CELL-CELL COUPLING OCCURS IN DORSAL MEDULLARY NEURONS AFTER MINIMIZING ANATOMICAL-COUPLING ARTIFACTS

Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to colle ctively as anatomical coupling, were identified in 20% of the solitary complex neurons rested in medullary tissue slices (120-350 mu m) prep ared from rat, postnatal day 1-18, using a modified amphotericin B-per forated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fif ty-eight per cent of anatomically coupled neurons exhibited electroton ic postsynaptic potential-like activity, which had peak-to-peak amplit udes of less than or equal to 7 mV, with the same polarity as action p otentials; increased and decreased in frequency during depolarizing an d hyperpolarizing current injection; was maintained during high Mg2+-l ow Ca2+ chemical synaptic blockade; and was measured only in anatomica lly coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lu cifer Yellow, Biocytin and ionic current used the same pathways of int ercellular communication, which were presumed to be gap junctions. Ana tomical coupling was attributed solely to the junctional transfer of L ucifer Yellow and Biocytin since potential sources of non-junctional s taining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip (less than or equa l to 3 mu m outer diameter) submerged in artificial cerebral spinal fl uid. Moreover, unintentional contact of the pipette tip with adjacent neurons than resulted in accidental staining, another source of non-ju nctional staining, was averted by continuously visualizing the tip pri or to tight seal formation with infrared video microscopy, used here f or the first time with Hoffman modulation contrast optics. During perf orated patch recording, which typically lasted for 1-3 h. Lucifer Yell ow was confined to the pipette, indicating that the amphotericin B pat ch was intact. However, once the patch was intentionally ruptured at t he end of recording, the viscous, lipophilic solution entired the neur on resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not comp romise tight seal formation with an exposed, cleaned soma, and resulte d in immediate (<1 min) steady-state perforation at 22-25 degrees C. T his adaptation of conventional perforated patch recording was termed ' 'rapid perforated patch recording''. The possible functional implicati on of cell-cell coupling in the dorsal medulla oblongata in central CO 2/H+ chemoreception for the cardiorespiratory control systems is discu ssed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57]. (C) 1997 IBRO. Published by Elsevier Science Ltd.