Highly dissimilar behaviors mediated by a multifunctional network in the marine mollusk Tritonia diomedea

Abstract

Several motor networks have now been found to be multifunctional, in which one group of neurons participates in the generation of multiple behavioral motor programs. Not surprisingly, the behaviors involved are frequently closely related, often using the same or similar muscle groups. Here we describe an interneuronal network in the marine mollusk Tritonia diomedea that is involved in producing two highly dissimilar behaviors, rhythmic, muscle-based escape swimming and nonrhythmic, cilia-mediated crawling. Several observations support this conclusion. First, the dorsal swim interneurons (DSIs) of the swim central pattern generator (CPG) directly excite Pedal neuron 21 (Pd21) and Pd5, the only identified cilia-activating efferent neurons in Tritonia. Second, stimulation of a single DSI elicits beating of the foot cilia in semi-intact preparations and crawling in intact animal treadmill preparations. Third, the DSIs fire at an elevated rate for nearly 1 hr after a swim motor program, which correlates reasonably well with the period freely behaving animals were found to crawl after they swam. Fourth, silencing the tonically active DSIs after a swim motor program substantially reduces or eliminates ongoing cilia neuron firing, indicating that the DSIs are major contributors to the synaptic input driving these cells. Finally, all of the other swim CPG neurons also connect to the cilia neurons, most monosynaptically. Taken together, these observations indicate that the Tritonia swim CPG network participates in producing both escape swimming and crawling. Given the extreme differences between these behaviors---rhythmic versus tonic, muscular versus ciliary, and brief versus prolonged--these findings reveal a striking versatility for a small multifunctional network.

Fig. 1.

The multifunctional Tritonianetwork. A, Diagram of synaptic connections, showing the afferent neurons (S), pre-CPG interneurons (Tr1, DRI), swim CPG neurons (DSI, C2, VSI-A, VSI-B), swim flexion neurons (DFN-A, DFN-B, VFN), and the locomotion cilia neurons (Pd21, Pd5; (Willows et al.; 1973; Getting, 1983a; Frost and Katz, 1996a; Frost et al.; 2001). The swim CPG consists of just six cells on each side of the brain (3 DSIs, 1 C2, 1 VSI-A, and 1 VSI-B).Bars, Excitatory synapses; circles, inhibitory synapses; combinations of barsand circles, multicomponent synaptic potentials.Solid lines, Monosynaptic connections; dashed lines, polysynaptic pathways, with the intervening interneurons yet to be identified. B, Firing responses of three of the swim CPG neurons during a three-cycle swim motor program elicited by a 2 sec, 10 Hz stimulus to pedal nerve 3. Note that DSI firing continues long after the end of the motor program, whereas VSI-B and C2 are silent before and after.

Fig. 2.

DSI activity elicits crawling. This experiment used an intact animal treadmill electrophysiology preparation, in which ciliary locomotion caused a drum positioned beneath the animal's foot to rotate. Sufficient depolarizing current was injected into a single DSI to elicit several seconds of firing (bottom trace). This produced a period of crawling that outlasted the DSI train by tens of seconds (top trace). With the treadmill apparatus, crawling, which is steady and nonrhythmic, is transduced into an oscillating voltage signal. The distance between two consecutivepeaks represents 2 mm of locomotion. The maximum rate of treadmill turning corresponded to 1 mm/sec of locomotion.

Fig. 3.

The DSIs directly excite the Pd21 and Pd5 cilia neurons. A, A DSI train evoked by intracellular stimulation produced excitation of the contralateral Pd21 in normal saline. B, Same result for Pd5. In this case, the duration of DSI stimulation is indicated by a bar.C, DSI produced constant latency, one-for-one EPSPs in Pd21 in high divalent cation saline. D, Same result for Pd5. R, Right; L, left.

Fig. 4.

DSI firing correlates with crawling and cilia neuron activity. A, Duration of postswim crawling in freely behaving animals. Twenty animals were transferred to a test arena, and their crawling was assessed once every 5 min for 6 hr. Eachpoint represents the mean of the six determinations of how many were crawling during the corresponding 30 min period. The transfer itself stimulated crawling, which declined over the first 3 hr. At that point, all animals were made to swim (time 0). For statistical analysis, each of the six postswim means was compared with the final pretest mean. The swim was followed by significantly enhanced crawling lasting 90 min. The dotted boxes in this and the subsequent graphs represent the duration of the significant effect. At 5 min after the swim, all animals that had their foot in contact with the substrate were crawling (13 of 13 animals). B, Duration of enhanced DSI firing after a swim motor program in the isolated brain. The motor program was elicited at time 0. DSI firing was significantly enhanced for 55 min (14 cells, 10 preparations).C, Duration of enhanced Pd21 firing. Pd21 firing was significantly enhanced for 75 min (8 cells, 7 preparations).D, Duration of enhanced Pd5 firing. Pd5 firing was significantly enhanced for 35 min (7 cells, 4 preparations).E, Example of DSI and Pd21 firing before and after a nerve stimulus-elicited swim motor program. Arrows inE and F indicate the time of the nerve stimulus. The individual cycles of the motor program cannot be clearly seen in the DSI trace at this time base but are visible as voltage oscillations in Pd21. F, Example of DSI and Pd5 firing before and after a nerve stimulus-elicited swim motor program. The statistics for B–D were only applied to the cells (numbers listed in Results) that were recorded for the full period shown on the graphs.

Fig. 5.

Tonic DSI firing actively maintains the elevated cilia neuron firing that follows the swim motor program. A, After a swim motor program (data not shown), the DSIs and cilia neurons fire at an elevated tonic rate. Hyperpolarizing two of the three contralateral DSIs during this period acted to eliminate the tonic firing in Pd21.B, In a similar experiment, hyperpolarizing all three contralateral DSIs repeatedly reduced or eliminated the elevated tonic firing in Pd5. R, Right; L, left.

Fig. 6.

Stimuli subthreshold for producing the swim nonetheless cause long-lasting DSI firing. A weak nerve stimulus administered to PdN3 at the arrow (10 Hz, 2 sec) elevated the rate of spontaneous DSI firing for at least 10 min.

Fig. 7.

CPG neuron VSI-A monosynaptically inhibits the cilia neurons. A, VSI-A action potentials elicited unitary, one-for-one IPSPs in the contralateral Pd5 in high divalent cation saline. The third current pulse caused two action potentials and two corresponding IPSPs in Pd5. B, In high divalent cation saline, a train of VSI-A spikes elicited one-for-one, summating IPSPs in the contralateral Pd21. C, The inhibitory VSI-A connection to Pd21 was sufficient to inhibit P21 firing in normal saline. VSI-A was driven via the intracellular electrode to fire two trains of action potentials. The slight depolarization of Pd21 during the inhibition was attributable to a reversal of the IPSP, which had initially been hyperpolarizing when Pd21 was penetrated.R, Right; L, left.

Fig. 8.

CPG neuron VSI-B polysynaptically inhibits Pd21.A, Driving VSI-B caused a decrease in the firing rate of the contralateral Pd21. B, In high divalent cation saline, a train of VSI-B spikes recruited IPSPs into Pd21 in a non-one-for-one manner, indicating that this inhibitory connection is indirect. L, Left; R, right.

Fig. 9.

Response of DSI and VSI-B to tactile skin stimulation. Poking the skin with a glass probe (arrow) elicited action potentials in VSI-B and inhibition in DSI.