Cat Petrosal Ganglion Research Articles

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 μM. ACh induces a fast, inactivating inward current, with a threshold between 10–50 μM, 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.

Effects of intermittent hypoxia on cat petrosal ganglion responses induced by acetylcholine, adenosine 5′-triphosphate and NaCN

Exposure to chronic intermittent hypoxia (CIH) for 4 days enhances the cat carotid body (CB) chemosensory responses to acute hypoxia. However, it is not known if CIH enhances the responses of the petrosal ganglion (PG) neurons that innervate the CB chemoreceptor cells. Accordingly, we studied the effects of the CB putative excitatory transmitter acetylcholine (ACh) and adenosine 5´-triphosphate (ATP), and the effects of citotoxic hypoxia (NaCN) applied to the isolated PG from cats exposed to CIH for 4 days. The dose-dependent curve parameters of the frequency of discharges evoked in the carotid sinus nerve by the application of ACh, ATP and NaCN to the isolated PG in control condition were not significantly modified in the CIH-treated cats. Present results suggest that CIH enhances the chemosensory responses to acute hypoxia acting primarily at the chemoreceptor cells, without major changes in the response of PG neurons evoked by the application of putative CB excitatory transmitters to their somata.

Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: Effects of cytisine and its bromo derivatives

Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although α4 and α7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing α4 or α7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and α-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5–10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 μM hexamethonium or 10 nM α-bungarotoxin. The order of potency of the agonists was 3-bromocytisine ≫ acetylcholine ≅ cytisine ≫ 5-bromocytisine, suggesting that homomeric α7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

Endothelins in the cat petrosal ganglion and carotid body: Effects and immunolocalization

In response to hypoxia, chemoreceptor cells of the carotid body (CB) release transmitters, which acting on the petrosal ganglion (PG) neuron terminals, increase the chemoafferent discharge. Additionally, vasoactive molecules produced within the CB may modulate hypoxic chemoreception by controlling blood flow and tissue PO 2. Endothelin-1 (ET-1) increases basal CB chemosensory discharges in situ, probably due to its vasoconstrictor action. However, the actions of ET-1 on PG neurons or its expression in the PG are not known. Using immunohistochemistry, we found that endothelin-like peptides are expressed in the cat PG and CB under normoxic conditions. Exogenous applications of ET-1 increased the chemosensory activity in the vascularly perfused CB but were ineffective on either the CB or PG superfused preparations, both of which are devoid of vascular control. Thus, our data indicate that the excitatory effect of ET-1 in the carotid chemoreceptor system appears to be mainly due to a vasoconstrictor effect in the CB blood vessels.

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.

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.

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 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.

Adenosine triphosphate-induced peripheral nerve discharges generated from the cat petrosal ganglion in vitro.

Since nucleotides have been postulated as transmitters between glomus cells and chemosensory nerve endings in the carotid body, we studied the effects of their application to the petrosal ganglion, where the perikarya of carotid (sinus) nerve are located. Cat petrosal ganglia were superfused in vitro, while electrical activities of their peripheral processes (carotid nerve and glossopharyngeal branch) were recorded simultaneously. Adenosine triphosphate (ATP) evoked dose-dependent bursts of impulses in carotid nerve, while those in glossopharyngeal branch were less intense and consistent. Adenosine monophosphate was less effective than ATP. ATP-induced carotid nerve responses presented no temporal desensitization and persisted after applying P(2Y) receptor blocker Reactive Blue 2 to the ganglion. The results indicate that ATP has an excitatory effect on the perikarya of the population of petrosal ganglion neurons projecting peripherally through the carotid nerve.

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 D 2 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.

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, Nω-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 μg–1 mg) increased the frequency of action potentials recorded from the CSN in a dose-dependent manner. SNP (10–50 μM) 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.

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.

The ionic basis of action potentials in petrosal ganglion cells of the cat.

The ionic conductances underlying the action potential and after-hyperpolarization of the cat petrosal ganglion neurones with myelinated axons in the carotid nerve were studied in vitro. Neurones were divided into two groups based on the presence or absence of an inflexion or hump on the spike falling phase. The application of tetrodotoxin (TTX, 3 X 10(-7)-3 X 10(-6) M) revealed the presence of a TTX-resistant component in spikes with a hump, which was abolished in Na+-free solution. The action potential without a hump was blocked by TTX. The spike hump decreased or was abolished when Ca2+-channel blockers (Mn2+, 3-4 mM or Co2+, 5 mM) or low-Ca2+ solutions (0.1-0.2 mM) were applied to the preparation. In neurones with a hump on the spike, regenerative responses were obtained in Na+-free, high-Ca2+ (8.8 mM) solution; these responses were antagonized by Mn2+, and their amplitude was proportional to the external Ca2+ concentration. It is concluded that the action potential with a hump was produced by an Na+ current, a part of which was TTX-resistant, and by a Ca2+ current which is responsible for the hump. Neurones without a hump had a TTX-sensitive Na+ spike. The spike with a hump was followed by a long-lasting after-hyperpolarization which reversed polarity at about -82 mV. During the hyperpolarization an increase in membrane conductance was observed. The amplitude and duration of the long hyperpolarizing potential decreased when Ca2+-channel blockers or low-Ca2+ solutions were applied. In Na+-free solution, regenerative responses were followed by a long hyperpolarization associated with an increase in membrane conductance. It is concluded that the long after-hyperpolarization is produced by activation of the Ca2+-dependent K+ conductance.