Hj. Zhong et al., SYNAPSE FORMATION AND HYPOXIC SIGNALING IN COCULTURES OF RAT PETROSALNEURONS AND CAROTID-BODY TYPE-1 CELLS, Journal of physiology, 503(3), 1997, pp. 599-612
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.