Rodrigo Varas

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.

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 microg-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 gamma-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.

Electrical and pharmacological properties of petrosal ganglion neurons that innervate the carotid body

The petrosal ganglion (PG) contains the somata of primary afferent neurons that innervate the chemoreceptor (glomus) cells in the carotid body (CB). The most accepted model of CB chemoreception states that natural stimuli trigger the release of one or more transmitters from glomus cells, which in turn acting on specific post-synaptic receptors increases the rate of discharge in the nerve endings of PG neurons. However, PG neurons that project to the CB represent only small fraction (roughly 20%) of the whole PG and their identification is not simple since their electrophysiological and pharmacological properties are not strikingly different as compared with other PG neurons, which project to the carotid sinus or the tongue. In addition, differences reported on the actions of putative transmitters on PG neurons may reflect true species differences. Nevertheless, some experimental strategies have contributed to identify and characterize the properties of PG neurons that innervate the CB. In this review, we examined the electrophysiological properties and pharmacological responses of PG neurons to putative CB excitatory transmitters, focusing on the methods of study and species differences. The evidences suggest that ACh and ATP play a major role in the fast excitatory transmission between glomus cells and chemosensory nerve endings in the cat, rat and rabbit. However, the role of other putative transmitters such as dopamine, 5-HT and GABA is less clear and depends on the specie studied.

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.

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.

Serotonin Receptors Expressed in Drosophila Mushroom Bodies Differentially Modulate Larval Locomotion

Drosophila melanogaster has been successfully used as a simple model to study the cellular and molecular mechanisms underlying behaviors, including the generation of motor programs. Thus, it has been shown that, as in vertebrates, CNS biogenic amines (BA) including serotonin (5HT) participate in motor control in Drosophila. Several evidence show that BA systems innervate an important association area in the insect brain previously associated to the planning and/or execution of motor programs, the Mushroom Bodies (MB). The main objective of this work is to evaluate the contribution of 5HT and its receptors expressed in MB to motor behavior in fly larva. Locomotion was evaluated using an automated tracking system, in Drosophila larvae (3rd-instar) exposed to drugs that affect the serotonergic neuronal transmission: alpha-methyl-L-dopa, MDMA and fluoxetine. In addition, animals expressing mutations in the 5HT biosynthetic enzymes or in any of the previously identified receptors for this amine (5HT1AR, 5HT1BR, 5HT2R and 5HT7R) were evaluated in their locomotion. Finally, RNAi directed to the Drosophila 5HT receptor transcripts were expressed in MB and the effect of this manipulation on motor behavior was assessed. Data obtained in the mutants and in animals exposed to the serotonergic drugs, suggest that 5HT systems are important regulators of motor programs in fly larvae. Studies carried out in animals pan-neuronally expressing the RNAi for each of the serotonergic receptors, support this idea and further suggest that CNS 5HT pathways play a role in motor control. Moreover, animals expressing an RNAi for 5HT1BR, 5HT2R and 5HT7R in MB show increased motor behavior, while no effect is observed when the RNAi for 5HT1AR is expressed in this region. Thus, our data suggest that CNS 5HT systems are involved in motor control, and that 5HT receptors expressed in MB differentially modulate motor programs in fly larvae.

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 alpha4 and alpha7 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 alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-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 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine >> acetylcholine approximately = cytisine >> 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

nAChR-induced octopamine release mediates the effect of nicotine on a startle response in Drosophila melanogaster.

Biogenic amines (BAs) play a central role in the generation of complex behaviors in vertebrates and invertebrates, including the fly Drosophila melanogaster. The comparative advantages of Drosophila as a genetic model to study the contribution of BAs to behaviors stumble upon the difficulty to access the fly brain to ask relevant physiological questions. For instance, it is not known whether the activation of nicotinic acetylcholine receptors (nAChRs) induces the release of BAs in fly brain, a phenomenon associated to several behaviors in vertebrates. Here, we describe a new preparation to study the efflux of BAs in the adult fly brain by in vitro chronoamperometry. Using this preparation we show that nAChR agonists including nicotine induce a fast, transient, dose‐dependent efflux of endogenous BAs, an effect mediated by α‐bungarotoxin‐sensitive nAChRs. By using different genetic tools we demonstrate that the BA whose efflux is induced by nAChR activation is octopamine (Oct). Furthermore, we show that the impairment of a mechanically induced startle response after nicotine exposure is not observed in flies deficient in Oct transmission. Thus, our data show that the efflux of BAs in Drosophila brain is increased by nAChR activation as in vertebrates, and that then AChR‐induced Oct release could have implications in a nicotine‐induced behavioral response.

Inhibition of rat carotid body glomus cells TASK-like channels by acute hypoxia is enhanced by chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) chemosensory responses to acute hypoxia. In spite of that, the primary molecular target of CIH in the CB remains unknown. A key step of the hypoxic response in the CB is the chemoreceptor cell depolarization elicited by the inhibition of K(+) channels. Thus, we tested the hypothesis that CIH potentiates the hypoxic-induced depolarization of rat CB chemoreceptor cells by enhancing the inhibition of a background K(+) TASK-like channel. Membrane potential, single channel and macroscopic currents were recorded in the presence of TEA and 4-aminopyridine in CB chemoreceptor cells isolated from adult rats exposed to CIH. The CIH treatment did not modify the resting membrane properties but the hypoxic-evoked depolarization increased by 2-fold. In addition, the hypoxic inhibition of the TASK-like channel current was larger and faster in glomus cells from CIH-treated animals. This novel effect of CIH may contribute to explain the enhancing effect of CIH on CB oxygen chemoreception.

Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.