Ganglionic amplification as assessed using intact ganglia and dynamic clamp in cell culture

Postganglionic sympathetic neurons have long been regarded as simple relays that transmit activity of their strong, supra-threshold input in a 1:1 fashion. Work from our laboratory has challenged this view, arguing that weak, sub-threshold synapses generate additional output via summation or their strengthening through metabotropic modulation of membrane excitability. Thus, sympathetic ganglia act as use-dependent amplifiers, increasing the gain of autonomic feedback loops. Recent microelectrode recordings from intact ganglia in-vitro and in-vivo indicate the presence of a third type of synapses with a strength that straddles threshold for AP-generation. Strengthening of these synapses by metabotropic modulation would largely impact ganglionic amplification. However, as microelectrodes create a shunting membrane leak, straddling synapses could be a recording artifact. Using a collagenase treatment to make neurons accessible for patch clamp electrodes, we have performed whole-cell recordings from intact superior cervical ganglion (SCG) in-vitro. These tight seal recordings confirm the presence of straddling synapses. Furthermore, the tonic firing pattern seen in most cells upon positive and the prominent sag seen upon negative current injection resemble whole-cell and perforated patch recordings from dissociated SCG neurons. Adding a leak conductance to dissociated SCG neurons using the dynamic clamp technique linearized the I-V relationship and resulted in a phasic firig pattern as seen with microelectrode recordings from intact ganglia. This suggests that microelectrode damage masks gain in-vivo. Finally, measuring the underlying synaptic currents in voltage clamp mode during whole-cell recordings from intact SCG allowed an assessment of the strength of weak, straddling and strong synapses. Together, this provides a framework for using dissociated SCG neurons and the dynamic clamp technique implementing virtual nicotinic synapses to study ganglionic amplfication with natural patterns of pre-ganglionic input and its modulation by metabotropic effects. This work was supported by NIH and by the University of Pittsburgh School of Medicine.