Petrosal Ganglion Research Articles

Characterization and developmental changes of Na+ currents of petrosal neurons with projections to the carotid body.

Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into an increase in spiking activity on the sinus nerve, and this response increases with postnatal age over the first week or two of life. Previous work from our laboratory has suggested a major role of axonal Na(+) channels in the initiation of afferent spiking activity. Using RT-PCR of the petrosal ganglia we identified Na(+) channel TTX-S isoforms Na(v)1.1, Na(v)1.6, and Na(v)1.7 and the TTX-resistant (TTX-R) isoforms Na(v)1.8 and Na(v)1.9 at high levels. Electrophysiologic recordings (at 3 ages: 3 days, 9 days, 18-20 days) of neurons that project to the carotid body exhibited predominantly fast-inactivating sodium currents, with a bimodal recovery from inactivation at -80 mV (fast component approximately 8 ms; slow component approximately 90 ms). Developmental age had little effect with no change in peak current density (approximately 1.4 nA/pF) and was associated with a slight, but significant increase in the speed of recovery from inactivation at -140 and -120 mV but not at other potentials. Assuming that the same Na(+) channel complement is present at the nerve terminal as at the soma, the association of a sensory modality (chemoreception) with a relatively uniform Na(+) channel profile suggests that the rapid kinetics of TTX-S channels may be essential for some aspects of chemoreceptor function beyond mediating simple axonal conduction.

Expression of Phox2 Transcription Factors and Induction of the Dopaminergic Phenotype in Primary Sensory Neurons

Cell-type-specific transcription factors may regulate phenotypic diversity by conferring selective responsiveness to relatively nonspecific environmental cues. To test this hypothesis, we examined whether the homeodomain transcription factors Phox2a/2b play a role in activity-dependent expression of the dopaminergic phenotype using petrosal ganglion (PG) sensory neurons as a model. The timing of Phox2a/2b expression is precisely correlated with the ability of PG neurons to express the dopamine-synthesizing enzyme, tyrosine hydroxylase (TH), in response to depolarizing stimuli. Phox2a/2b expression is highest at embryonic day 16.5, when virtually all PG neurons exhibit activity-dependent TH induction, and subsequently falls in parallel with the loss of activity-dependent TH induction. Expression is maintained, however, in all dopaminergic neurons. Physiologic stimulation of PG neurons in vivo induces TH expression exclusively in Phox2a/2b + cells. Our data suggest that constitutive expression of Phox2a/2b defines the potential of neurons to become dopaminergic in response to membrane depolarization during a critical window of phenotypic plasticity.

Chronic hypoxia upregulates connexin43 expression in rat carotid body and petrosal ganglion.

Recent studies have demonstrated that oxygen-sensitive type I cells in the carotid body express the gap junction-forming protein connexin43 (Cx43). In the present study, we examined the hypothesis that chronic exposure to hypoxia increases Cx43 expression in type I cells as well as in chemoafferent neurons in the petrosal ganglion. Immunocytochemical studies in tissues from normal rats revealed diffuse and granular Cx43-like immunoreactivity in the cytoplasm of type I cells and dense punctate spots of immunoreactive product at the margins of type I cells and near the borders of chemosensory cell lobules. Cx43-like immunoreactivity was not detectable in petrosal ganglion neurons from normal animals. After a 2-wk exposure to hypobaric (380 Torr) hypoxia, Cx43 immunostaining was substantially enhanced in and around type I cells. Moreover, chronic hypoxia elicited the expression of Cx43-like immunoreactivity in the cytoplasm of afferent neurons throughout the petrosal ganglion. Quantitative RT-PCR studies indicate that chronic hypoxia evokes a substantial increase in Cx43 mRNA levels in the carotid body, along with a marked elevation of Cx43 expression in the petrosal ganglion. Increased Cx43 expression and gap junction formation in type I cells and sensory neurons may contribute to carotid body adaptation during sustained stimulation in extreme physiological conditions.

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro.

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.

Acetylcholine sensitivity of cat petrosal ganglion neurons.

We investigated if neuronal nicotinic acetylcholine receptors (nAChRs) are localized in chemoreceptor afferent neurons in the cat petrosal ganglion (PG) and if acetylcholine (ACh) excites chemoreceptor afferent neurons. Immunocytochemistry revealed that a majority of PG neurons expressed alpha 4 and/or alpha 7 subunits of neuronal nAChRs, and a part of them were tyrosine hydroxylase positive. Excitability of cultured PG neurons was studied with patch clamp techniques (whole cell configuration). ACh and nicotine evoked both inward and outward currents. The inward current was partially blocked by removal of extracellular calcium and by antagonists for alpha 4 beta 2 (dihydro-beta-erythroidine) or alpha 7 nAChRs (methyllycaconitine). Outward current was blocked by 4-aminopyridine (4-AP) and sometimes by atropine. ACh-induced membrane potential changes were well correlated with ACh-induced currents. Depolarization and hyperpolarization occurred in response to ACh. Occasionally depolarization was followed by a train of action potentials. The results suggest that heterogeneous neuronal nAChRs are widely distributed in both chemoreceptor and other PG neurons. In some neurons nAChRs may be functionally coupled with outward K+ channels. Further studies are required to determine whether chemoreceptor neurons have a distinct distribution pattern of nAChRs and K+ channels.

Responses of petrosal ganglion neurons in vitro to hypoxic stimuli and putative transmitters.

Responses of petrosal ganglion neurons in vitro to hypoxic stimuli and putative transmitters.

Nitric oxide modulation of carotid chemoreception.

In response to hypoxia, the glomus cells of the carotid body (CB) are expected to release an excitatory transmitter which in turn initiates the chemosensory discharge in the nerve terminals of the petrosal ganglion chemosensory neurons (Eyzaguirre & Zapata 1984). In addition to excitatory transmitters, other molecules produced within the CB may modulate chemoreceptor activity. Recently, it was advanced that gaseous molecules like carbon monoxide (in low concentration) and nitric oxide (NO) act as modulatory inhibitors of chemosensory responses induced by hypuxichypoxia (Chugh et al. 1994, Prabhakar et al. 1993, Wang et al. 1994, 1995). In fact, the administration of the NO precursor L-arginine and NO donors reduced chemosensory responses to hypoxia in the cat CB perfused or superfused in vitro (Katayama et al. 1994, Wang et al. 1994). On the other hand, inhibition of nitric oxide synthase enzyme (NOS) increased basal chemosensory discharges (Chugh et al. 1994, Prabhakar et al. 1993, Wang et al. 1994, 1995) and enhanced chemosensory responses to hypoxic-hypoxia in vitro (Wang et al. 1994, 1995).

Expression of 5-HT 3 receptors in primary sensory neurons of the petrosal ganglion of adult rats

By using a specific antiserum, expression of the 5-HT 3 receptor was examined in the petrosal ganglion (PG) of adult male rats. We found that the 5-HT 3 receptors are widely distributed in the PG. This finding was confirmed by RT-PCR detection of the 5-HT 3 receptor mRNA in the tissue. Unlike the distribution patterns of tyrosine hydroxylase (TH), which occurred in limited regions of PG, the 5-HT 3 receptors seemed to distribute throughout the ganglion. As many TH-positive neurons in PG innervate type I cells in the carotid body, the coexistence of 5-HT 3 receptor and TH in some neurons suggests that this receptor may play a role in carotid body chemoreception.

Prenatal nicotine affects catecholamine gene expression in newborn rat carotid body and petrosal ganglion.

Nicotine exposure modifies the expression of catecholamine and opioid neurotransmitter systems involved in attenuation of hypoxic chemosensitivity. We used in situ hybridization histochemistry to determine the effect of prenatal and early postnatal nicotine exposure on tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), preproenkephalin (PPE), and D2-dopamine receptor mRNA levels in the rat carotid body and petrosal ganglion during postnatal development. In the carotid body, nicotine increased TH mRNA expression in animals at 0 and 3 postnatal days (both, P < 0.05 vs. control) without affecting TH mRNA levels at 6 and 15 days. At 15 postnatal days, DbetaH mRNA levels were increased in the carotid body of nicotine-exposed animals. Dopamine D2-receptor mRNA levels in the carotid body increased with postnatal age but were unaffected by nicotine exposure. PPE was not expressed in the carotid body at any of the ages studied in control or treated animals. In the petrosal ganglion, nicotine increased the number of ganglion cells expressing TH mRNA in animals at 3 days (P < 0.01 vs. control). DbetaH mRNA expression was not induced nor was PPE mRNA expression increased in the petrosal ganglion in treated animals. Prenatal nicotine exposure upregulates mRNAs involved in the synthesis of two inhibitory neuromodulators, dopamine and norepinephrine, in peripheral arterial chemoreceptors, which may contribute to abnormalities in cardiorespiratory control observed in nicotine exposed animals.

Voltage-gated K + channels in chemoreceptor sensory neurons of rat petrosal ganglion

A subpopulation of sensory neurons in the petrosal ganglion transmits information between peripheral chemoreceptors (glomus cells) in the carotid body and relay neurons in the nucleus of the solitary tract. Expression of voltage-gated K + channels in these neurons was characterized by immunohistochemical localization. Five members of the Kv1 family, Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 and members of two other families, Kv2.1 and Kv4.3, were identified in over 90% of the chemoreceptor neurons. Although the presence of these channel proteins was consistent throughout the population, individual neurons showed considerable variation in K + current profiles.

Ionic currents and action potential induced by electrical stimulation, ACh and ATP in petrosal ganglion neurons

The glossopharyngeal nerve contains the sensory fibers of the petrosal ganglion (PG) neurons that innervate the carotid body and sinus, through the carotid sinus nerve, and the tongue and the pharynx, through the glossopharyngeal branch. Based on the shape of the action potential, two populations of PG neurons can be recognized. However, little is known about the ionic currents underlying these action potentials. The application of ACh and ATP, transmitters putatively involved in the communication between the glomus cells and PG neuron terminals, to the isolated PG increase the frequency of discharge of the carotid sinus nerve, while only ATP increases discharges in the glossopharyngeal branch. The above suggests the presence of at least two neuronal populations. Petrosal ganglia were excised from cats anesthetized with sodium pentobarbitone (40 mg/kg, ip), dissociated, and the neurons cultured for 3–16 days. Using whole-cell patch-clamp technique, we recorded from isolated PG neurons both action potentials and ionic currents evoked by electrical stimulation, and by application of ACh and ATP. Neurons presented either fast action potentials (F-type) or slower spikes with an inflexion in the repolarizing phase (H-type). The F-type neurons had fast inward and a sustained outward current. The spike and inward currents were reversibly blocked when choline replaced Na+ or in the presence of 3 μM tetrodotoxin (TTX). The outward current was partly blocked by 5 mM tetraethylammonium (TEA). The H-type neurons presented an inward current with at least two components, and an outward current with a fast -partly inactivating- and a slower sustained component. When external Na+ was replaced by choline, or when 3 μM TTX was added, the amplitude of the spike and the inward current were reversibly decreased. Similarly, when intracellular K+ was replaced by Cs+, or when 5 mM TEA was added, the amplitude and duration of the spike increased and the outward current was reduced. Under voltage-clamp, ATP induced a dose-dependent inward current that was partly desensitized during 20 s application pulses. The ATP-induced current had a threshold of 100 nM, and saturated between 20–50 μM. ACh also induced a fast, inactivating inward current, with a threshold between 10–50 μM, and saturated about 1–5 mM. From 34 neurons tested for ACh and ATP, 9% responded only to ACh, 24% to ATP only, 50% responded to both agents, and the remaining 17% were unresponsive. In current-clamp, ATP and ACh depolarized the neurons and may induce action potentials. Our results show that H- and F-type PG neurons express different voltage-gated ionic and receptor-gated currents, which are activated either by ACh or ATP, with a half population responding to both putative transmitters.

Prenatal nicotine exposure up-regulates catecholaminergic traits in peripheral arterial chemoreceptors of newborn rat pups

Infants born to smoking mothers have depressed hypoxic arousal responses, reduced respiratory drive, and blunted ventilatory responses to hypoxia. These abnormalities in respiratory control are believed to be features of infants at risk for Sudden Infant Death Syndrome (SIDS). The peripheral arterial chemoreceptors in the carotid body are extensively involved in modulating respiratory responses to hypoxia, and arousal responses during sleep. Similar to findings in infants exposed prenatally to tobacco smoke, prenatal exposure to nicotine, a major component of tobacco smoke, alters acute ventilatory responses to short exposures to hypoxia and hyperoxia (Dejours test), manipulations that are used as a test of peripheral arterial chemoreceptor in function in newborn rats. Nicotine, through binding to cholinergic receptors, causes depolarization and catecholamine and opioid release from cells and neurons. Chemosensory type 1 cells in the carotid body and a subset of chemoreceptor sensory neurons in the petrosal ganglion are rich in catecholamines and contain nicotinic receptors. Physiology and pharmacology data also suggest that the carotid body contains met-enkephalins. Release of dopamine and norepineph-rine from chemosensory type 1 cells with subsequent binding to inhibitory dopaminergic and adrenergic receptors on carotid sinus nerve fibers results in diminished neuronal output from the carotid body leading to depressed hypoxic ventilatory responses. Similarly, the release of met-enkephalins from type 1 cells and binding to delta-opioid receptors on the carotid sinus nerve decreases hypoxic chemosensitivity. We used in situ hybridization histochemistry to determine the effect of prenatal nicotine exposure on the change in the levels of mRNAs for the enzymes regulating dopamine and norepinephrine synthesis, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DβH), respectively and preproenkephalin (PPE) mRNA in the rat carotid body and petrosal ganglion during postnatal development. We also determined the change in the level of D2-dopamine receptor mRNA expression in these tissues induced by prenatal exposure to nicotine. In the carotid body, prenatal nicotine exposure increased THmRNA expression in animals at 0 and 3 postnatal days (both, P < 0.05 vs control) without affecting THmRNA levels at 6 and 15 days. However, at 15 postnatal days, DβH mRNA levels were increased in the carotid body of animals prenatally exposed to nicotine. D2-dopamine receptor mRNA expression in the carotid body increased with postnatal age and was unaffected by nicotine exposure. In the petrosal ganglion, prenatal nicotine exposure increased the number of ganglion cells expressing THmRNA in animals at 3 days (P < 0.01 vs control). DβH mRNA expression was not induced nor was PPE mRNA expression up-regulated in the petrosal ganglion in treated animals. PPE was not expressed in the carotid body in control or prenatally treated animals. In conclusion, prenatal nicotine exposure up-regulates mRNAs for synthesizing enzymes for two inhibitory neuromodulators, dopamine and norepinephrine, in peripheral arterial chemoreceptors that might contribute to abnormalities in cardiorespiratory control in animals prenatally exposed to nicotine. A possible role for brain-derived neurotrophic factor (BDNF) in nicotine induced up-regulation of catecholaminergic traits in peripheral arterial chemoreceptors will be discussed.

Prenatal hypoxia impairs the early postnatal development of the carotid chemoafferent pathway

Rat carotid bodies (CB) are considered as the main component that initiates the Hypoxic Ventilatory Response (HVR). Most of these chemoafferent fibres project into discrete areas of the medulla oblongata via the petrosal ganglion (PG), mainly in the caudal part of the nucleus Tractus Solitarius (NTS), and, to a lesser extent, in the ventrolateral medulla (VLM) [1]. Both medullary areas contain two major respiratory cell groups: the dorsal respiratory group, in the ventrolateral subset of the solitary tract, and the ventral respiratory group, in the VLM. These two medullary respiratory groups are closely associated with catecholaminergic neurones that belong to, the A2C2 cell group and the A1C1 cell group respectively. There is growing evidence that medullary cate-cholaminergic neurones participate in the chemoreflex response to systemic hypoxia. During the two first weeks of life, the sensitivity of the carotid bodies adapts to the comparatively hypoxic environment of the foetus and then reset to the higher PaO2 of the new-born. This environmental change in oxygen leads to an increase in the carotid body sensitivity, due in part to a decrease in the release of dopamine [2]. The postnatal maturation of the carotid body sensitivity depends on the level of environmental oxygen during the early postnatal period. The aim of the present study is to investigate the effects of prenatal hypoxia (last 15 days of gestation in 10% O2) on the neurochemical and functional development (postnatal day 0, 3, 7, 14, 21 and 68) of the chemoafferent pathway. We thus assessed the development of in vivo tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in the catecholamine synthesis [3], in the CB, PG and the A1C1, A2C2 cell groups. In the same way, TH mRNA was evaluated in the CB, PG structures. Moreover, we evaluated the functional maturity of the chemoreflex pathway by measuring the HVR. We attempted to address four main questions. First, can prenatal hypoxia induce a neurochemical impairment of the CB resetting? Second, could the neurochemical impairment be related to the mRNA level? Third, are central and peripheral structures affected in the same manner? Fourth, are the neuronal impairments reflected at the functional level? Our results show that 1) prenatal hypoxia amplifies the neurochemical resetting of the peripheral chemoreceptors; 2) a part of the neurochemical impairment is explained at the mRNA level; 3) central and peripheral structures exhibit opposite impairment; 4) prenatal hypoxia modifies the HVR pattern. In conclusion, prenatal hypoxic exposure during the two last weeks of gestation impairs the carotid body chemoreflex pathway at the functional, the neurochemical and the molecular level, affecting the CB resetting process.

Pharmacological and immunochemical evidence of the dopamine D3 receptor in the goat carotid body.

Dopamine (DA) represents the predominant amine in the carotid body (CB) of several species. Over 3 decades, numerous studies of the effects of DA on carotid chemoreceptor function have produced a variety of results, whether conducted in vivo or in vitro. The reported effects of DA on carotid chemoreflex function are mostly inhibitory, although several investigators have reported excitatory effects, or both inhibitory and excitatory effects1. DA D2 receptors have been found on the CB type I cells, petrosal ganglion and in afferent nerves2,3,4. It is possible that multiple DA receptor types may exist within the carotid chemoreflex pathway.

Muscarinic receptors influence catecholamine release from the cat carotid body during hypoxia.

Many frontiers in the modeling of the ventilatory system and its control are at the cellular and molecular levels. The carotid body’s response to hypoxia is no exception to this. A commonly accepted model of the carotid body’s chemotransduction of hypoxia presents the transmitter-laden glomus cell being depolarized by the hypoxic challenge in as-yet-to-be­determined processes. This depolarization activates voltage-gated calcium channels with the subsequent entrance of extracellular calcium. This event, in turn, precipitates the release of several transmitters. Many investigators have reported the hypoxia-generated release of catecholamines from the carotid body. And recently we have reported the release of acetylcholine (ACh) from the cat carotid body during hypoxia1. These transmitters are presumed to bind to “postsynaptic” receptors located on the apposed sensory afferent fibers which have their cell bodies in the petrosal ganglion with their axons terminating in nucleus tractus solitarii. The transmitters are also presumed to bind to “presynaptic” autoreceptors on the glomus cells. Over the last decade our working hypothesis has been that ACh is the (or at least one) critically important excitatory transmitter. And in the mid 1990s we generated some neurophysiological recordings2 which suggested the presence of both M1 and M2 muscarinic receptors in the carotid body. The data could not answer whether the receptors were located postsynaptically, presynaptically, or both. Since then we have reported preliminary results suggesting that they are at least located on the glomus cell.

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10–100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.

Acetylcholine sensitivity in sensory neurons dissociated from the cat petrosal ganglion

The petrosal ganglia contain the somata of the sensory fibers of the glossopharyngeal nerves, innervating structures of the tongue, pharynx, carotid sinus and carotid body. Petrosal ganglia were excised from adult cats and their neurons were dissociated and kept in tissue culture for 7–12 days. Intracellular recordings were obtained through conventional microelectrodes. In response to depolarizing pulses, most cells (41/60) presented a ‘hump’ in the falling phase of their action potentials (H-type), while the remaining neurons lack such hump (F-type). The two types of cells had no differences in resting membrane potential or action potential amplitude. Acetylcholine (ACh) applied locally elicited responses in nearly two thirds of both H-type and F-type neurons tested. Most H-type neurons (17/19) responded with a slow long lasting depolarization, while the remaining (2) did so by generating spikes. In contrast, half of F-type neurons (6/12) responded with one or more spikes and the other half only with a slow depolarization. These results indicate that ACh receptors are present in the soma of many petrosal ganglion neurons subjected to tissue culture, thus supporting the idea that — under normal conditions — their peripheral sensory processes may be excited by ACh.

Differential expression of A 2a, A 1-adenosine and D 2-dopamine receptor genes in rat peripheral arterial chemoreceptors during postnatal development

The sensitivity of peripheral arterial chemoreceptors in the carotid body to hypoxia increases with postnatal maturation. Carotid sinus nerve activity is augmented by adenosine binding to A 2a-adenosine receptors and attenuated by dopamine binding to D 2-dopamine receptors. In this study, we used in situ hybridization histochemistry to determine the change in the levels of mRNA expression for A 2a and A 1-adenosine receptors and D 2-dopamine receptors in the rat carotid body. We also investigated the cellular distribution and possible colocalization of these receptor mRNAs and tyrosine hydroxylase (TH) mRNAs during the first 2 weeks of postnatal development. By using immunohistocytochemistry, we detected A 2a-adenosine receptor protein in the carotid body and petrosal ganglion. We found that A 2a-adenosine receptor mRNA and protein are expressed in the carotid body in animals at 0, 3, 6 and 14 postnatal days. The level of A 2a-adenosine receptor mRNA expression significantly decreased by 14 postnatal days ( P<0.02 vs. day 0) while D 2-dopamine receptor mRNA levels significantly increased by day 3 and remained greater than D 2-dopamine receptor mRNA levels at day 0 ( P<0.001 all ages vs. day 0). TH mRNA was colocalized in cells in the carotid body with A 2a adenosine receptor and D 2-dopamine receptor mRNAs. A 1-adenosine receptor mRNA was not expressed in the carotid body at any of the ages examined. In the petrosal ganglion, A 1-adenosine receptor mRNA was abundantly expressed in numerous cells, A 2a-adenosine receptor mRNA was expressed in a moderate number of cells while D 2-dopamine receptor mRNA was seen in a few cells in the rostral petrosal ganglion. In conclusion, using in situ hybridization histochemistry, we have shown that mRNA for both the excitatory, A 2a-adenosine receptor, and the inhibitory, D 2-dopamine receptor, is developmentally regulated in presumably type I cells in the carotid body which may contribute to the maturation of hypoxic chemosensitivity. Furthermore, the presence A 1-adenosine receptor mRNAs in cell bodies of the petrosal ganglion suggests that adenosine might also have an inhibitory role in hypoxic chemotransmission.

Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors.

Using functional co-cultures of rat carotid body (CB) O2 chemoreceptors and 'juxtaposed' petrosal neurones (JPNs), we tested whether ATP and ACh acted as co-transmitters. Perforated-patch recordings from JPNs often revealed spontaneous and hypoxia-evoked (PO2 approximately 5 mmHg) excitatory postsynaptic responses. The P2X purinoceptor blocker, suramin (50 microM) or a nicotinic ACh receptor (nAChR) blocker (hexamethonium, 100 microM; mecamylamine, 1 microM) only partially inhibited these responses, but together, blocked almost all activity. Under voltage clamp (-60 mV), fast perfusion of 100 microM ATP over hypoxia-responsive JPNs induced suramin-sensitive (IC50 = 73 microM), slowly-desensitizing, inward currents (IATP) with time constant of activation tauon = 30.6 +/- 4. 8 ms (n = 7). IATP reversed at 0.33 +/- 3.7 mV (n = 4), and the dose-response curve was fitted by the Hill equation (EC50 = 2.7 microM; Hill coefficient approximately 0.9). These purinoceptors contained immunoreactive P2X2 subunits, but their activation by alpha,beta-methylene ATP (alpha,beta-meATP; EC50 = 2.1 microM) suggests they are P2X2/P2X3 heteromultimers. Suramin and nAChR blockers inhibited the extracellular chemosensory discharge in the intact rat carotid body-sinus nerve preparation in vitro. Further, P2X2 immunoreactivity was widespread in rat petrosal ganglia in situ, and co-localized in neurones expressing the CB chemo-afferent marker, tyrosine hydroxylase (TH). P2X2 labelling in the CB co-localized with nerve-terminal markers, and was intimately associated with TH-positive type 1 cells. Thus ATP and ACh are co-transmitters during chemotransduction in the rat carotid body.

Single-unit recordings of arterial chemoreceptors from mouse petrosal ganglia in vitro.

A preparation was developed that allows for the recording of single-unit chemoreceptor activity from mouse carotid body in vitro. An anesthetized mouse was decapitated, and each carotid body was harvested, along with the sinus nerve, glossopharyngeal nerve, and petrosal ganglia. After exposure to collagenase/trypsin, the cleaned complex was transferred to a recording chamber where it was superfused with oxygenated saline. The ganglia was searched for evoked or spontaneous unit activity by using a glass suction electrode. Single-unit action potentials were 57 +/- 10 (SE) (n = 16) standard deviations above the recording noise, and spontaneous spikes were generated as a random process. Decreasing superfusate PO(2) to near 20 Torr caused an increase in spiking activity from 1. 3 +/- 0.4 to 14.1 +/- 1.9 Hz (n = 16). The use of mice for chemoreceptor studies may be advantageous because targeted gene deletions are well developed in the mouse model and may be useful in addressing unresolved questions regarding the mechanism of chemotransduction.