Synapse Formation and Hypoxic Signalling in Co-Cultures of Rat Petrosal Neurones and Carotid Body Type 1 Cells
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1. To investigate synaptic mechanisms mediating chemosensory signalling 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(-)-or Hepes-buffered medium at approximately 35 degrees C. 2. When cultured without type 1 cells, PNs were almost always quiescent (n = 104) and unresponsive to hypoxia (Po2 = 5-25 mmHg) during perforated patch, whole-cell recordings of membrane potential or voltage-activated currents; in contrast, many PNs (77 out of 170) that were juxtaposed to type 1 cell clusters in co-culture displayed spontaneous activity, comprising spikes and subthreshold potentials (SSPs) that resembled synaptic potentials. 3. Additional tests suggested that de novo chemical synapses developed between PNs and type 1 cell clusters in vitro. For example: (i) the spontaneous activity was reversibly suppressed by substituting low calcium-high magnesium in the bath; (ii) SSPs had variable amplitudes and persisted following action potential blockade with TTX (1 microM); (iii) the interval distribution between successive spontaneous events appeared random; and (iv) the frequency of spontaneous potentials was diminished (reversibly) by the nicotinic antagonist hexamethonium (100 microM), suggesting contributions from the spontaneous release of ACh. 4. Many complexes of 'juxtaposed' PNs and type 1 clusters were physiologically functional, since exposure to hypoxia caused a reversible depolarization and/or increased spike discharge in approximately 30% of such neurones (n = 140). The hypoxia-induced spike discharge persisted in the presence of the dopamine D2 receptor blocker spiperone (10-50 microM; n = 5); however, this discharge was reversibly inhibited by 100-200 microM hexamethonium, suggesting that it was mediated, at least in part, by ACh acting through nicotinic receptors. 5. The hypoxia-induced spike discharge and frequency of spontaneous potentials in co-cultured PNs were reversibly suppressed when the buffer was switched from CO2-HCO3- to Hepes (10 mM) at pH 7.4; further, 'functional' PNs that displayed spontaneous activity and/or hypoxia-induced responses in co-culture were encountered more frequently in CO2-HCO3- (> or = 40%) than in Hepes (< or = 26%) buffer. 6. We conclude that functional chemical synapses can develop de novo in cultures of carotid body type 1 cells and PNs and that ACh is probably an important excitatory neurotransmitter secreted from type 1 cells during hypoxic chemotransduction in the rat carotid body.
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