Analysis of intrinsic connectivity in a multifunctional central pacemaker nucleus in vertebrates

Gymnotiform fish emit electric organ discharges (EODs) for both active electroreception and electrocommunication. In pulse-type species, EODs are generated by a hierarchical electromotor network controlled by a medullary pacemaker nucleus (PN), which comprises intrinsic pacemaker cells (PM-cells) and projecting relay cells (R-cells). Active electroreception requires the emission of stereotyped EODs, an electromotor output that implies a functional PN configuration based on the synchronous rhythmic discharge of PM-cells, a high-safety-factor feedforward transmission to R-cells, and the subsequent synchronous activation of the R-cell population. To evaluate if this functional organization implies an intrinsic PN connectivity grounded in electrical coupling (EC) between neurons, we examined the PN of Gymnotus omarorum in brainstem slices using electrophysiological recordings, immunohistochemical labeling, and dye-coupling analysis. Homotypic connections (PM-PM and R-R) exhibited low-magnitude, bidirectional EC with symmetrical, low-pass filter properties, supporting synchronous yet adaptable pacemaker activity and coordinated descending commands. Heterotypic connections (PM-R) also displayed bidirectional, symmetrical steady state coupling but revealed an apparent direction-dependent filtering behavior. During spontaneous PN activity, forward transmission of action potentials from PM- to R-cells appears to be favored over the propagation of slower signals, while retrograde R-to-PM transmission exhibits low-pass filter characteristics. Together with precise PM-to-R discharge timing, functional direction-dependent filtering suggests a role of PM-cell axons in shaping signal flow at heterotypic connections. Complementary dye-coupling and immunohistochemical evidence indicate that PN neurons are interconnected via gap junctions, likely formed by connexin 35, providing the structural substrate for the observed electrotonic interactions.