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH (pHi) was followed in individual neurons at 37 degrees C with the use of a fluorescence imaging system. Most neurons from the NTS and VLM did not exhibit pHi recovery when CO2 was increased from 5 to 10% at constant extracellular HCO3- concentration [extracellular pH (pHo) decreased approximately 0.3 pH unit] (hypercapnic acidosis). However, when CO2 was increased from 5 to 10% at constant pHo (isohydric hypercapnia), pHi recovery was seen. In contrast, all neurons from the IO and Hyp exhibited pHi recovery during hypercapnic acidosis. All pHi recovery in the four areas studied was inhibited by 1 mM amiloride and unaffected by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. These data indicate that 1) pHi regulation differs between neurons in chemosensitive (NTS and VLM) and nonchemosensitive (IO and Hyp) areas of the medulla, 2) pHi recovery is due solely to Na+/H+ exchange in all four areas, and 3) Na+/H+ exchange is more sensitive to inhibition by extracellular acidosis in NTS and VLM neurons than in IO and Hyp neurons.

A fluorescence technique to measure intracellular pH of single neurons in brainstem slices.

We have developed a technique to measure the pH, of single neurons in brainstem slices using a fluorescence imaging system. Slices were loaded with the pH-sensitive fluorescent dye BCECF and fluorescence was visualized by exciting the slices alternately at 500 and 440 nm. The emitted fluorescence at 530 nm was directed through an MTI GenIISys image intensifier and MT1 CCD72 camera. The images were processed by image-1/FL software. The ratio of emitted fluorescence at excitation wavelengths of 500 and 440 nm was measured and converted to pH by constructing a calibration curve using high K+/nigericin solutions at pH values ranging from 5.8 to 8.6. BCECF-loaded slices showed distinct spheres of intense fluorescence and diffuse background fluorescence. Slices labeled with a neuron-specific antibody, neuron-specific enolase, showed staining that correlated with the spheres of intense fluorescence of BCECF-loaded cells. Slices labeled with a glial-specific antibody, glial fibrillary acidic protein, showed a diffuse, background staining. Neurons that were retrograde-labeled with rhodamine beads fluoresced as large spheres that exactly correlated with the fluorescence from BCECF-loaded cells. Further, large fluorescent spheres had membrane potentials of about -60 mV and generated action potentials. These findings indicate that the large fluorescent spheres are neurons. pHi was measured in these large spheres (neurons) in the dorsal and ventral medullary chemosensitive regions, and was 7.32 +/- 0.02 (n = 110) and 7.38 +/- 0.02 (n = 85), respectively.

Regional interactions between thermosensitive neurons in diencephalic slices.

Rat brain slices were used to investigate regional interactions between thermosensitive neurons in different diencephalic regions. Horizontal tissue slices rested over three thermodes. This permitted independent thermal stimulation of rostral, middle, and caudal regions. Thermocouples measured tissue temperatures in these three locations, and extracellular recordings measured neuronal responses to temperature changes both locally (at the site of the recorded neuron) and in remote regions of the slice. Many of the neurons that were sensitive to remote temperatures were located near the lateral border of the diencephalic nuclei, especially in the perifornical area. All neurons displaying remote thermosensitivity also displayed local thermosensitivity. These neurons usually showed opposite responses to remote and local temperatures; i.e., most of these neurons were locally warm sensitive but showed cold sensitivity to remote temperatures. These findings indicate that thermosensitive synaptic networks extend throughout the diencephalon and may explain the effect of temperature on a variety of homeostatic systems.