Figure 3.

The giant dopaminergic pedal neuron of Biomphalaria glabrata. (A) Intracellular recording from LPeD1 (lower trace) of B. glabrata; isolated CNS in normal saline solution. The recording exhibits alternating phases of activity and quiescence. A putative cardio-respiratory motor neuron in the visceral ganglion (VJ Mn, upper trace) was observed to burst out of phase with LPeD1. Calibration bars: 5 s, 20 mV. (B) Changing the bathing medium to a raised divalent saline solution (Hi-Di) eliminated polysynaptic signaling, enabling resolution of direct one-to-one inhibitory postsynaptic potential (IPSPs, upper record). Each IPSP occurred with a brief and constant latency following each LPeD1 impulse (lower record). Calibration bars = 1 s; 5 mV, upper record; 20 mV, lower record. (C) Pharmacological evidence for direct dopaminergic signaling from LPeD1 to VJ Mn. Control: Depolarizing current pulses were passed into LPeD1 producing sequentially greater number of impulses (lower record). The IPSPs produced in the VJ Mn became progressively larger as more impulses were stimulated. When the dopaminergic (D2) antagonist sulpiride was added to the solution (100 uM), the IPSPs were blocked. They returned following approximately 15 min Wash (right panel). Calibration bars = 0.5 s, 20 mV, 20 mV. (D-F) Morphological confirmation of LPeD1 and VJ Mn identity. (D) Neurobiotin was injected into LPeD1, a second neighboring cell in the pedal ganglion, and the VJ Mn. The three injected neurons were visualized with Avidin 546 (false color magenta). The axon of LPeD1 can be seen descending through the left pleural ganglion (arrow), while the neighboring cell projects to the contralateral pedal ganglion (arrowhead). (E) When the preparation was processed for TH-like immunoreactivity and viewed with avidin 488 (green), only the LPeD1 neuron and its descending axon (arrow) were labeled. (F) When the fill (D) and immunohistochemical (E) panels were overlaid, only the LPeD1 neuron and its descending axon (arrow) appear white (colocalization). The other injected cells, VJ Mn and the neighboring pedal neuron (arrowhead), appear magenta. Calibration bar = 50 μm, applies to D-F. Reprinted from Vallejo et al., 2014, J. Comp. Neurol. 522: 2532-2552, with permission from John Wiley & Sons, Inc.