Synaptic mechanisms during re-innervation of rat arterial chemoreceptors in co-culture

The carotid body plays a key role in the control of ventilation during hypoxia, a stimulus that releases catecholamines and other neurotransmitters from chemoreceptor (type 1) cells. Using co-cultures of rat type 1 clusters and 'juxtaposed' petrosal neurons (JPN), we recently showed that hypoxic chemotransmission is mediated via co-release of ACh and ATP. Recordings from JPN at functional, regenerated 'synapses' in vitro revealed spontaneous activity consisting of random e.p.s.p.'s and/or action potentials. This activity depended on chemical transmission since it was inhibited by extracellular solutions containing low Ca2+/high Mg2+, or blockers of nicotinic (e.g. 1-2 microM mecamylamine) and/or P2 purinergic (suramin or reactive blue 2; 10-50 microM) receptors. These solutions also inhibited hypoxia-evoked responses in JPN. The newly formed 'synapses' appeared stable, allowing repeated demonstration of hypoxic chemotransmission in the same JPN after at least a approximately 24-h re-incubation period. Immunofluorescence studies in situ revealed positive staining of P2X2 and P2X3 purinoceptor subunits in chemoafferent nerve terminals, but not type 1 cells; in contrast, both elements were immunopositive for the synaptic vesicle antigen (SV2). These data further support a co-transmitter role for ATP and the involvement of heteromeric P2X2/P2X3 purinoceptors in carotid body function.