Possible Role of TRP Channels in Rat Glomus Cells.

Carotid body (CB) glomus cells depolarize in response to hypoxia, causing a [Ca(2+)](i) increase, at least in part, through activation of voltage-dependent channels. Recently, Turner et al. (2013) showed that mouse glomus cells with knockout of TASK1/3(-/-) channels have near-normal [Ca(2+)](i) response to hypoxia. Thus, we postulated that TRP channels may provide an alternate calcium influx pathway which may be blocked by the TRP channel antagonist, 2-APB (2-aminoethoxydiphenylborane). We confirmed that 2-APB inhibited the afferent nerve response to hypoxia, as previously reported (Lahiri S, Patel G, Baby S, Roy A (2009) 2-APB mediated effects on hypoxic calcium influx in rat carotid body glomus cells. FASEB 2009, Abstract, LB157; Kumar P, Pearson S, Gu Y (2006) A role for TRP channels in carotid body chemotransduction? FASEB J 20:A12-29). To examine the mechanism for this inhibition, we examined dissociated rat CB glomus cells for [Ca(2+)](i) responses to hypoxia, anoxia (with sodium dithionite), 20 mM K(+), NaSH, NaCN, and FCCP in absence/presence of 2-APB (100 µM). Also the effect of 2-APB on hypoxia and/or anoxia were investigated on NADH and mitochondria (MT) membrane potential. Our findings are as follows: (1) 2-APB significantly blocked the [Ca(2+)](i) increase in response to hypoxia and anoxia, but not the responses to 20 mM K(+). (2) The [Ca(2+)](i) responses NaSH, NaCN, and FCCP were significantly blocked by 2-APB. (3) Hypoxia-induced increases in NADH/NAD(+) and MT membrane depolarization were not effected by 2-APB. Thus TRP channels may provide an important pathway for calcium influx in glomus cells in response to hypoxia.

Postnatal hyperoxia impairs acute oxygen sensing of rat glomus cells by reduced membrane depolarization.

Previous work demonstrated that hyperoxia (30-60% O(2)) exposure in the post-natal period reduces the ventilatory response to acute hypoxia and this impairment may continue considerably beyond the period of hyperoxia exposure. Previous work from our laboratory demonstrated that 1-2 weeks of hyperoxia (60% O(2)) starting between P1 and P14: reduced the single chemoreceptor unit response to hypoxia, reduced the rise in glomus cell calcium caused by acute hypoxia and reduced hypoxia-induced catecholamine release (Donnelly 05, Donnelly 09). The present study asked whether the impairment extended to hypoxia-induced membrane depolarization, an earlier step in the transduction cascade. Perforated patch, whole-cell recordings were obtained from rat glomus cells exposed to hyperoxia from P0-P8 or P8-P15 and age-matched control groups. In both cases, hypoxia-induced membrane depolarization was significantly less in the hyperoxia treated groups compared to controls, while depolarization to 20 mM K(+) was not significantly affected. Resting membrane potential and input resistance were also not different in the hyperoxia treated groups. Whole carotid body quantitative real time PCR showed that TASK-1, TASK-3 and L-type Ca(2+) channel expression was significantly down-regulated at Hyper 8-15 compared to controls. We conclude that 1 week of postnatal hyperoxia during the early and late stage of CB maturation impairs organ function by affecting the coupling between hypoxia and glomus cell depolarization. This may be caused by altered expression of TASK1, TASK3 or L-type Ca(2+) channel gene expression. We speculate that an identification of cellular changes caused by hyperoxia may yield unique insights to the mechanism of oxygen sensing by the carotid bodies.

Purinergic modulation of carotid body glomus cell hypoxia response during postnatal maturation in rats.

Carotid body (CB) glomus cells respond to hypoxia by releasing neurotransmitters, such as ATP, which are believed to stimulate excitatory receptors on apposed nerve endings of the carotid sinus nerves as well as bind to autoreceptors on the glomus cell membrane to modulate response magnitude. The CB response to hypoxia is small at birth and increases during postnatal maturation in mammals. As ATP has been shown to inhibit the glomus cell response to hypoxia via an autoreceptor mechanism, we hypothesized that ATP-mediated inhibition may vary with age and play a role in postnatal development of the hypoxia response magnitude. The effects of ATP on CB glomus cell intracellular calcium ([Ca(2+)](i)) responses to hypoxia were studied at two ages, P0-1 and P14-18. The inhibitory effect of ATP or a stable ATP analog on the glomus cell response to hypoxia was greater in newborn rats compared to the more mature age group. Use of selective P2Y receptor agonists and antagonists suggests that the inhibitory effect of ATP on the glomus cell [Ca(2+)](i) response to hypoxia may be mediated by a P2Y12 receptor. Thus, developmental changes in ATP-mediated glomus cell inhibition may play a role in carotid chemoreceptor postnatal maturation.

Chronic compression of mouse dorsal root ganglion alters voltage-gated sodium and potassium currents in medium-sized dorsal root ganglion neurons.

Chronic compression (CCD) of the dorsal root ganglion (DRG) is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. Previously, we examined electrophysiological changes in small-diameter lumbar level 3 (L3) and L4 DRG neurons treated with CCD; the present study extends these observations to medium-sized DRG neurons, which mediate additional sensory modalities, both nociceptive and non-nociceptive. Whole-cell patch-clamp recordings were obtained from medium-sized somata in the intact DRG in vitro. Compared with neurons from unoperated control animals, CCD neurons exhibited a decrease in the current threshold for action potential generation. In the CCD group, current densities of TTX-resistant and TTX-sensitive Na(+) current were increased, whereas the density of delayed rectifier voltage-dependent K(+) current was decreased. No change was observed in the transient or "A" current after CCD. We conclude that CCD in the mouse produces hyperexcitability in medium-sized DRG neurons, and the hyperexcitability is associated with an increased density of Na(+) current and a decreased density of delayed rectifier voltage-dependent K(+) current.

Increased Na+ and K+ currents in small mouse dorsal root ganglion neurons after ganglion compression.

We investigated the effects of chronic compression (CCD) of the L3 and L4 dorsal root ganglion (DRG) on pain behavior in the mouse and on the electrophysiological properties of the small-diameter neuronal cell bodies in the intact ganglion. CCD is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. On days 1, 3, 5, and 7 after the onset of compression, there was a significant decrease from preoperative values in the threshold mechanical force required to elicit a withdrawal of the foot ipsilateral to the CCD (tactile allodynia). Whole cell patch-clamp recordings were obtained, in vitro, from small-sized somata and, for the first time, in the intact DRG. Under current clamp, CCD neurons exhibited a significantly lower rheobase compared with controls. A few CCD but no control neurons exhibited spontaneous action potentials. CCD neurons showed an increase in the density of TTX-resistant and TTX-sensitive Na(+) current. CCD neurons also exhibited an enhanced density of voltage-dependent K(+) current, due to an increase in delayed rectifier K(+) current, without a change in the transient or "A" current. We conclude that CCD in the mouse produces a model of radicular pain, as we have previously demonstrated in the rat. While the role of enhanced K(+) current remains to be elucidated, we speculate that it represents a compensatory neuronal response to reduce ectopic or aberrant levels of neuronal activity produced by the injury.

Altered functional properties of satellite glial cells in compressed spinal ganglia.

The cell bodies of sensory neurons in the dorsal root ganglion (DRG) are enveloped by satellite glial cells (SGCs). In an animal model of intervertebral foraminal stenosis and low-back pain, a chronic compression of the DRG (CCD) increases the excitability of neuronal cell bodies in the compressed ganglion. The morphological and electrophysiological properties of SGCs were investigated in both CCD and uninjured, control lumbar DRGs. SGCs responded within 12 h of the onset of CCD as indicated by an increased expression of glial fibrillary acidic protein (GFAP) in the compressed DRG but to lesser extent in neighboring or contralateral DRGs. Within 1 week, coupling through gap junctions between SGCs was significantly enhanced in the compressed ganglion. Under whole-cell patch clamp recordings, inward and outward potassium currents, but not sodium currents, were detected in individual SGCs. SGCs enveloping differently sized neurons had similar electrophysiological properties. SGCs in the compressed vs. control DRG exhibited significantly reduced inwardly rectifying potassium currents (Kir), increased input resistances and positively shifted resting membrane potentials. The reduction in Kir was greater for nociceptive medium-sized neurons compared to non-nociceptive neurons. Kir currents of SGCs around spontaneously active neurons were significantly reduced 1 day after compression but recovered by 7 days. These data demonstrate rapid alterations in glial membrane currents and GFAP expression in close temporal association with the development of neuronal hyperexcitability in the CCD model of neuropathic pain. However, these alterations are not fully sustained and suggest other mechanisms for the maintenance of the hyperexcitable state.

Developmental maturation of chemosensitivity to hypoxia of peripheral arterial chemoreceptors--invited article.

Peripheral arterial chemoreceptors, particularly the carotid body chemoreceptors, are the primary sites for the detection of hypoxia and reflexly increase ventilatory drive and behavioral arousal during hypoxic or asphyxial events. Newborn infants are at risk for hypoxic and asphyxial events during sleep, yet, the strength of the chemoreceptor responses is low or absent at birth and then progressively increases with early postnatal development. This review summarizes the available data showing that even though the "oxygen sensor" in the glomus cells has not been unequivocally identified, it is clear that development affects many of the other properties of the chemoreceptor unit (glomus cell, afferent nerve fibers and neurotransmitter profile at the synapse) that are necessary and essential for the propagation of the "sensing" response, and exposure to hypoxia, hyperoxia and nicotine can modify normal development of each of the components leading to altered peripheral chemoreceptor responses.

Spontaneous action potential generation due to persistent sodium channel currents in simulated carotid body afferent fibers.

The mechanism by which action potentials (APs) are generated in afferent nerve fibers in the carotid body is unknown, but it is generally speculated to be release of an excitatory transmitter and synaptic depolarizing events. However, previous results suggested that Na(+) channels in the afferent nerve fibers play an important role in this process. To better understand the potential mechanism by which Na(+) channels may generate APs, a mathematical model of chemoreceptor nerve fibers that incorporated Hodgkin-Huxley-type Na(+) channels with kinetics of activation and inactivation, as determined previously from recordings of petrosal chemoreceptor neurons, was constructed. While the density of Na(+) channels was kept constant, spontaneous APs arose in nerve terminals as the axonal diameter was reduced to that in rat carotid body. AP excitability and pattern were similar to those observed in chemoreceptor recordings: 1) a random pattern at low- and high-frequency discharge rates, 2) a high sensitivity to reductions in extracellular Na(+) concentration, and 3) a variation in excitability that increased with AP generation rate. Taken together, the results suggest that an endogenous process in chemoreceptor nerve terminals may underlie AP generation, a process independent of synaptic depolarizing events.

Cortical delta-opioid receptors potentiate K+ homeostasis during anoxia and oxygen-glucose deprivation.

Central neurons are extremely vulnerable to hypoxic/ischemic insult, which is a major cause of neurologic morbidity and mortality as a consequence of neuronal dysfunction and death. Our recent work has shown that delta-opioid receptor (DOR) is neuroprotective against hypoxic and excitotoxic stress, although the underlying mechanisms remain unclear. Because hypoxia/ischemia disrupts ionic homeostasis with an increase in extracellular K(+), which plays a role in neuronal death, we asked whether DOR activation preserves K(+) homeostasis during hypoxic/ischemic stress. To test this hypothesis, extracellular recordings with K(+)-sensitive microelectrodes were performed in mouse cortical slices under anoxia or oxygen-glucose deprivation (OGD). The main findings in this study are that (1) DOR activation with [D-Ala(2), D-Leu(5)]-enkephalinamide attenuated the anoxia- and OGD-induced increase in extracellular K(+) and decrease in DC potential in cortical slices; (2) DOR inhibition with naltrindole, a DOR antagonist, completely abolished the DOR-mediated prevention of increase in extracellular K(+) and decrease in DC potential; (3) inhibition of protein kinase A (PKA) with N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide dihydrochloride had no effect on the DOR protection; and (4) inhibition of protein kinase C (PKC) with chelerythrine chloride reduced the DOR protection, whereas the PKC activator (phorbol 12-myristate 13-acetate) mimicked the effect of DOR activation on K(+) homeostasis. These data suggest that activation of DOR protects the cortex against anoxia- or ODG-induced derangement of potassium homeostasis, and this protection occurs via a PKC-dependent and PKA-independent pathway. We conclude that an important aspect of DOR-mediated neuroprotection is its early action against derangement of K(+) homeostasis during anoxia or ischemia.

Lamotrigine and phenytoin, but not amiodarone, impair peripheral chemoreceptor responses to hypoxia.

Amiodarone, lamotrigine, and phenytoin, common antiarrhythmic and antiepileptic drugs, inhibit a persistent sodium current in neurons (I(NaP)). Previous results from our laboratory suggested that I(NaP) is critical for functionality of peripheral chemoreceptors. In this study, we determined the effects of therapeutic levels of amiodarone, lamotrigine, and phenytoin on peripheral chemoreceptor and ventilatory responses to hypoxia. Action potentials (APs) of single chemoreceptor afferents were recorded using suction electrodes advanced into the petrosal ganglion of an in vitro rat peripheral chemoreceptor complex. AP frequency (at Po(2) approximately 150 Torr and Po(2) approximately 90 Torr), conduction time, duration, and amplitude were measured before and during perfusion with therapeutic dosages of the drug or vehicle. Hypoxia-induced catecholamine secretion within the carotid body was measured using amperometry. With the use of whole body plethysmography, respiration was measured in unanesthesized rats while breathing room air, 12% O(2), and 5% CO(2), before and after intraperitoneal administration of amiodarone, lamotrigine, phenytoin, or vehicle. Lamotrigine (10 microM) and phenytoin (5 microM), but not amiodarone (5 microM), decreased chemoreceptor AP frequency without affecting other AP parameters or magnitude of catecholamine secretion. Similarly, lamotrigine (5 mg/kg) and phenytoin (10 mg/kg) blunted the hypoxic but not the hypercapnic ventilatory response. In contrast, amiodarone (2.5 mg/kg) did not alter the ventilatory response to hypoxia or hypercapnia. We conclude that lamotrigine and phenytoin at therapeutic levels impair peripheral chemoreceptor function and ventilatory response to acute hypoxia. These are consistent with I(NaP) serving an important function in AP generation and may be clinically important in the care of patients using these drugs.

An important functional role of persistent Na+ current in carotid body hypoxia transduction.

Systemic hypoxia in mammals is sensed and transduced by the carotid body into increased action potential (AP) frequency on the sinus nerve, resulting in increased ventilation. The mechanism of hypoxia transduction is not resolved, but previous work suggested that fast Na(+) channels play an important role in determining the rate and timing of APs (Donnelly, DF, Panisello JM, and Boggs D. J Physiol. 511: 301-311, 1998). We speculated that Na(+) channel activity between APs, termed persistent Na(+) current (I(NaP)), is responsible for AP generation that and riluzole and phenytoin, which inhibit this current, would impair organ function. Using whole cell patch clamp recording of intact petrosal neurons with projections to the carotid body, we demonstrated that I(NaP) is present in chemoreceptor afferent neurons and is inhibited by riluzole. Furthermore, discharge frequencies of single-unit, chemoreceptor activity, in vitro, during normoxia (Po(2) 150 Torr) and during acute hypoxia (Po(2) 90 Torr) were significantly reduced by riluzole concentrations at or above 5 microM, and by phenytoin at 100 microM, without significant affect on nerve conduction time, AP magnitude (inferred from extracellular field), and AP duration. The effect of both drugs appeared solely postsynaptic because hypoxia-induced catecholamine release in the carotid body was not altered by either drug. The respiratory response of unanesthetized, unrestrained 2-wk-old rats to acute hypoxia (12% inspired O(2) fraction), which was measured with whole body plethysmography, was significantly reduced after treatment with riluzole (2 mg/kg ip) and phenytoin (20 mg/kg ip). We conclude that I(NaP) is present in chemoreceptor afferent neurons and serves an important role in peripheral chemoreceptor function and, hence, in the ventilatory response to hypoxia.

Upregulation of the hyperpolarization-activated cation current after chronic compression of the dorsal root ganglion.

A chronic compression of the DRG (CCD) produces cutaneous hyperalgesia and an enhanced excitability of neuronal somata in the compressed ganglion. The hyperpolarization-activated current (I(h)), present in the somata and axons of DRG neurons, acts to induce a depolarization after a hyperpolarizing event and, if upregulated after CCD, may contribute to enhanced neuronal excitability. Whole-cell patch-clamp recordings were obtained from acutely dissociated, retrogradely labeled, cutaneous, adult rat DRG neurons of medium size. Neurons were dissociated from L4 and L5 control DRGs or DRGs that had each been compressed for 5-7 d by L-shaped rods inserted into the intervertebral foramina. I(h), consisting of a slowly activating inward current during a step hyperpolarization, was recorded from every labeled, medium-sized neuron and was blocked by 1 mm cesium or 15 microm ZD7288. Compared with control, CCD increased the current density and rate of activation significantly without changing its reversal potential, voltage dependence of activation, or rate of deactivation. Because I(h) activation provides a depolarizing current to the neuron, thus enhancing neuronal excitability, our results are consistent with the hypothesis that I(h) contributes to hyperalgesia after CCD-induced nerve injury.

Perinatal hyperoxia for 14 days increases nerve conduction time and the acute unitary response to hypoxia of rat carotid body chemoreceptors.

Hyperoxia in the immediate perinatal period, but not in adult life, is associated with a life-long impairment of the ventilatory response to acute hypoxia. This effect is attributed to a functional impairment of peripheral chemoreceptors, including a reduction in the number of chemoreceptor afferent fibers and a reduction in "whole nerve" afferent activity. The purpose of the present study was to assess the activity levels of single chemoreceptor units in the immediate posthyperoxic period to determine whether functional impairment extended to single chemoreceptor units and whether the impairment was only induced by hyperoxia exposure in the immediate postnatal period. Two groups of rat pups were exposed to 60% inspired O2 fraction for 2 wk at ages 0-14 days and 14-28 days, at which time single-unit activities were isolated and recorded in vitro. Compared with control pups, hyperoxia-treated pups had a 10-fold reduction in baseline (normoxia) spiking activity. Peak unit responses to 12, 5, and 0% O2 were reduced and nerve conduction time was significantly slower in both hyperoxia-treated groups compared with control groups. We conclude that 1) hyperoxia greatly reduces single-unit chemoreceptor activities during normoxia and acute hypoxia, 2) the treatment effect is not limited to the immediate newborn period, and 3) at least part of the impairment may be due to changes in the afferent axonal excitability.

Development of carotid body/petrosal ganglion response to hypoxia.

Carotid body chemoreceptors undergo significant maturational changes in the post-natal period over a period of days to weeks. This is likely initiated by the rise in Pa(O2) at the time of birth and reflects the changing value of "normoxia" from 25 Torr to near 100 Torr. Chemoreceptors in the newborn period have a lower absolute discharge frequency and the dynamic response to acute hypoxia is less compared to the adult. This maturation change appears due to changes occurring presynaptically to the afferent nerve fibers. Hypoxia-induced secretion from the glomus cell (catecholamine and other constituents of dense cored vesicles) is enhanced whilst constitutive (non-hypoxia-dependent) release is reduced with age. On the post-synaptic side, the number of afferent synaptic sites increases four- to five-fold in the post-natal period and there may be an increase in afferent nerve excitability. Both of these changes are subject to environmental perturbations in which post-natal exposure to chronic hypoxia or hyperoxia leads to significantly reduced organ sensitivity and function. Thus, developmental changes and environmental factors may significantly change the ability of an animal to detect and respond to hypoxic insults, perhaps leading to periods of heightened vulnerability to hypoxic stresses.

Mitochondrial function and carotid body transduction.

Carotid body chemoreceptors respond to a decrease in arterial oxygen tension by increasing spiking activity on the sinus nerve. Our understanding of the oxygen-transducing ability of the organ arose from studies in the 1930s intended to understand how metabolic poisons stimulated breathing. Since that time, an intimate link between energy state and hypoxia sensing has been assumed and forms the basis of the metabolic hypothesis of oxygen sensing. This hypothesis is supported by studies demonstrating a loss of mitochondrial potential in carotid body cells at oxygen tensions that cause no change in cells from other tissues. Although the nature of the coupling between mitochondrial function and nerve excitation remains unresolved, experimental evidence supports roles for (1) release of mitochondrial calcium stores, (2) modulation of membrane channels that are linked to mitochondrial complexes I and IV, and (3) generation of signaling intermediates, such as reactive oxygen species (ROS) from complex I and III of the electron transport chain. If the mitochondrion is the oxygen-sensing site for peripheral chemoreceptors, then there exists the potential ability to manipulate, perhaps pharmacologically, the sensing function by alterations in expression of uncoupler proteins or chemicals that can alter the affinity of cytochrome oxidase for oxygen. Such manipulation may be useful for the treatment of hypoventilation syndromes or high altitude accommodation.

Non-additive interactions between mitochondrial complex IV blockers and hypoxia in rat carotid body responses.

The metabolic hypothesis of carotid body chemoreceptor hypoxia transduction proposes an impairment of ATP production as the signal for activation. We hypothesized that mitochondrial complex IV blockers and hypoxia would act synergistically in exciting afferent nerve activity. Following a pre-treatment with low dosage sodium cyanide (10-20µM), the hypoxia-induced nerve response was significantly reduced along with hypoxia-induced catecholamine release. However, in isolated glomus cells, the intracellular calcium response was enhanced as initially predicted. This suggests a cyanide-mediated impairment in the step between the glomus cell intracellular calcium rise and neurotransmitter release from secretory vesicles. Administration of a PKC blocker largely reversed the inhibitory actions of cyanide on the neural response. We conclude that the expected synergism between cyanide and hypoxia occurs at the level of glomus cell intracellular calcium but not at downstream steps due to a PKC-dependent inhibition of secretion. This suggests that at least one regulatory step beyond the glomus cell calcium response may modulate the magnitude of chemoreceptor responsiveness.

Voltage-gated Na(+) channels in chemoreceptor afferent neurons--potential roles and changes with development.

Carotid body chemoreceptors increase their action potential (AP) activity in response to a decrease in arterial oxygen tension and this response increases in the post-natal period. The initial transduction site is likely the glomus cell which responds to hypoxia with an increase in intracellular calcium and secretion of multiple neurotransmitters. Translation of this secretion to AP spiking levels is determined by the excitability of the afferent nerve terminals that is largely determined by the voltage-dependence of activation of Na(+) channels. In this review, we examine the biophysical characteristics of Na(+) channels present at the soma of chemoreceptor afferent neurons with the assumption that similar channels are present at nerve terminals. The voltage dependence of this current is consistent with a single Na(+) channel isoform with activation around the resting potential and with about 60-70% of channels in the inactive state around the resting potential. Channel openings, due to transitions from inactive/open or closed/open states, may serve to amplify external depolarizing events or generate, by themselves, APs. Over the first two post-natal weeks, the Na(+) channel activation voltage shifts to more negative potentials, thus enhancing the amplifying action of Na(+) channels on depolarization events and increasing membrane noise generated by channel transitions. This may be a significant contributor to maturation of chemoreceptor activity in the post-natal period.

Perinatal hyperoxia exposure impairs hypoxia-induced depolarization in rat carotid body glomus cells.

Chronic post-natal hyperoxia reduces the hypoxic ventilatory response by reducing the carotid body sensitivity to acute hypoxia as demonstrated by a reduced afferent nerve response, reduced calcium response of carotid body glomus cells and reduced catecholamine secretion in response to acute hypoxia. The present study examined whether hyperoxia alters the electrophysiological characteristics of glomus cells. Rats were treated with hyperoxia for 1 week starting at P1 or P7 and for 2 weeks starting at P1 followed by harvesting and dissociation of their carotid bodies for whole cell, perforated-patch recording. As compared to glomus cells from normoxia animals, hyperoxia treated cells showed a significant reduction in the magnitude of depolarization in response to hypoxia and anoxia, despite little change in the depolarizing response to 20 mM K(+). Resting cell membrane potential in glomus cells from rats exposed to hyperoxia from P1 to P15 and studied at P15 was slightly depolarized compared to other treatment groups and normoxia-treated cells, but conductance normalized to cell size was not different among groups. We conclude that postnatal hyperoxia impairs carotid chemoreceptor hypoxia transduction at a step between hypoxia sensing and membrane depolarization. This occurs without a major change in baseline electrophysiological characteristics, suggesting altered signaling or alterations in the relative abundance of different leak channel isoforms.

Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording.

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 µM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 µM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 µM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 µM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.