Electrophysiology and transmitter sensitivities of isolated rat petrosal neurons: Synapse formation and hypoxic signaling in co-culture with carotid body chemoreceptors.

摘要

The mammalian carotid body (CB), located at the carotid bifurcation, is a peripheral chemosensory organ which senses arterial Po{dollar}sb2{dollar}, Pco{dollar}sb2{dollar} and pH, and controls breathing via reflex responses. Chemoreceptor (glomus) cells in the CB contain various neurotransmitters which are released in response to chemosensory stimuli onto petrosal sensory nerve endings, but the underlying synaptic mechanisms are unclear. In this study, a combination of electrophysiological and pharmacological techniques were used to characterize membrane currents and sensitivity of cultured rat petrosal neurons (PN) to putative CB neurotransmitters. Further, co-cultures of PN and glomus cells were used to test for de novo functional synapse formation, and to elucidate the neurotransmitter mechanisms that mediate hypoxic signaling.; Conventional whole-cell and perforated patch recordings revealed that many PN contained, in addition to previously-characterized voltage-gated Na{dollar}sp+{dollar}, K{dollar}sp+{dollar}, and Ca{dollar}sp{lcub}2+{rcub}{dollar} currents, a cation non-selective inward rectifier (I{dollar}sb{lcub}rm h{rcub}){dollar} which was activated at hyperpolarized membrane potentials, and appears to regulate spike frequency during chemosensory signaling. A subpopulation of PN contained a fast inactivating outward current (I{dollar}rmsb{lcub}A{rcub}),{dollar} which also regulates spike frequency in neurons. The membrane properties of PN were largely unaffected by the natural CB stimulus, hypoxia (Po{dollar}sb2{dollar} = {dollar}sim{dollar}25 mm Hg). Acetylcholine (ACh), a putative CB neurotransmitter, was excitatory and depolarized {dollar}sim{dollar}68% of petrosal neurons (n = 109) via hexamethonium-sensitive, nicotinic ACh receptors (nAChR). 5-HT, another putative CB neurotransmitter, was also excitatory and depolarized {dollar}sim{dollar}43% of PN (n = 123), in association with a conductance increase; this response was mediated by MDL72222-sensitive 5-HT{dollar}sb3{dollar} receptors. More than one-half of CB glomus cells (n = 20) were also excited by 5-HT, but in contrast, this response was associated with a conductance decrease, and mediated by 5-HT{dollar}sb2{dollar} receptors. Dopamine depolarized approximately 30% PN (n = 69), but inhibited spike activity triggered by depolarizing stimuli (n = 4). Similarly, GABA also caused depolarization in {dollar}sim{dollar}84% of PN (n = 90), but inhibited spike activity via GABA{dollar}rmsb{lcub}A{rcub}{dollar} receptors.; In co-cultures of dissociated rat PN and CB cells, synaptic-like activity was recorded from some neurons that were juxtaposed to glomus cell clusters. This activity, absent in PN cultured alone (n = 104), was observed in 77 out of 170 co-cultured neurons, and comprised spikes and subthreshold potentials (SSP) that resembled postsynaptic potentials seen at chemical synapses. Moreover, and especially in HCO{dollar}sp-sb3{dollar}/CO{dollar}sb2{dollar}-buffered medium, many chemosensory complexes were functional, since a hypoxic stimulus (Po{dollar}sb2{dollar} = {dollar}sim{dollar}25 mm Hg) was transduced and relayed (as a depolarization and/or increased spike discharge) to {dollar}sim{dollar}30% 'juxtaposed' neurons (n = 140). These spike discharges and SSP were reversibly inhibited by 200 {dollar}mu{dollar}M hexamethonium (n = 12), suggesting that functional chemical synapses can develop de novo in co-culture and that ACh is likely an important excitatory neurotansmitter in CB chemoreception. Substance P (SP) increased spontaneous spike activity in co-cultured PN (21/36) via SP NK-1 receptors, and this may be related to its ability to modulate the transient I{dollar}rmsb{lcub}A{rcub}{dollar} current in these neurons. Thus, these co-cultures have provided new insights into the synaptic and neurotransmitter mechanisms underlying CB chemoreception during hypoxia.