The Role of Ca2+ and BK Channels of Locus Coeruleus (LC) Neurons as a Brake to the CO2 Chemosensitivity Response of Rats.

The cellular mechanisms by which LC neurons respond to hypercapnia are usually attributed to an "accelerator" whereby hypercapnic acidosis causes an inhibition of K+ channels or activation of Na+ and Ca+2 channels to depolarize CO2-sensitive neurons. Nevertheless, it is still unknown if this "accelerator" mechanism could be controlled by a brake phenomenon. Whole-cell patch clamping, fluorescence imaging microscopy and plethysmography were used to study the chemosensitive response of the LC neurons. Hypercapnic acidosis activates L-type Ca2+ channels and large conductance Ca-activated K+ (BK) channels, which function as a "brake" on the chemosensitive response of LC neurons. Our findings indicate that both Ca2+ and BK currents develop over the first 2 weeks of postnatal life in rat LC slices and that this brake pathway may cause the developmental decrease in the chemosensitive firing rate response of LC neurons to hypercapnic acidosis. Inhibition of this brake by paxilline (BK channel inhibitor) returns the magnitude of the chemosensitive firing rate response from LC neurons in rats older than P10 to high values similar to those in LC neurons from younger rats. Inhibition of BK channels in LC neurons by bilateral injections of paxilline into the LC results in a significant increase in the hypercapnic ventilatory response of adult rats. Our findings indicate that a BK channel-based braking system helps to determine the chemosensitive respiratory drive of LC neurons and contributes to the hypercapnic ventilatory response. Perhaps, abnormalities of this braking system could result in hypercapnia-induced respiratory disorders and panic responses.

Theoretical perspectives on central chemosensitivity: CO2/H+-sensitive neurons in the locus coeruleus.

Central chemoreceptors are highly sensitive neurons that respond to changes in pH and CO2 levels. An increase in CO2/H+ typically reflects a rise in the firing rate of these neurons, which stimulates an increase in ventilation. Here, we present an ionic current model that reproduces the basic electrophysiological activity of individual CO2/H+-sensitive neurons from the locus coeruleus (LC). We used this model to explore chemoreceptor discharge patterns in response to electrical and chemical stimuli. The modeled neurons showed both stimulus-evoked activity and spontaneous activity under physiological parameters. Neuronal responses to electrical and chemical stimulation showed specific firing patterns of spike frequency adaptation, postinhibitory rebound, and post-stimulation recovery. Conversely, the response to chemical stimulation alone (based on physiological CO2/H+ changes), in the absence of external depolarizing stimulation, showed no signs of postinhibitory rebound or post-stimulation recovery, and no depolarizing sag. A sensitivity analysis for the firing-rate response to the different stimuli revealed that the contribution of an applied stimulus current exceeded that of the chemical signals. The firing-rate response increased indefinitely with injected depolarizing current, but reached saturation with chemical stimuli. Our computational model reproduced the regular pacemaker-like spiking pattern, action potential shape, and most of the membrane properties that characterize CO2/H+-sensitive neurons from the locus coeruleus. This validates the model and highlights its potential as a tool for studying the cellular mechanisms underlying the altered central chemosensitivity present in a variety of disorders such as sudden infant death syndrome, depression, and anxiety. In addition, the model results suggest that small external electrical signals play a greater role in determining the chemosensitive response to changes in CO2/H+ than previously thought. This highlights the importance of considering electrical synaptic transmission in studies of intrinsic chemosensitivity.

Ventilatory and chemoreceptor responses to hypercapnia in neonatal rats chronically exposed to moderate hyperoxia.

Rats reared in hyperoxia hypoventilate in normoxia and exhibit progressive blunting of the hypoxic ventilatory response, changes which are at least partially attributed to abnormal carotid body development. Since the carotid body also responds to changes in arterial CO2/pH, we tested the hypothesis that developmental hyperoxia would attenuate the hypercapnic ventilatory response (HCVR) of neonatal rats by blunting peripheral and/or central chemoreceptor responses to hypercapnic challenges. Rats were reared in 21% O2 (Control) or 60% O2 (Hyperoxia) until studied at 4, 6-7, or 13-14days of age. Hyperoxia rats had significantly reduced single-unit carotid chemoafferent responses to 15% CO2 at all ages; CO2 sensitivity recovered within 7days after return to room air. Hypercapnic responses of CO2-sensitive neurons of the caudal nucleus tractus solitarius (cNTS) were unaffected by chronic hyperoxia, but there was evidence for a small decrease in neuronal excitability. There was also evidence for augmented excitatory synaptic input to cNTS neurons within brainstem slices. Steady-state ventilatory responses to 4% and 8% CO2 were unaffected by developmental hyperoxia in all three age groups, but ventilation increased more slowly during the normocapnia-to-hypercapnia transition in 4-day-old Hyperoxia rats. We conclude that developmental hyperoxia impairs carotid body chemosensitivity to hypercapnia, and this may compromise protective ventilatory reflexes during dynamic respiratory challenges in newborn rats. Impaired carotid body function has less of an impact on the HCVR in older rats, potentially reflecting compensatory plasticity within the CNS.

Microglial Acid Sensing Regulates Carbon Dioxide-Evoked Fear.

BACKGROUND: Carbon dioxide (CO2) inhalation, a biological challenge and pathologic marker in panic disorder, evokes intense fear and panic attacks in susceptible individuals. The molecular identity and anatomic location of CO2-sensing systems that translate CO2-evoked fear remain unclear. We investigated contributions of microglial acid sensor T cell death-associated gene-8 (TDAG8) and microglial proinflammatory responses in CO2-evoked behavioral and physiological responses. METHODS: CO2-evoked freezing, autonomic, and respiratory responses were assessed in TDAG8-deficient ((-/-)) and wild-type ((+/+)) mice. Involvement of TDAG8-dependent microglial activation and proinflammatory cytokine interleukin (IL)-1ß with CO2-evoked responses was investigated using microglial blocker, minocycline, and IL-1ß antagonist IL-1RA. CO2-chemosensitive firing responses using single-cell patch clamping were measured in TDAG8(-/-) and TDAG8(+/+) mice to gain functional insights. RESULTS: TDAG8 expression was localized in microglia enriched within the sensory circumventricular organs. TDAG8(-/-) mice displayed attenuated CO2-evoked freezing and sympathetic responses. TDAG8 deficiency was associated with reduced microglial activation and proinflammatory cytokine IL-1ß within the subfornical organ. Central infusion of microglial activation blocker minocycline and IL-1ß antagonist IL-1RA attenuated CO2-evoked freezing. Finally, CO2-evoked neuronal firing in patch-clamped subfornical organ neurons was dependent on acid sensor TDAG8 and IL-1ß. CONCLUSIONS: Our data identify TDAG8-dependent microglial acid sensing as a unique chemosensor for detecting and translating hypercapnia to fear-associated behavioral and physiological responses, providing a novel mechanism for homeostatic threat detection of relevance to psychiatric conditions such as panic disorder.

A HCO(3)(-)-dependent mechanism involving soluble adenylyl cyclase for the activation of Ca²âº currents in locus coeruleus neurons.

Hypercapnic acidosis activates Ca²âº channels and increases intracellular Ca²âº levels in neurons of the locus coeruleus, a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca²âº-activated K⁺ channels, in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in locus coeruleus neurons. Here, we present evidence that the Ca²âº current is activated by a HCO(3)(-)-sensitive pathway. The increase in HCO(3)(-) associated with hypercapnia activates HCO(3)(-)-sensitive adenylyl cyclase (soluble adenylyl cyclase). This results in an increase in cyclic adenosine monophosphate levels and activation of Ca²âº channels via cyclic adenosine monophosphate-activated protein kinase A. We also show the presence of soluble adenylyl cyclase in the cytoplasm of locus coeruleus neurons, and that the cyclic adenosine monophosphate analogue db-cyclic adenosine monophosphate increases Ca²âºi. Disrupting this pathway by decreasing HCO(3)(-) levels during acidification or inhibiting either soluble adenylyl cyclase or protein kinase A, but not transmembrane adenylyl cyclase, can increase the magnitude of the firing rate response to hypercapnia in locus coeruleus neurons from older neonates to the same extent as inhibition of K⁺ channels. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Substance P differentially modulates firing rate of solitary complex (SC) neurons from control and chronic hypoxia-adapted adult rats.

NK1 receptors, which bind substance P, are present in the majority of brainstem regions that contain CO2/H(+)-sensitive neurons that play a role in central chemosensitivity. However, the effect of substance P on the chemosensitive response of neurons from these regions has not been studied. Hypoxia increases substance P release from peripheral afferents that terminate in the caudal nucleus tractus solitarius (NTS). Here we studied the effect of substance P on the chemosensitive responses of solitary complex (SC: NTS and dorsal motor nucleus) neurons from control and chronic hypoxia-adapted (CHx) adult rats. We simultaneously measured intracellular pH and electrical responses to hypercapnic acidosis in SC neurons from control and CHx adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. Substance P significantly increased the basal firing rate in SC neurons from control and CHx rats, although the increase was smaller in CHx rats. However, substance P did not affect the chemosensitive response of SC neurons from either group of rats. In conclusion, we found that substance P plays a role in modulating the basal firing rate of SC neurons but the magnitude of the effect is smaller for SC neurons from CHx adult rats, implying that NK1 receptors may be down regulated in CHx adult rats. Substance P does not appear to play a role in modulating the firing rate response to hypercapnic acidosis of SC neurons from either control or CHx adult rats.

Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats.

The locus coeruleus (LC) plays an important role in central chemoreception. In young rats (P9 or younger), 85% of LC neurons increase firing rate in response to hypercapnia vs. only about 45% of neurons from rats P10 or older. Carbenoxolone (CARB - gap junction blocker) does not affect the % of LC neurons responding in young rats but it decreases the % responding by half in older animals. We evaluated the participation of gap junctions in the CO2 ventilatory response in unanesthetized adult rats by bilaterally microinjecting CARB (300µM, 1mM or 3mM/100nL), glycyrrhizic acid (GZA, CARB analog, 3mM) or vehicle (aCSF - artificial cerebrospinal fluid) into the LC of Wistar rats. Bilateral gap junction blockade in LC neurons did not affect resting ventilation; however, the increase in ventilation produced by hypercapnia (7% CO2) was reduced by ∼25% after CARB 1mM or 3mM injection (1939.7±104.8mLkg(-1)min(-1) for the aCSF group and 1468.3±122.2mLkg(-1)min(-1) for 1mM CARB, P<0.05; 1939.7±104.8mLkg(-1)min(-1) for the aCSF group and 1540.9±68.4mLkg(-1)min(-1) for the 3mM CARB group, P<0.05) due largely to a decrease in respiratory frequency. GZA injection or CARB injection outside the LC (peri-LC) had no effect on ventilation under any conditions. The results suggest that gap junctions in the LC modulate the hypercapnic ventilatory response of adult rats.

Temperature influences neuronal activity and CO2/pH sensitivity of locus coeruleus neurons in the bullfrog, Lithobates catesbeianus.

The locus coeruleus (LC) is a chemoreceptive brain stem region in anuran amphibians and contains neurons sensitive to physiological changes in CO2/pH. The ventilatory and central sensitivity to CO2/pH is proportional to the temperature in amphibians, i.e., sensitivity increases with increasing temperature. We hypothesized that LC neurons from bullfrogs, Lithobates catesbeianus, would increase CO2/pH sensitivity with increasing temperature and decrease CO2/pH sensitivity with decreasing temperature. Further, we hypothesized that cooling would decrease, while warming would increase, normocapnic firing rates of LC neurons. To test these hypotheses, we used whole cell patch-clamp electrophysiology to measure firing rate, membrane potential (V(m)), and input resistance (R(in)) in LC neurons in brain stem slices from adult bullfrogs over a physiological range of temperatures during normocapnia and hypercapnia. We found that cooling reduced chemosensitive responses of LC neurons as temperature decreased until elimination of CO2/pH sensitivity at 10°C. Chemosensitive responses increased at elevated temperatures. Surprisingly, chemosensitive LC neurons increased normocapnic firing rate and underwent membrane depolarization when cooled and decreased normocapnic firing rate and underwent membrane hyperpolarization when warmed. These responses to temperature were not observed in nonchemosensitive LC neurons or neurons in a brain stem slice 500 µm rostral to the LC. Our results indicate that modulation of cellular chemosensitivity within the LC during temperature changes may influence temperature-dependent respiratory drive during acid-base disturbances in amphibians. Additionally, cold-activated/warm-inhibited LC neurons introduce paradoxical temperature sensitivity in respiratory control neurons of amphibians.

Transient outwardly rectifying A currents are involved in the firing rate response to altered CO2 in chemosensitive locus coeruleus neurons from neonatal rats.

The effect of hypercapnia on outwardly rectifying currents was examined in locus coeruleus (LC) neurons in slices from neonatal rats [postnatal day 3 (P3)-P15]. Two outwardly rectifying currents [4-aminopyridine (4-AP)-sensitive transient current and tetraethyl ammonium (TEA)-sensitive sustained current] were found in LC neurons. 4-AP induced a membrane depolarization of 3.6 ± 0.6 mV (n = 4), while TEA induced a smaller membrane depolarization of 1.2 ± 0.3 mV (n = 4). Hypercapnic acidosis (HA) inhibited both currents. The maximal amplitude of the TEA-sensitive current was reduced by 52.1 ± 4.5% (n = 5) in 15% CO2 [extracellular pH (pHo) 7.00, intracellular pH (pHi) 6.96]. The maximal amplitude of the 4-AP-sensitive current was reduced by 34.5 ± 3.0% (n = 6) in 15% CO2 (pHo 7.00, pHi 6.96), by 29.4 ± 6.8% (n = 6) in 10% CO2 (pHo 7.15, pHi 7.14), and increased by 29.0 ± 6.4% (n = 6) in 2.5% CO2 (pHo 7.75, pHi 7.35). 4-AP completely blocked hypercapnia-induced increased firing rate, but TEA did not affect it. When LC neurons were exposed to HA with either pHo or pHi constant, the 4-AP-sensitive current was inhibited. The data show that the 4-AP-sensitive current (likely an A current) is inhibited by decreases in either pHo or pHi. The change of the A current by various levels of CO2 is correlated with the change in firing rate induced by CO2, implicating the 4-AP-sensitive current in chemosensitive signaling in LC neurons.

Postnatal development and activation of L-type Ca2+ currents in locus ceruleus neurons: implications for a role for Ca2+ in central chemosensitivity.

Little is known about the role of Ca(2+) in central chemosensitive signaling. We use electrophysiology to examine the chemosensitive responses of tetrodotoxin (TTX)-insensitive oscillations and spikes in neurons of the locus ceruleus (LC), a chemosensitive region involved in respiratory control. We show that both TTX-insensitive spikes and oscillations in LC neurons are sensitive to L-type Ca(2+) channel inhibition and are activated by increased CO(2)/H(+). Spikes appear to arise from L-type Ca(2+) channels on the soma whereas oscillations arise from L-type Ca(2+) channels that are distal to the soma. In HEPES-buffered solution (nominal absence of CO(2)/HCO(3)(-)), acidification does not activate either oscillations or spikes. When CO(2) is increased while extracellular pH is held constant by elevated HCO(3)(-), both oscillation and spike frequency increase. Furthermore, plots of both oscillation and spike frequency vs. intracellular [HCO(3)(-)]show a strong linear correlation. Increased frequency of TTX-insensitive spikes is associated with increases in intracellular Ca(2+) concentrations. Finally, both the appearance and frequency of TTX-insensitive spikes and oscillations increase over postnatal ages day 3-16. Our data suggest that 1) L-type Ca(2+) currents in LC neurons arise from channel populations that reside in different regions of the neuron, 2) these L-type Ca(2+) currents undergo significant postnatal development, and 3) the activity of these L-type Ca(2+) currents is activated by increased CO(2) through a HCO(3)(-)-dependent mechanism. Thus the activity of L-type Ca(2+) channels is likely to play a role in the chemosensitive response of LC neurons and may underlie significant changes in LC neuron chemosensitivity during neonatal development.

The caudal solitary complex is a site of central CO(2) chemoreception and integration of multiple systems that regulate expired CO(2).

The solitary complex is comprised of the nucleus tractus solitarius (NTS, sensory) and dorsal motor nucleus of the vagus (DMV, motor), which functions as an integrative center for neural control of multiple systems including the respiratory, cardiovascular and gastroesophageal systems. The caudal NTS-DMV is one of the several sites of central CO(2) chemoreception in the brain stem. CO(2) chemosensitive neurons are fully responsive to CO(2) at birth and their responsiveness seems to depend on pH-sensitive K(+) channels. In addition, chemosensitive neurons are highly sensitive to conditions such as hypoxia (e.g., neural plasticity) and hyperoxia (e.g., stimulation), suggesting they employ redox and nitrosative signaling mechanisms. Here we review the cellular and systems physiological evidence supporting our hypothesis that the caudal NTS-DMV is a site for integration of respiratory, cardiovascular and gastroesophageal systems that work together to eliminate CO(2) during acute and chronic respiratory acidosis to restore pH homeostasis.

Hyperoxic stimulation of synchronous orthodromic activity and induction of neural plasticity does not require changes in excitatory synaptic transmission.

The first study, described in the companion article, reports that acute exposure of rat hippocampal slices to either hyperbaric oxygen (HBO: 2.84 and 4.54 atmospheres absolute, ATA) or normobaric reoxygenation (NBOreox; i.e., normobaric hyperoxia: 0.6 or 0.0→0.95 ATA) stimulates synchronous orthodromic activity in CA1 neurons, which includes activation of O2-induced potentiation (OxIP) and, in some cases, hyperexcitability (secondary population spikes, sPS). In this second study we tested the hypothesis that HBO and NBOreox increase orthodromic activity of CA1 neurons (oPS, orthodromic population spike) and OxIP via a combination of both increased excitatory synaptic transmission (field excitatory postsynaptic potential, fEPSP) and intrinsic excitability (antidromic population spike, aPS). HBO and NBOreox increased the oPS but rarely increased or potentiated the fEPSP. HBO exposure produced epileptiform antidromic activity, which was abolished during inhibition of fast GABAergic and glutamatergic synaptic transmission. Decreasing O2 from 0.95 ATA (control) to 0.6 ATA (intermediate O2) or 0.0 ATA (hypoxia) reversibly abolished the fEPSP, and reoxygenation rarely induced potentiation of the fEPSP or aPS. Intracellular recordings and antidromic field potential recordings, however, revealed that synaptic transmission and neuronal excitability were preserved, albeit at lower levels, in 0.60 ATA O2. Together, these data indicate that 1) the changes in excitatory postsynaptic activity are not required for stimulation of the oPS during and HBO/NBOreox or for activation of OxIP, suggesting the latter is a form of intrinsic plasticity; 2) HBO disinhibits spontaneous synaptic transmission to induce epileptiform activity; and 3) although synchronous synaptic activation of the CA1 neuronal population requires hyperoxia (i.e., 0.95 ATA O2), synaptic activation of individual CA1 neurons does not.

Hyperbaric hyperoxia and normobaric reoxygenation increase excitability and activate oxygen-induced potentiation in CA1 hippocampal neurons.

Breathing hyperbaric oxygen (HBO) is common practice in hyperbaric and diving medicine. The benefits of breathing HBO, however, are limited by the risk of central nervous system O2 toxicity, which presents as seizures. We tested the hypothesis that excitability increases in CA1 neurons of the rat hippocampal slice (400 microm) over a continuum of hyperoxia that spans normobaric and hyperbaric pressures. Amplitude changes of the orthodromic population spike were used to assess neuronal O2 sensitivity before, during, and following exposure to 0, 0.6, 0.95 (control), 2.84, and 4.54 atmospheres absolute (ATA) O2. Polarographic O2 electrodes were used to measure tissue slice PO2 (PtO2). In 0.95 ATA O2, core PtO2 at 200 microm deep was 115±16 Torr (mean±SE). Increasing O2 to 2.84 and 4.54 ATA increased core PtO2 to 1,222±77 and 2,037±157 Torr, respectively. HBO increased the orthodromic population spike amplitude and usually induced hyperexcitability (i.e., secondary population spikes) and, in addition, a long-lasting potentiation of the orthodromic population spike that we have termed "oxygen-induced potentiation" (OxIP). Exposure to 0.60 ATA O2 and hypoxia (0.00 ATA) decreased core PtO2 to 84±6 and 20±4 Torr, respectively, and abolished the orthodromic response. Reoxygenation from 0.0 or 0.6 ATA O2, however, usually produced a response similar to that of HBO: hyperexcitability and activation of OxIP. We conclude that CA1 neurons exhibit increased excitability and neural plasticity over a broad range of PtO2, which can be activated by a single, hyperoxic stimulus. We postulate that transient acute hyperoxia stimulus, whether caused by breathing HBO or reoxygenation following hypoxia (e.g., disordered breathing), is a powerful stimulant for orthodromic activity and neural plasticity in the CA1 hippocampus.

The locus coeruleus and central chemosensitivity.

The locus coeruleus (LC) lies in the dorsal pons and supplies noradrenergic (NA) input to many regions of the brain, including respiratory control areas. The LC may provide tonic input for basal respiratory drive and is involved in central chemosensitivity since focal acidosis of the region stimulates ventilation and ablation reduces CO(2)-induced increased ventilation. The output of LC is modulated by both serotonergic and glutamatergic inputs. A large percentage of LC neurons are intrinsically activated by hypercapnia. This percentage and the magnitude of their response are highest in young neonates and decrease dramatically after postnatal day P10. The cellular bases for intrinsic chemosensitivity of LC neurons are comprised of multiple factors, primary among them being reduced extracellular and intracellular pH, which inhibit inwardly rectifying and voltage-gated K(+) channels, and activate L-type Ca(2+) channels. Activation of K(Ca) channels in LC neurons may limit their ultimate response to hypercapnia. Finally, the LC mediates central chemosensitivity and contains pH-sensitive neurons in amphibians, suggesting that the LC has a long-standing phylogenetic role in respiratory control.

CO2 chemoreception in cardiorespiratory control.

Considerable progress has been made elucidating the cellular signals and ion channel targets involved in the response to increased CO2/H+ of brain stem neurons from chemosensitive regions. Intracellular pH (pHi) does not exhibit recovery from an acid load when extracellular pH (pHo) is also acid. This lack of pHi recovery is an essential but not unique feature of all chemosensitive neurons. These neurons have pH-regulating transporters, especially Na+/H+ exchangers, but some may also contain HCO3--dependent transporters as well. Studies in locus ceruleus (LC) neurons have shown that firing rate will increase in response to decreased pHi or pHo but not in response to increased CO2 alone. A number of K+ channels, as well as other channels, have been suggested to be targets of these pH changes with a fall of pH inhibiting these channels. In neurons from some regions it appears that multiple signals and multiple channels are involved in their chemosensitive response while in neurons from other regions a single signal and/or channel may be involved. Despite the progress, a number of key issues remain to be studied. A detailed study of chemosensitive signaling needs to be done in neurons from more brain stem regions. Fully describing the chemosensitive signaling pathways in brain stem neurons will offer new targets for therapies to alter the strength of central chemosensitivity and will yield new insights into the reason why there are multiple central chemoreceptive sites.

pH regulating transporters in neurons from various chemosensitive brainstem regions in neonatal rats.

We studied the membrane transporters that mediate intracellular pH (pH(i)) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pH(i) recovery from an NH(4)Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO(2)) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na(+)/H(+) exchange (NHE). Recovery in RTN neurons was blocked approximately 50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO(3)-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked approximately 65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na(+) and increased by external HCO(3)(-). On the basis of these findings, pH(i) recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO(3)-dependent transporter in LC neurons. Thus, pH(i) recovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons.

Chronic hypoxia suppresses the CO2 response of solitary complex (SC) neurons from rats.

We studied the effect of chronic hypobaric hypoxia (CHx; 10-11% O(2)) on the response to hypercapnia (15% CO(2)) of individual solitary complex (SC) neurons from adult rats. We simultaneously measured the intracellular pH and firing rate responses to hypercapnia of SC neurons in superfused medullary slices from control and CHx-adapted adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. We found that CHx caused the percentage of SC neurons inhibited by hypercapnia to significantly increase from about 10% up to about 30%, but did not significantly alter the percentage of SC neurons activated by hypercapnia (50% in control vs. 35% in CHx). Further, the magnitudes of the responses of SC neurons from control rats (chemosensitivity index for activated neurons of 166+/-11% and for inhibited neurons of 45+/-15%) were the same in SC neurons from CHx-adapted rats. This plasticity induced in chemosensitive SC neurons by CHx appears to involve intrinsic changes in neuronal properties since they were the same in synaptic blockade medium.

Chemosensory responses to CO2 in multiple brain stem nuclei determined using a voltage-sensitive dye in brain slices from rats.

We used epifluorescence microscopy and a voltage-sensitive dye, di-8-ANEPPS, to study changes in membrane potential during hypercapnia with or without synaptic blockade in chemosensory brain stem nuclei: the locus coeruleus (LC), the nucleus of the solitary tract, lateral paragigantocellularis nucleus, raphé pallidus, and raphé obscurus and, in putative nonchemosensitive nuclei, the gigantocellularis reticular nucleus and the spinotrigeminal nucleus. We studied the response to hypercapnia in LC cells to evaluate the performance characteristics of the voltage-sensitive dye. Hypercapnia depolarized many LC cells and the voltage responses to hypercapnia were diminished, but not eradicated, by synaptic blockade (there were intrinsically CO2-sensitive cells in the LC). The voltage response to hypercapnia was substantially diminished after inhibiting fast Na+ channels with tetrodotoxin. Thus action potential-related activity was responsible for most of the optical signal that we detected. We systematically examined CO2 sensitivity among cells in brain stem nuclei to test the hypothesis that CO2 sensitivity is a ubiquitous phenomenon, not restricted to nominally CO2 chemosensory nuclei. We found intrinsically CO2 sensitive neurons in all the nuclei that we examined; even the nonchemosensory nuclei had small numbers of intrinsically CO2 sensitive neurons. However, synaptic blockade significantly altered the distribution of CO2-sensitive cells in all of the nuclei so that the cellular response to CO2 in more intact preparations may be difficult to predict based on studies of intrinsic neuronal activity. Thus CO2-sensitive neurons are widely distributed in chemosensory and nonchemosensory nuclei and CO2 sensitivity is dependent on inhibitory and excitatory synaptic activity even within brain slices. Neuronal CO2 sensitivity important for the behavioral response to CO2 in intact animals will thus be determined as much by synaptic mechanisms and patterns of connectivity throughout the brain as by intrinsic CO2 sensitivity.