The tail-elicited tail withdrawal reflex of Aplysia is mediated centrally at tail sensory-motor synapses and exhibits sensitization across multiple temporal domains

Abstract

The defensive withdrawal reflexes of Aplysia californica have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the tail-elicited tail withdrawal reflex (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have identified the induction requirements and molecular basis of different temporal phases of synaptic facilitation that underlie sensitization in this system. They have also permitted more recent studies elucidating the role of synaptic and nuclear signaling during synaptic facilitation. Here we report the development of a novel, compartmentalized semi-intact T-TWR preparation that allows examination of the unique contributions of processing in the SN somatic compartment (the pleural ganglion) and the SN-MN synaptic compartment (the pedal ganglion) during the induction of sensitization. Using this preparation we find that the T-TWR is mediated entirely by central connections in the synaptic compartment. Moreover, the reflex is stably expressed for at least 24 h, and can be modified by tail shocks that induce sensitization across multiple temporal domains, as well as direct application of the modulatory neurotransmitter serotonin. This preparation now provides an experimentally powerful system in which to directly examine the unique and combined roles of synaptic and nuclear signaling in different temporal domains of memory formation.

Figure 1.

Synaptic activity in the pedal but not the pleural CNS compartment is required for behavioral expression in the “split-bath” tail-elicited tail withdrawal reflex (T-TWR) reduced behavioral preparation. (A) Diagram of an intact Aplysia overlaid with critical CNS reflex circuitry (red). The ipsilateral pleural and pedal ganglia, tail nerve (P9), and exterior tissues (dark gray; a = training site, b = reflex initiation site, and c = withdrawal response output) preserved through surgery to produce the semi-intact behavioral preparation. (B) Arrangement within the experimental chamber of the training site, reflex initiation (test), and behavioral output sites of the isolated tail attached through a single P9 nerve to the CNS in an inner isolated chamber, and (C) the main circuit elements of the preserved pleural (SN soma) and pedal (SN-MN synapse, MN cell body) compartments. SN = sensory neuron, MN = motor neuron, 5HT = serotonergic neurons. (D) Bath exchange of artificial seawater (ASW) for high Mg²⁺ ASW (Mg-ASW) in the pedal chamber (containing the tail SN-MN synapses) reversibly blocks the T-TWR, which recovers when ASW is reintroduced (top). Bath exchange with Mg-ASW in the pleural chamber (containing the tail SN cell bodies, but not the tail SN-MN synapse) does not disrupt the tail withdrawal response (bottom). T-TWR traces were obtained using a force transducer which tracked tension (in grams of force) on the tail across time.

Figure 2.

The T-TWR remains stable across 24 h. (A) Representative traces and (B) group averages of the mean of three tests administered 24 h after determination of baseline T-TWR (n = 6). Data are presented as mean ± SEM. Duration (Dur), amplitude (Amp), time to peak amplitude (Tpk).

Figure 3.

A single training shock induces a transient sensitization of the T-TWR. A single 1.5-sec shock induces a short-term sensitization at 10 min that is absent in subsequent post-tests at 30 min, 1 h, and 24 h. (A) Representative traces of animals receiving a single shock and tested at 10 min, 30 min, or 1 h and at 24 h, and (B) summary data. Asterisk (*) indicates P < 0.05.

Figure 4.

Two spaced training shocks (ISI = 45 min) induce intermediate- and long-lasting sensitization of the T-TWR. The T-TWR shows a similar sensitivity to trial number as previously described for the T-SWR reflex. (A) Representative traces from a single animal tested across all time points, and (B) summary data. Asterisk (*) indicates P < 0.05.

Figure 5.

Electric shocks to the reduced T-TWR preparation trigger immediate 5HT release within the vicinity of tail SN cell bodies and SN-MN synapses. (A) 5HT release was measured with carbon fiber electrodes inserted into the CNS near the tail SN cell bodies (pleural recording site) or at tail SN-MN synapses (pedal recording site). (B) Representative traces of 5HT release detected near the tail SN cell bodies (pleural release) and tail SN-MN synapses (pedal release) immediately after a 1.5-sec 100-mA shock to the training site. A stimulus artifact, caused by introduction of the shocking electrode to the preparations and the shock itself, has been removed in the representative traces.

Figure 6.

A single pulse of 5HT to the CNS results in a transient sensitization of the T-TWR, while multiple 5HT pulses induce intermediate- and long-term sensitization. Sample T-TWR recordings and summary data showing response amplitude and duration after a single 5-min pulse of 5HT (A) and five 5-min pulses of 5HT (B). Ten minutes after a single pulse of 5HT, or after the first of five pulses of 5HT (data from these two groups were combined for the 10-min analysis), the T-TWR showed an increase in amplitude and shortening of duration that was not present at 1 h or 24 h. After repeated 5HT pulses, the T-TWR expressed sensitization in the form of enhanced response amplitude and lengthened duration at 1 h and 24 h after 5HT. Asterisk (*) indicates P < 0.05.