Acetylcholine contributes to hypoxic chemotransmission in co-cultures of rat type 1 cells and petrosal neurons

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Abstract

The neurotransmitter mechanisms that mediate chemosensory transmission in the mammalian carotid body (CB), i.e. the primary arterial PO2 detector, are controversial. Given the inherent difficulty of recording from afferent terminals in situ, the authors have adopted an alternative approach based on co-culture of dissociated CB receptor (type 1) cell clusters and petrosal neurons (PN) from 8–14-day-old rat pups. Electrophysiological, perforated patch recordings from petrosal somas, juxtaposed to type 1 clusters, revealed the development of a high incidence of functional ‘synapses’ in vitro. Recent evidence has strengthened the case for acetylcholine (ACh) as a co-released transmitter: (i) cultured type 1 cells express several cholinergic markers including the vesicular ACh transporter (VAChT), intracellular acetylcholinesterase (AChE), and occasional clear cored vesicles (≈50 nm diameter); (ii) the frequency of spontaneous synaptic activity, as well as the hypoxia-induced depolarization recorded in ‘juxtaposed’ PN in co-culture, were partially suppressed by the nicotinic ACh receptor (nAChR) blocker, mecamylamine (2 μM); (iii) consistent with the presence of extracellular AChE, ACh-mediated membrane noise in type 1 cells as well as the hypoxia-evoked PN response in co-culture were potentiated in a few cases by the AChE inhibitor, eserine (100 μM). Thus, since many PN and type 1 cells express mecamylamine-sensitive nAChR, released ACh may act presynaptically on type 1 cell autoreceptors and/or postsynaptically on petrosal terminals. Other CB transmitter candidates (e.g. 5-HT and ATP) were found to excite PN, though their potential role as co-released sensory transmitters requires further investigation.

Introduction

The synaptic mechanisms that mediate the carotid body (CB) chemoexcitatory response to hypoxia have been a controversial subject for over 50 years. While there appears to be a consensus that CB type 1 cells are the actual sensory transducers during hypoxaemia (González et al., 1994, López-Barneo, 1996, see however Sun and Reis, 1994), the nature of the excitatory transmitter(s) that translate type 1 cell depolarization into a train of action potentials in carotid sinus nerve (CSN) remains arguable. Supportive evidence for acetylcholine (ACh) as a major excitatory transmitter in CB chemosensory signalling emerged and accumulated since the 1930s (Schweitzer and Wright, 1938, Hollinshead and Sawyer, 1945, Eyzaguirre and Zapata, 1968). However, this idea was challenged in the intervening years (Douglas, 1954, Sampson, 1971, McQueen, 1977), only to undergo a recent resurgence (Fitzgerald et al., 1995, Fitzgerald et al., 1997, Zhong et al., 1997). Other neurotransmitter candidates, including dopamine and substance P, have also been proposed as mediators of CB chemoexcitation (Prabhakar et al., 1993, González et al., 1994, Prabhakar, 1994).

The most direct resolution to this problem requires the recording of synaptic events from sinus nerve terminals opposed to type 1 receptor cells in situ during hypoxic chemotransduction. This approach has met with limited success due to access difficulties, poor visibility, and unstable recording conditions (Hayashida et al., 1980). Alternative approaches which rely solely on recording of extracellular CSN activity suffer from the disadvantage that subthreshold synaptic events are not recorded, thereby precluding a clear interpretation of the mechanisms mediating chemical transmission (Katz, 1969). The authors have had recent success with a compromise approach, based on co-culture of dispersed rat CB type 1 cell clusters and dissociated petrosal ganglia (Zhong et al., 1997), which contain the cell bodies of chemoafferent CSN fibers (McDonald, 1981, Finley et al., 1992). In these co-cultures, subthreshold events can be recorded from a substantial proportion of neurons located close to a type 1 cell cluster, with which they appear to form de novo chemical synapses. Most importantly, at some of these complexes consisting of a type 1 cluster and a juxtaposed neuron, a hypoxic stimulus is transduced in the type 1 cells and the information relayed to the neuron as a depolarization and/or increased action potential frequency (Zhong et al., 1997).

Here, the potential usefulness of this co-culture preparation as a model system to aid resolution of the ‘transmitter question’ in the carotid body is reviewed, and additional evidence in support of ACh as an excitatory co-transmitter in chemosensory signalling is provided.

Section snippets

Culture techniques and electrophysiology

The procedures for obtaining cultures of dispersed type 1 cell clusters, with and without petrosal neurons (PN) from 8–14-day-old rat pups have been described in detail elsewhere (Nurse, 1987, Nurse, 1990, Stea and Nurse, 1992, Zhong et al., 1997). Recordings of membrane potential under current clamp (zero current) conditions were carried out with the nystatin perforated-patch technique to minimize disturbance of the intracellular milieu, as previously described (Zhong and Nurse, 1997a, Zhong

Results

The most widely accepted markers for type 1 or glomus cells relate to their adrenergic phenotype, characterized by the expression of catecholaminergic enzymes (e.g. tyrosine hydroxylase), catecholamine histofluorescence, and the presence of large cytoplasmic dense cored vesicles (McDonald, 1981, González et al., 1994). However, these cells appear to express a broad transmitter repertoire, including several biogenic amines and neuropeptides (Wang et al., 1992, González et al., 1994, Prabhakar,

Discussion

In this communication the authors review and extend the evidence for ACh as a major carotid body excitatory co-transmitter, released from type 1 receptor cells, during chemosensory signalling. In reviewing this evidence, it is instructive to ask at the outset whether ACh meets the requirements for a ‘classical’ transmitter in the carotid body, analogous to its established role at the skeletal neuromuscular junction (Katz, 1969). A first requirement, that type 1 cells possess the ability to

Acknowledgements

The authors are indebted to the expert technical assistance of Cathy Vollmer for preparing and maintaining the cultures and for carrying out the immunofluorescence procedures. The authors thank Bert Visheau for help with the electron microscopy, and Dr Huijun Zhong for his participation in the earlier experiments. This work was supported by the Medical Research Council of Canada.

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