SYNAPSE FORMATION AND HYPOXIC SIGNALING IN COCULTURES OF RAT PETROSALNEURONS AND CAROTID-BODY TYPE-1 CELLS

1. To investigate synaptic mechanisms mediating chemosensory signallin g in the carotid body, we developed co-cultures of chemoreceptor type 1 cell clusters and dissociated petrosal neurones (PNs) from 7- to 14- day-old rat pups and tested for functional connectivity in CO2-HCO3--o r Hepes-buffered medium at similar to 35 degrees C. 2. When cultured w ithout type 1 cells, PNs were almost always quiescent (n = 104) and un responsive to hypoxia (P-O2 5-25 mmHg) during perforated patch, whole- cell recordings of membrane potential or voltage-activated currents, i n contrast, many PNs (77 out of 170) that were juxtaposed to type 1 ce ll clusters in co-culture displayed spontaneous activity, comprising s pikes and subthreshold potentials (SSPs) that resembled synaptic poten tials. 3. Additional tests suggested that de novo chemical synapses de veloped between PNs and type 1 cell clusters in vitro. For example: (i ) the spontaneous activity was reversibly suppressed by substituting l ow calcium-high magnesium in the bath; (ii) SSPs had variable amplitud es and persisted following action potential blockade with TTX (1 mu M) ; (iii) the interval distribution between successive spontaneous event s appeared random; and (iv) the frequency of spontaneous potentials wa s diminished (reversibly) by the nicotinic antagonist hexamethonium (1 00 mu M), suggesting contributions from the spontaneous release of ACh .4. Many complexes of 'juxtaposed' PNs and type 1 clusters were physio logically functional, since exposure to hypoxia caused a reversible de polarization and/or increased spike discharge in similar to 30% of suc h neurones (n = 140). The hypoxia-induced spike discharge persisted in the presence of the dopamine D-2 receptor blocker spiperone (10-50 mu M; n = 5); however, this discharge was reversibly inhibited by 100-20 0 mu M hexamethonium, suggesting that it was mediated, at least in par t, by ACh acting through nicotinic receptors. 5. The hypoxia-induced s pike discharge and frequency of spontaneous potentials in co-cultured PNs were reversibly suppressed when the buffer was switched from CO2-H CO3- to Hepes (10 mM) at pH 7.4; further; 'functional' PNs that displa yed spontaneous activity and/or hypoxia-induced responses in co-cultur e were encountered more frequently in CO2-HCO3- (greater than or equal to 40%) than in Hepes (less than or equal to 26%) buffer. 6. We concl ude that functional chemical synapses can develop de novo in cultures of carotid body type 1 cells and PNs and that ACh is probably an impor tant excitatory neurotransmitter secreted from type 1 cells during hyp oxic chemotransduction in the rat carotid body.