Distribution of voltage-gated potassium and hyperpolarization-activated channels in sensory afferent fibers in the rat carotid body

The chemosensory glomus cells of the carotid body (CB) detect changes in O2 tension. Carotid sinus nerve fibers, which originate from peripheral sensory neurons located within the petrosal ganglion, innervate the CB. Release of transmitter from glomus cells activates the sensory afferent fibers to transmit information to the nucleus of the solitary tract in the brainstem. The ion channels expressed within the sensory nerve terminals play an essential role in the ability of the terminal to initiate action potentials in response to transmitter-evoked depolarization. However, with a few exceptions, the identity of ion channels expressed in these peripheral nerve fibers is unknown. This study addresses the expression of voltage-gated channels in the sensory fibers with a focus on channels that set the resting membrane potential and regulate discharge patterns. By using immunohistochemistry and fluorescence confocal microscopy, potassium channel subunits and HCN (hyperpolarization-activated) family members were localized both in petrosal neurons that expressed tyrosine hydroxylase and in the CSN axons within the carotid body. Channels contributing to resting membrane potential, including HCN2 responsible in part for I(h) current and the KCNQ2 and KCNQ5 subunits thought to underlie the neuronal "M current," were identified in the sensory neurons and their axons innervating the carotid body. In addition, the results presented here demonstrate expression of several potassium channels that shape the action potential and the frequency of discharge, including Kv1.4, Kv1.5, Kv4.3, and K(Ca) (BK). The role of these channels should be considered in interpretation of the fiber discharge in response to perturbation of the carotid body environment.     

Cytospecific properties of arterial chemosensory neurons

The carotid body of the cat was reinnervated either by the carotid branch or by the glossal branch of the IXth nerve and evaluated histologically and neurophysiologically. Regenerating foreign fibers re-established 90% fewer specialized terminals on glomus cells and displayed a greatly diminished chemosensory response, compared to axons of the regenerating carotid branch. Regenerating sensory neurons appear to develop chemosensitivity as a consequence of contact with glomus cells.     

Axonal transport of labeled material into sensory nerve endings of cat carotid body

The origin of nerve endings on glomus cells in the carotid body has been the subject of much controversy in recent years. Specifically, the problem is whether these nerve endings, which contain clear-core (“synaptic”) vesicles and mitochondria, arise from sensory neurons in the petrosal ganglion or from efferent neurons located in the brain stem or elsewhere. To study this problem, [³H]proline was applied to cat petrosal ganglia, the animals were allowed to survive for 3 h-7 days, and the peripheral distribution of the label was analyzed by sample oxidation/scintillation counting and by EM autoradiography. The time courses of distribution of label along the nerves and the accumulation of label in the carotid body indicated the presence of fast, intermediate and slow components of axonal flow. EM autoradiographs of carotid bodies showed the label localized almost exclusively to nerve fibers and to nerve terminals on glomus cells. As much as 60–90% of the nerve terminals were labeled in a given ultrathin-section autoradiograph. Passive movement of label from the injection site, or fibers of passage (efferent) through the ganglion, did not contribute to the labeled material since administration of [³H]proline onto the desheathed nerve away from the ganglion was ineffective in labeling the carotid body. The results suggest that most, if not all, nerve terminals on glomus cells in cat carotid body arise from neurons in the petrosal ganglion.     

ATP- and ACh-induced responses in isolated cat petrosal ganglion neurons

Chemoreceptor (glomus) cells of the carotid body are synaptically connected to the sensory nerve endings of petrosal ganglion (PG) neurons. In response to natural stimuli, the glomus cells release transmitters, which acting on the nerve terminals of petrosal neurons increases the chemosensory afferent discharge. Among several transmitter molecules present in glomus cells, acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are considered to act as excitatory transmitter in this synapse. To test if ACh and ATP play a role as excitatory transmitters in the cat CB, we recorded the electrophysiological responses from PG neurons cultured in vitro. Under voltage clamp, ATP induces a concentration-dependent inward current that partially desensitizes during 20-30 s application pulses. The ATP-induced current has a threshold near 100 nM and saturates between 20-50 muM. ACh induces a fast, inactivating inward current, with a threshold between 10-50 muM, and saturates around 1 mM. A large part of the population of PG neurons (60%) respond to both ATP and ACh. Present results support the hypothesis that ACh and ATP act as excitatory transmitters between cat glomus cells and PG neurons.     

Endothelins and nitric oxide: vasoactive modulators of carotid body chemoreception

The carotid body (CB) is the main arterial chemoreceptor that senses arterial PO2, PCO2 and pH. The structural unit of the CB is the glomoid, which is formed by clusters of chemoreceptor (glomus) cells located around the capillaries. The glomus cells are synaptically connected to nerve terminals of petrosal ganglion (PG) neurons and surrounded by sustentacular cells. The most accepted model of CB chemoreception states that glomus cells are the primary sensors. In response to hypoxia, hypercapnia and acidosis, glomus cells release one or more transmitters, which acting on the nerve terminals of sensory PG neurons, increase the chemosensory discharge. The CB has a high blood flow and an elevated metabolism that correlate to its oxygen-sensing function. Thus, vasoactive molecules produced within the CB may modulate the chemosensory process by controlling the CB blood flow and tissue PO2. In this review, we examine recent evidence supporting the idea that endothelins (ETs) and nitric oxide (NO) modulate the CB function acting upon chemoreceptor cells and chemosensory neurons or by regulating the blood flow through the CB parenchyma.     

Neurons synthesizing nitric oxide innervate the mammalian carotid body

The carotid body is an arterial chemoreceptor organ sensitive to blood levels of O₂, CO₂ and pH. The present immunocytochemical and neurochemical study has demonstrated the presence of an extensive plexus of nitric oxide (NO)-synthesizing nerve fibers in this organ. These nitric oxide synthase (NOS)-containing axons are closely associated with parenchymal type I cells and with blood vessels in the carotid body. Denervation and retrograde tracing experiments have revealed that these fibers arise from NOS-immunoreactive and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neuronal cell bodies located in the petrosal ganglion and the carotid body, and dispersed along the glossopharyngeal and carotid sinus nerves (CSN). Within the petrosal ganglion, these neurons are topographically segregated from the catecholaminergic cells, and they contain the neuropeptide, substance P. NOS-positive autonomic microganglial cells in the carotid body and CSN also exhibit choline acetyltransferse (ChAT) immunoreactivity. Our results suggest that nitric oxide may be a novel neuronal messenger in the mammalian carotid body involved in the modulation of chemosensory transduction and transmission in this organ. © 1993 Wiley-Liss, Inc.     

Selective activation of carotid nerve fibers by acetylcholine applied to the cat petrosal ganglion in vitro

The petrosal ganglion innervates carotid body chemoreceptors through the carotid (sinus) nerve. These primary sensory neurons are activated by transmitters released from receptor (glomus) cells, acetylcholine (ACh) having been proposed as one of the transmitters involved in this process. Since the perikarya of primary sensory neurons share several properties with peripheral sensory endings, we studied the electrical responses of the carotid nerve and glossopharyngeal branch to ACh locally applied to the cat petrosal ganglion superfused in vitro. Ganglionar applications of AChCl (1 μg−1 mg) generated bursts of action potentials conducted along the carotid nerve, while only a few spikes were exceptionally recorded from the glossopharyngeal branch in response to the largest doses. Carotid nerve responses to ACh were dose-dependent, the higher doses inducing transient desensitization. Application of nicotine to the petrosal ganglion also evoked dose-dependent excitatory responses in the carotid nerve. Responses to ACh were reversibly antagonized by adding hexamethonium to the superfusate, more intense and prolonged block of ACh responses being produced by mecamylamine. Ganglionar applications of γ-amino butyric acid and serotonin, in doses of up to 5 mg, did not induce firing of action potentials in any of the branches of the glossopharyngeal nerve. Our results indicate that petrosal ganglion neurons projecting through the carotid nerve are selectively activated by ACh acting on nicotinic ACh receptors located in the somata of these neurons. Thus, cholinosensitivity would be shared by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.     

Regeneration of taste buds by nongustatory nerve fibers

Previous cross-reinnervation studies in situ by other investigators have demonstrated that cutaneous sensory and motor axons are incapable of trophically supporting mammalian taste buds. The present experiments examined the gustatory trophic potency of chemosensory and barosensory axons of the carotid sinus nerve. We report here that morphologically normal taste buds appeared on cat circumvallate papillae at 2 to 19 months after cross-anastomosis of the carotid sinus and lingual nerves, branches of the IXth cranial (glossopharyngeal) nerve. However, neurophysiologic and histologic data also indicated that, despite microsurgical procedures designed to direct regenerating lingual nerve fibers toward the carotid body and carotid sinus, some lingual axons escaped the anastomosis and subsequently grew within their native distal stump. The principal objective of this study was thus to determine whether foreign innervation of taste buds did indeed occur, or regenerated lingual nerve fibers were instead responsible for the newly formed buds. Our results showed that stray lingual fibers were not responsible for the reappearance of taste buds because transection of the original proximal lingual nerve stump (cross-anastomosed to the distal carotid sinus nerve stump) did not reduce the incidence of taste buds or the accumulation of radiolabeled material axoplasmically transported from the petrosal (sensory) ganglion. Autoradiography of labeled tissue samples showed that more than 90% of the taste buds were labeled at 8 and 9 days after lingual nerve transection. These data support the hypothesis that sensory axons in the carotid sinus nerve share an important trophic chemistry with gustatory neurons.     

Expression of histamine receptors and effect of histamine in the rat carotid body chemoafferent pathway

Chemosensory information from peripheral arterial oxygen sensors in the carotid body is relayed by petrosal ganglion neurons to the respiratory networks in the medulla oblongata. Biogenic amines, including histamine, released from glomus (type I) cells of the carotid body are considered to be primary transmitters in hypoxic chemosensitivity. Immunocytochemistry at light-and electron-microscopical levels, and RT-PCR, revealed the expression of histamine receptors 1 and 3 as well as histidine decarboxylase in the rat carotid body glomus cells and petrosal ganglion neurons. Histamine receptors 1 and 3, but not histidine decarboxylase, were also observed in the ventrolateral, intermediate and commissural subnuclei of the nucleus tractus solitarii in the medulla oblongata. In order to examine the possible role of histamine in the afferent branch of the respiratory system, we applied histamine receptor 1 and 3 agonists to the carotid body, which caused a mildly increased phrenic nerve activity in a working heart-brainstem preparation. Moreover, microinjection of antagonists of histamine receptors 1 and 3 into the nucleus tractus solitarii caused significant changes in the inspiratory timing and the chemoreceptor response. Our data show that histamine acting via histamine receptors 1 and 3 plays an important neuromodulatory role in the afferent control of chemosensitivity.     

Dopamine inhibits ATP-induced responses in the cat petrosal ganglion in vitro

The petrosal ganglion (PG) provides sensory innervation to the carotid sinus and carotid body through the carotid (sinus) nerve (CN). Application of either acetylcholine (ACh) or adenosine 5'-triphosphate (ATP) to the PG superfused in vitro activates CN fibers. Dopamine (DA) modulates the effects of ACh. We have previously shown that DA when applied to the PG modulates the effects of ACh on carotid sinus nerve fibers. We currently report the effects of DA on the ATP-induced responses in the isolated PG in vitro. While DA had no effect on the basal activity recorded from the CN, it reduced ATP-induced responses in a dose-dependent manner, when preceding ATP applications by 30 s. Our results suggest that DA-a transmitter present in a group of PG neurons and in carotid body cells-may act as an inhibitory modulator of ATP-evoked responses in PG neurons.     

Responses to hypoxia of petrosal ganglia in vitro

NaCN is a classical stimulus used to elicit discharges from carotid body chemoreceptors. The effect is assumed to be mediated by glomus (type I) cells, which release an excitatory transmitter for the excitation of carotid nerve endings. Since the sensory perikarya of the glossopharyngeal nerve (from which the carotid nerve branches) are located in the petrosal ganglion, we tested whether application of this drug to the petrosal ganglion superfused in vitro elicits antidromic discharges in the carotid nerve. NaCN did indeed cause an intense and prolonged burst of nerve impulses in the carotid nerve, while provoking a less intense and much briefer burst of discharges in the glossopharyngeal branch. Carotid nerve responses to NaCN were reduced and shortened by prior or following application of dopamine to the ganglion. Sodium azide applied to the petrosal ganglion evoked a less intense and much briefer burst of impulses in the carotid nerve. Ganglionar application of 2,4-dinitrophenol did not induce discharges in the carotid nerve. Switching the superfusion of the ganglion from a normoxic to a hypoxic solution did not evoke discharges in the carotid nerve. Therefore, the perikarya of carotid nerve neurons are sensitive to NaCN, but are not excited by reducing the pO(2) of the superfusing solution.     

Modulatory effect of nitric oxide on acetylcholine-induced activation of cat petrosal ganglion neurons in vitro

The inhibitory effect of nitric oxide (NO) on carotid chemosensory responses to hypoxia has been attributed in part to an antidromic inhibition of chemoreceptor cells activity. However, NO may also modulate the activity of the primary sensory neurons because NO is produced in the soma of these neurons located in the petrosal ganglion. Since a population of petrosal neurons is selectively activated by acetylcholine (ACh), we studied the effects of NO-donor, sodium nitroprusside (SNP), and the NO-synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), on the responses evoked in the carotid sinus nerve (CSN) by ACh applied to the petrosal ganglion in vitro. ACh (1 microgram-1 mg) increased the frequency of action potentials recorded from the CSN in a dose-dependent manner. SNP (10-50 microM) reduced the sensibility and amplitude of the CSN response to ACh, although the maximal response appears less affected. The withdrawal of SNP from the superfusion medium increased the sensibility of the responses to ACh. l-NAME (1-2 mM) slightly increased the sensibility of the ACh-induced responses, effect that persisted after l-NAME withdrawal. These results suggest that NO may play a role as modulator in this autonomic primary sensory ganglion.     

Cat carotid bodies reinnervated by normal or foreign nerves

The carotid body of the cat was reinnervated with either its native nerve, the carotid sinus nerve (CSN, re-anastomosis), or a foreign nerve, the lingual branch of the IXth cranial nerve (LN, cross-anastomosis). In both types of preparations, regenerating axons from the LN or CSN readily penetrated carotid body parenchymal tissue, as demonstrated by axoplasmic transport of radiolabeled material from the petrosal (sensory) ganglion. Electron microscopy revealed nearly normal fiber invasion into lobules of glomus (type I) and sustentacular (type II) cells following reinnervation by either the foreign or native nerve. However, while the regenerated CSN fibers formed a normal complement of specialized axon terminals in contact with type I cells, the incidence of such terminals in LN reinnervated carotid bodies was reduced by over 90% (2-19 months survival time). This low incidence of specialized LN endings was correlated with reductions in the magnitude of the chemosensory discharge elicited in these preparations by asphyxia, NaCN or acetylcholine. These data suggest that chemosensitivity depends upon intimate association between glomus cells and afferent nerve endings; and that the ability to form such contacts may reside in particular axons whose incidence is higher in the CSN than in the LN.     

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.     

Dopamine modulates carotid nerve responses induced by acetylcholine on the cat petrosal ganglion in vitro

We have recently reported that application of acetylcholine (ACh) or nicotine to the petrosal ganglion—the sensory ganglion of the glossopharyngeal nerve—elicits a burst of discharges in the carotid nerve branch, innervating the carotid body and sinus, but not in the glossopharyngeal branch, innervating the tongue and pharynx. Thus, the perikarya of sensory neurons for the carotid bifurcation exhibit selective cholinosensitivity. Since dopamine (DA) modulates carotid nerve chemosensory activity, we searched for the presence of DA sensitivity at the perikarya of these neurons in the cat petrosal ganglion superfused in vitro. Applications of DA in doses of up to 5 mg to the ganglion did not modify the rate of spontaneous discharges in the carotid nerve. However, if DA was applied 30 s before ACh injections, ACh-evoked reactions were modified: low doses of DA enhanced the subsequent responses to ACh, while high doses of DA depressed the responses to ACh. This depressant effect of DA on ACh responses was partially antagonized by adding spiroperone to the superfusate. Our results show that the response to ACh of petrosal ganglion neurons projecting through the carotid nerve is modulated by DA acting on D₂ receptors located in the somata of these neurons. Thus, dopaminergic modulation of cholinosensitivity could be shared also by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.     

Neurocalcin-immunoreactive neurons in the petrosal ganglion innervate the taste bud

The distribution and origin of neurocalcin-immunoreactive (NC-ir) nerve fibers in the taste bud and carotid body were examined by an immunofluorescence method. In the circumvallate papilla of the tongue, NC-ir nerve fibers made subepithelial nerve plexuses and occasionally penetrated the taste bud. However, the carotid body was devoid of ir nerve fibers. In the petrosal ganglion, 32% of neurons were immunoreactive for NC. Such neurons were mostly medium-sized to large, and scattered throughout the ganglion. In the superior cervical and intralingual ganglia, numerous ir varicose fibers surrounded postsynaptic neurons. However, NC-ir could not be detected in cell bodies of these neurons. The retrograde tracing method indicated that NC-ir petrosal neurons innervated taste buds in the circumvallate papilla. NC-ir neurons may have a gustatory function in the petrosal ganglion.     

ACh and ATP mediate excitatory transmission in cat carotid identified chemoreceptor units in vitro

Several molecules have been proposed as excitatory transmitters between glomus (type 1) cells and nerve terminals of petrosal ganglion (PG) neurons in the carotid body (CB). We tested whether ACh and ATP have a role to play as excitatory transmitters in the cat CB by recording intracellularly from identified PG neurons functionally connected to the CB in vitro. PG neurons projecting to the CB were classified according to their intracellular responses as: (a) neurons with humped action potentials (hAP neurons) responding phasically to long-lasting depolarizing pulses (53/67), and (b) neurons with smooth action potentials (non-hAP neurons) that fire tonically during long-lasting depolarizations (14/67). CB stimulation by stop flow and/or acidosis induced activity in 28 of 39 hAP-type neurons, being classified as chemosensory, but in none of the non-hAP neurons. Hexamethonium (10 microM) and suramin (100 microM) reversibly abolished the increased discharges evoked in chemosensory neurons (8/9) by stop flow or acidosis. Moreover, 24 of 27 chemosensory neurons responded to ganglionar application of ACh and ATP, while two neurons responded only to ACh and one to ATP. Mechanical deformation of the carotid sinus induced firing activity in 10 of 13 non-hAP neurons, but in none of the hAP neurons tested. Interestingly, 4/10 non-hAP neurons, which responded to carotid sinus mechanical stimulation also responded to ganglionar application of ATP, but were insensitive to ACh. Present results favor the hypothesis that ACh and ATP are excitatory transmitters in the cat CB, acting-at least-on the PG neuron terminals in the CB.     

Presence and coexistence of putative neurotransmitters in carotid sinus baro- and chemoreceptor afferent neurons

The presence and coexistence of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) were studied in the petrosal and jugular neurons innervating the carotid body and carotid sinus of the rat. The retrograde labeling of the carotid sinus nerve with Fluoro-gold (FG) demonstrated that most (94.5%) FG-labeled ganglionic neurons were observed in the petrosal ganglion. Fewer (5.2%) FG-labeled neurons were seen in the jugular ganglion and very few (0.3%) were observed in the nodose ganglion. Immunohistochemistry revealed that subpopulations of TH-, VIP-, CGRP-, SP- and GAL-immunoreactive (-ir) neurons in the petrosal ganglion projected to the carotid sinus nerve. Approximately 4% of FG-labeled neurons contained TH-ir and were predominantly found in the caudal portion of the petrosal ganglion. Nearly 90% of total TH-ir neurons in the petrosal ganglion were labeled with FG. Less than 1% of FG-labeled neurons were immunoreactive for VIP in this ganglion. In the petrosal ganglion, 25% of FG-labeled neurons contained CGRP-ir, and 16.7% of FG-labeled neurons contained SP-ir. 30% of CGRP-ir or SP-ir neurons in the petrosal ganglion were labeled with FG. In the jugular ganglion, no TH- or VIP-ir neurons projected to the carotid sinus nerve and only small populations of CGRP- or SP-ir neurons projected to the carotid sinus nerve. Many FG-labeled and GAL-ir neurons were observed in the petrosal and jugular ganglia. The double-immunofluorescence method revealed the coexistence of CGRP- and SP-ir in carotid sinus nerve-projecting neurons in the petrosal and jugular ganglia. Likewise, GAL-ir coexisted with CGRP- and SP-ir in these ganglionic neurons. There was no coexistence of TH-ir and VIP-ir in carotid sinus nerve projections. The present study demonstrates the presence of multiple putative transmitters in baro- and chemoreceptor afferent neurons of the carotid sinus nerve. These neurochemicals are likely to contribute to transmission of signals from the carotid body and carotid sinus to neurons of the brainstem.     

Expression of D2 dopamine receptor mRNA in the arterial chemoreceptor afferent pathway.

Dopamine is a major neurotransmitter in the arterial chemoreceptor pathway. In the present study we wished to determine if messenger RNAs for dopamine D1 and D2 receptor are expressed in carotid body (type I cells), in sensory neurons of the petrosal ganglion which innervate the carotid body and in sympathetic neurons of the superior cervical ganglion. We failed to detect D1 receptor mRNA in any of these tissues. However, we found that D2 receptor mRNA was expressed by dopaminergic carotid body type I cells. D2 receptor mRNA was also found in petrosal ganglion neurons that innervated the carotid sinus and carotid body. In addition, a large number of sympathetic postganglionic neurons in the superior cervical ganglion expressed D2 receptor mRNA.     

Bioelectric potentials in the carotid body

Simultaneous recordings of focal slow potentials (sVs) and chemosensory discharges were made from cat carotid body-nerve preparations in situ. Chemoreceptor stimulants (100% N2, asphyxia, NaCN, ACh and nicotine), and depressants (100% O2, spontaneous gasps and dopamine) changed receptor polarization. sVs evoked by stimulants had a negative polarity whereas depressants elicited positive deflections. There was a direct correlation between maximal frequency of chemosensory discharges and peak sV amplitude when NaCN injections or N2 inhalation were used. However, cholinergic agents, dopamine and substance P evoked sVs which lacked correlation in time-course, amplitude or polarity with changes in sensory frequency. After a 6-day carotid nerve crush, different stimuli still evoked sVs even in the absence of sensory discharges. Both sVs and chemosensory discharges were abolished after 1 h ischemia produced by ligature of carotid body blood vessels. Thus, sVs from carotid body chemoreceptors probably include a neuronal component (the generator potential) directly responsible for the origin of chemosensory discharges, and a non-neuronal component (receptor or secretory potentials) probably originating in glomus and/or sustentacular cells.