Capture rates of Eptesicus fuscus increase following white-nose syndrome across the eastern US.

Emerging infectious diseases threaten wildlife globally. While the effects of infectious diseases on hosts with severe infections and high mortality rates often receive considerable attention, effects on hosts that persist despite infection are less frequently studied. To understand how persisting host populations change in the face of disease, we quantified changes to the capture rates of Eptesicus fuscus (big brown bats), a persisting species susceptible to infection by the invasive fungal pathogen Pseudogymnoascus destructans (Pd; causative agent for white-nose syndrome), across the eastern US using a 30-year dataset. Capture rates of male and female E. fuscus increased from preinvasion to pathogen establishment years, with greater increases to the capture rates of females than males. Among females, capture rates of pregnant and post-lactating females increased by pathogen establishment. We outline potential mechanisms for these broad demographic changes in E. fuscus capture rates (i.e., increases to foraging from energy deficits created by Pd infection, increases to relative abundance, or changes to reproductive cycles), and suggest future research for identifying mechanisms for increasing capture rates across the eastern US. These data highlight the importance of understanding how populations of persisting host species change following pathogen invasion across a broad spatial scale. Understanding changes to population composition following pathogen invasion can identify broad ecological patterns across space and time, and open new avenues for research to identify drivers of those patterns.

Long-term spring through fall capture data of Eptesicus fuscus in the eastern USA before and after white-nose syndrome.

Emerging infectious diseases threaten wildlife populations. Without well monitored wildlife systems, it is challenging to determine accurate population and ecosystem losses following disease emergence. North American temperate bats present a unique opportunity for studying the broad impacts of wildlife disease emergence, as their federal monitoring programs were prioritized in the USA throughout the 20th century and they are currently threatened by the invasive fungal pathogen, Pseudogymnoascus destructans (Pd), which causes white-nose syndrome. Here we provide a long-term dataset for capture records of Eptesicus fuscus (big brown bat) across the eastern USA, spanning 16 years before and 14 years after Pd invasion into North America. These data represent 30,496 E. fuscus captures across 3,567 unique sites. We encourage the use of this dataset for quantifying impacts of wildlife disease and other threats to wildlife (e.g., climate change) with the incorporation of other available data. We welcome additional data contributions for E. fuscus captures across North and Central America as well as the inclusion of other variables into the dataset that contribute to the quantification of wildlife health.

Characterization of laryngeal motor neuron properties in the American bullfrog, Lithobates catesbieanus.

Motor neurons represent the final output from the central respiratory network. American bullfrogs, Lithobates catesbieanus, have provided insight into development and plasticity of the breathing control system, yet cellular aspects of bullfrog motor neurons are not well-described. In this study, we characterized properties of laryngeal motor neurons that produce motor outflow to the glottal dilator, a muscle that gates airflow to the lungs of anurans. To this end, we measured several intrinsic membrane properties of labeled laryngeal motor neurons in brain slices. Using unsupervised clustering analyses, we identified two broad classes of motor neurons: those with high firing rates and strong adaptation (∼70 %), and those with lower firing rates and less adaptation (∼30 %). These results suggest that two neuronal cell types innervate the glottal dilator, roughly aligning with the composition of fast and slower twitch fibers of this muscle. In sum, these data reinforce the need to consider cell-type when assessing motor neuron function in the respiratory network.

Noradrenergic modulation determines respiratory network activity during temperature changes in the in vitro brainstem of bullfrogs.

Among vertebrate ectotherms, air breathing frequency is generally constrained across warmer temperatures, but decreases during cooling. The brainstem mechanisms that give rise to this ventilatory strategy are unclear. Neuromodulation has recently been shown to stabilize motor circuit output across temperatures. Therefore, we tested the hypothesis that an important neuromodulatory system in respiratory control network, norepinephrine, produces this pattern of respiratory motor activity across temperatures. To this end, we used in vitro brainstem-spinal cord preparations from adult bullfrogs, Lithobates catesbeianus, to assess the role of noradrenergic signaling in shaping the frequency response of the respiratory network during temperature changes. We identified that noradrenergic signaling through the α1 adrenergic receptor constrains motor output from the respiratory network across warm temperatures. In contrast, the α2 adrenergic receptor actively inhibits respiratory motor output during cooling. These results indicate that noradrenergic tuning, rather than passive thermal responses, produces temperature responses of the respiratory circuits.

Analysis of the respiratory component of heart rate variability in the Cururu toad Rhinella schneideri.

Beat-to-beat variation in heart rate (f H ) has been used as a tool for elucidating the balance between sympathetic and parasympathetic modulation of the heart. A portion of the temporal changes in f H is evidenced by a respiratory influence (cardiorespiratory interaction) on heart rate variability (HRV) with heartbeats increasing and decreasing within a respiratory cycle. Nevertheless, little is known about respiratory effects on HRV in lower vertebrates. By using frequency domain analysis, we provide the first evidence of a ventilatory component in HRV similar to mammalian respiratory sinus arrhythmia in an amphibian, the toad Rhinella schneideri. Increases in the heartbeats arose synchronously with each lung inflation cycle, an intermittent breathing pattern comprised of a series of successive lung inflations. A well-marked peak in the HRV signal matching lung inflation cycle was verified in toads whenever lung inflation cycles exhibit a regular rhythm. The cardiac beat-to-beat variation evoked at the moment of lung inflation accounts for both vagal and sympathetic influences. This cardiorespiratory interaction may arise from interactions between central and peripheral feedback mechanisms governing cardiorespiratory control and may underlie important cardiorespiratory adjustments for gas exchange improvement especially under extreme conditions like low oxygen availability.

Synaptic up-scaling preserves motor circuit output after chronic, natural inactivity.

Neural systems use homeostatic plasticity to maintain normal brain functions and to prevent abnormal activity. Surprisingly, homeostatic mechanisms that regulate circuit output have mainly been demonstrated during artificial and/or pathological perturbations. Natural, physiological scenarios that activate these stabilizing mechanisms in neural networks of mature animals remain elusive. To establish the extent to which a naturally inactive circuit engages mechanisms of homeostatic plasticity, we utilized the respiratory motor circuit in bullfrogs that normally remains inactive for several months during the winter. We found that inactive respiratory motoneurons exhibit a classic form of homeostatic plasticity, up-scaling of AMPA-glutamate receptors. Up-scaling increased the synaptic strength of respiratory motoneurons and acted to boost motor amplitude from the respiratory network following months of inactivity. Our results show that synaptic scaling sustains strength of the respiratory motor output following months of inactivity, thereby supporting a major neuroscience hypothesis in a normal context for an adult animal.

Activation of respiratory muscles does not occur during cold-submergence in bullfrogs, Lithobates catesbeianus.

Semiaquatic frogs may not breathe air for several months because they overwinter in ice-covered ponds. In contrast to many vertebrates that experience decreased motor performance after inactivity, bullfrogs, Lithobates catesbeianus, retain functional respiratory motor processes following cold-submergence. Unlike mammalian hibernators with unloaded limb muscles and inactive locomotor systems, respiratory mechanics of frogs counterintuitively allow for ventilatory maneuvers when submerged. Thus, we hypothesized that bullfrogs generate respiratory motor patterns during cold-submergence to avoid disuse and preserve motor performance. Accordingly, we measured activity of respiratory muscles (buccal floor compressor and glottal dilator) via electromyography in freely behaving bullfrogs at 20 and 2°C. Although we confirm that ventilation cycles occur underwater at 20°C, bullfrogs did not activate either respiratory muscle when submerged acutely or chronically at 2°C. We conclude that cold-submerged bullfrogs endure respiratory motor inactivity, implying that other mechanisms, excluding underwater muscle activation, maintain a functional respiratory motor system throughout overwintering.

Effect of temperature on chemosensitive locus coeruleus neurons of savannah monitor lizards, Varanus exanthematicus.

Savannah monitor lizards (Varanus exanthematicus) are unusual among ectothermic vertebrates in maintaining arterial pH nearly constant during changes in body temperature in contrast to the typical α-stat regulating strategy of most other ectotherms. Given the importance of pH in the control of ventilation, we examined the CO2/H+ sensitivity of neurons from the locus coeruleus (LC) region of monitor lizard brainstems. Whole-cell patch-clamp electrophysiology was used to record membrane voltage in LC neurons in brainstem slices. Artificial cerebral spinal fluid equilibrated with 80% O2, 0.0-10.0% CO2, balance N2, was superfused across brainstem slices. Changes in firing rate of LC neurons were calculated from action potential recordings to quantify the chemosensitive response to hypercapnic acidosis. Our results demonstrate that the LC brainstem region contains neurons that can be excited or inhibited by, and/or are not sensitive to CO2 in V. exanthematicus While few LC neurons were activated by hypercapnic acidosis (15%), a higher proportion of the LC neurons responded by decreasing their firing rate during exposure to high CO2 at 20°C (37%); this chemosensitive response was no longer exhibited when the temperature was increased to 30°C. Further, the proportion of chemosensitive LC neurons changed at 35°C with a reduction in CO2-inhibited (11%) neurons and an increase in CO2-activated (35%) neurons. Expressing a high proportion of inhibited neurons at low temperature may provide insights into mechanisms underlying the temperature-dependent pH-stat regulatory strategy of savannah monitor lizards.

Environmentally induced return to juvenile-like chemosensitivity in the respiratory control system of adult bullfrog, Lithobates catesbeianus.

KEY POINTS: The degree to which developmental programmes or environmental signals determine physiological phenotypes remains a major question in physiology. Vertebrates change environments during development, confounding interpretation of the degree to which development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) produces phenotypes. Tadpoles mainly breathe water for gas exchange and frogs may breathe water or air depending on their environment and are, therefore, exemplary models to differentiate the degree to which life-stage vs. environmental context drives developmental phenotypes associated with neural control of lung breathing. Using isolated brainstem preparations and patch clamp electrophysiology, we demonstrate that adult bullfrogs acclimatized to water-breathing conditions do not exhibit CO2 and O2 chemosensitivity of lung breathing, similar to water-breathing tadpoles. Our results establish that phenotypes associated with developmental stage may arise from plasticity per se and suggest that a developmental trajectory coinciding with environmental change obscures origins of stage-dependent physiological phenotypes by masking plasticity. ABSTRACT: An unanswered question in developmental physiology is to what extent does the environment vs. a genetic programme produce phenotypes? Developing animals inhabit different environments and switch from one to another. Thus a developmental time course overlapping with environmental change confounds interpretations as to whether development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) generates phenotypes. Tadpoles of the American bullfrog, Lithobates catesbeianus, breathe water at early life-stages and minimally use lungs for gas exchange. As adults, bullfrogs rely on lungs for gas exchange, but spend months per year in ice-covered ponds without lung breathing. Aquatic submergence, therefore, removes environmental pressures requiring lung breathing and enables separation of adulthood from environmental factors associated with adulthood that necessitate control of lung ventilation. To test the hypothesis that postmetamorphic respiratory control phenotypes arise through permanent developmental changes vs. reversible environmental signals, we measured respiratory-related nerve discharge in isolated brainstem preparations and action potential firing from CO2 -sensitive neurons in bullfrogs acclimatized to semi-terrestrial (air-breathing) and aquatic-overwintering (no air-breathing) habitats. We found that aquatic overwintering significantly reduced neuroventilatory responses to CO2 and O2 involved in lung breathing. Strikingly, this gas sensitivity profile reflects that of water-breathing tadpoles. We further demonstrated that aquatic overwintering reduced CO2 -induced firing responses of chemosensitive neurons. In contrast, respiratory rhythm generating processes remained adult-like after submergence. Our results establish that phenotypes associated with life-stage can arise from phenotypic plasticity per se. This provides evidence that developmental time courses coinciding with environmental changes obscure interpretations regarding origins of stage-dependent physiological phenotypes by masking plasticity.

Control of lung ventilation following overwintering conditions in bullfrogs, Lithobates catesbeianus.

Ranid frogs in northern latitudes survive winter at cold temperatures in aquatic habitats often completely covered by ice. Cold-submerged frogs survive aerobically for several months relying exclusively on cutaneous gas exchange while maintaining temperature-specific acid-base balance. Depending on the overwintering hibernaculum, frogs in northern latitudes could spend several months without access to air, the need to breathe or the chemosensory drive to use neuromuscular processes that regulate and enable pulmonary ventilation. Therefore, we performed experiments to determine whether aspects of the respiratory control system of bullfrogs, Lithobates catesbeianus, are maintained or suppressed following minimal use of air breathing in overwintering environments. Based on the necessity for control of lung ventilation in early spring, we hypothesized that critical components of the respiratory control system of bullfrogs would be functional following simulated overwintering. We found that bullfrogs recently removed from simulated overwintering environments exhibited similar resting ventilation when assessed at 24°C compared with warm-acclimated control bullfrogs. Additionally, ventilation met resting metabolic and, presumably, acid-base regulation requirements, indicating preservation of basal respiratory function despite prolonged disuse in the cold. Recently emerged bullfrogs underwent similar increases in ventilation during acute oxygen lack (aerial hypoxia) compared with warm-acclimated frogs; however, CO2-related hyperventilation was significantly blunted following overwintering. Overcoming challenges to gas exchange during overwintering have garnered attention in ectothermic vertebrates, but this study uncovers robust and labile aspects of the respiratory control system at a time point correlating with early spring following minimal to no use of lung breathing in cold-aquatic overwintering habitats.

Reassessment of chemical control of breathing in undisturbed bullfrogs, Lithobates catesbeianus, using measurements of pulmonary ventilation.

Despite the importance of bullfrogs (Lithobates catesbeianus) as models in respiratory control, chemical control of breathing in conscious bullfrogs has never been assessed with methods that measure the tidal volume (VT). This has precluded the calculation of important respiratory variables like minute ventilation (V.E) and air convection requirement. To address this, we adapted airflow pneumotachography for use in bullfrogs and reassessed chemical control of breathing. We show that V.E measured with pneumotachography produces breathing pattern and metabolism values consistent with anurans. Second, we confirm that bullfrogs have small ventilatory responses to hypercarbia that include increases in tidal volume and a post-hypercarbic hyperpnea. We observed that the magnitude of the post-hypercarbic hyperpnea does not depend on ventilatory responses during hypercarbia. Finally, we showed that increases in breathing frequency and VT during hypoxia are differentially regulated with time. These findings comprise the first complete assessment of hypercarbic and hypoxic V.E responses in intact bullfrogs and emphasize the importance of measuring VT.

Activation state of the hyperpolarization-activated current modulates temperature-sensitivity of firing in locus coeruleus neurons from bullfrogs.

Locus coeruleus neurons of anuran amphibians contribute to breathing control and have spontaneous firing frequencies that, paradoxically, increase with cooling. We previously showed that cooling inhibits a depolarizing membrane current, the hyperpolarization-activated current (I h) in locus coeruleus neurons from bullfrogs, Lithobates catesbeianus (Santin JM, Watters KC, Putnam RW, Hartzler LK. Am J Physiol Regul Integr Comp Physiol 305: R1451-R1464, 2013). This suggests an unlikely role for I h in generating cold activation, but led us to hypothesize that inhibition of I h by cooling functions as a physiological brake to limit the cold-activated response. Using whole cell electrophysiology in brain slices, we employed 2 mM Cs(+) (an I h antagonist) to isolate the role of I h in spontaneous firing and cold activation in neurons recorded with either control or I h agonist (cyclic AMP)-containing artificial intracellular fluid. I h did not contribute to the membrane potential (V m) and spontaneous firing at 20°C. Although voltage-clamp analysis confirmed that cooling inhibits I h, its lack of involvement in setting baseline firing and V m precluded its ability to regulate cold activation as hypothesized. In contrast, neurons dialyzed with cAMP exhibited greater baseline firing frequencies at 20°C due to I h activation. Our hypothesis was supported when the starting level of I h was enhanced by elevating cAMP because cold activation was converted to more ordinary cold inhibition. These findings indicate that situations leading to enhancement of I h facilitate firing at 20°C, yet the hyperpolarization associated with inhibiting a depolarizing cation current by cooling blunts the net V m response to cooling to oppose normal cold-depolarizing factors. This suggests that the influence of I h activation state on neuronal firing varies in the poikilothermic neuronal environment.

Acute and chronic temperature effects on cardiovascular regulation in the red-eared slider (Trachemys scripta).

Acute and chronic changes in ambient temperature alter several aspects of reptilian physiology. We investigated the effects of each type of temperature change on reptilian cardiovascular regulation in red-eared slider turtles (Trachemys scripta), a species known to experience marked seasonal changes in ambient temperature. Turtles were instrumented with occlusive catheters in the femoral artery and vein. Following an acclimation period of 10 days at 13 °C (13(1)), cardiovascular responses to adrenaline, and the cardiac limb of the baroreflex were quantified. Ambient temperature was then reduced 1 °C day(-1) until 3 °C was reached (3(1)). Turtles were maintained at this temperature for 1-week before cardiovascular responses were reassessed. Turtles were then gradually (1 °C day(-1)) returned to an ambient temperature of 13 °C, (13(2)). After a 1-week re-acclimation period, cardiovascular responses were again determined. Finally, 1-week post-pharmacological manipulation of turtles in the 13(2) treatment, ambient temperature was reduced to 3 °C over 24 h (3(2)), and cardiovascular responses were again assessed. Temperature reduction from 13(1) to 3(1) decreased mean arterial blood pressure (P(m)) and heart rate (f(H)) by ~38 and ~63%, respectively. Acute temperature reduction, from 13(2) to 3(2), decreased f(H) similarly, ~66%; however, while P(m) decreased ~28%, this was not significantly different than P(m) at 13(2). The adrenaline injections increased f(H) ranging from 90 to 170% at 13 °C which was a greater change than that observed at 3 °C ranging from a 40 to 70% increase. The increase in P m at the lowest dose of adrenaline did not differ across the temperature treatment groups. The operational point (set-point) P(m) of the baroreflex was decreased similarly by both methods of temperature reduction (3(1) or 3(2)). Further, a hypertensive cardiac baroreflex was absent in the majority of the animals studied independent of temperature. Baroreflex gain and normalized gain based on individual estimates of the relationship were decreased by temperature reduction similarly. Collectively, the data suggest that red-eared slider turtles modulate (down-regulate) some cardiovascular control mechanisms during reduced ambient temperature.

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.

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.

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.

The chemosensitive response of neurons from the locus coeruleus (LC) to hypercapnic acidosis with clamped intracellular pH.

Currently, a change in pH(i) is believed to be the major signal in the chemosensitive (CS) response of brainstem neurons to hypercapnia; however, multiple factors (e.g., Ca2+, CO2, pH(i), and pHo) have been suggested to contribute to this increase in firing rate. While there is evidence for a significant role of pH(i) in the CS response, we hypothesize that hypercapnic acidosis (HA) can increase firing rate even with no change in pH(i). We tested several methods to clamp pH(i), including high intracellular buffer and the use of rapid diffusion of weak bases or weak acids through the cell membrane. We were able to clamp pH(i) during hypercapnic exposure using weak acids. We observed a CS response to HA, with pH(i) clamped, indicating that intracellular acidification, while sufficient to increase firing rate, is not required for the response of CS neurons. The CS response to HA without a change in pH(i) is most likely due to extracellular acidification and/or increased CO2 and strongly supports the multiple factors model of chemosensitive signaling.

Intrinsic chemosensitivity of individual nucleus tractus solitarius (NTS) and locus coeruleus (LC) neurons from neonatal rats.

Chemosensitive (CS) neurons are found in discrete brainstem regions, but whether the CS response of these neurons is due to intrinsic chemosensitivity of individual neurons or is mediated by changes in chemical and/or electrical synaptic input is largely unknown. We studied the effect of synaptic blockade (11.4 mM Mg2+/0.2mM Ca2+) solution (SNB) and a gap junction uncoupling agent carbenoxolone (CAR--100 microM) on the response of neurons from two CS brainstem regions, the NTS and the LC. In NTS neurons, SNB decreased spontaneous firing rate (FR). We calculated the magnitude of the FR response to hypercapnic acidosis (HA; 15% CO2) using the Chemosensitivity Index (CI). The percentage of NTS neurons activated and CI were the same in the absence and presence of SNB. Blocking gap junctions with CAR did not significantly alter spontaneous FR. CAR did not alter the CI in NTS neurons and resulted in a small decrease in the percentage of activated neurons, which was most evident in NTS neurons from rats younger than postnatal day 10. In LC neurons, SNB resulted in an increase in spontaneous FR. As with NTS neurons, SNB did not alter the percentage of activated neurons or the CI in LC neurons. CAR resulted in a small increase in spontaneous FR in LC neurons. In contrast, CAR had a marked effect on the response of LC neurons to HA: a reduced percentage of CS LC neurons and decreased CI. In summary, both NTS and LC neurons appear to contain intrinsically CS neurons. CS neurons from the two regions receive different tonic input in slices (excitatory for NTS and inhibitory for LC); however, blocking chemical synaptic input does not affect the CS response in either region. In NTS neurons, gap junction coupling plays a small role in the CS response, but gap junctions play a major role in the chemosensitivity of many LC neurons.