Contingent-dependent enhancement of rhythmic motor patterns: an in vitro analog of operant conditioning

Abstract

Operant conditioning is characterized by the contingent reinforcement of a designated behavior. Previously, feeding behavior in Aplysia has been demonstrated to be modified by operant conditioning, and a neural pathway (esophageal nerve; E n.) that mediates some aspects of reinforcement has been identified. As a first step toward a cellular analysis of operant conditioning, we developed an in vitro buccal ganglia preparation that expressed the essential features of operant conditioning. Motor patterns that represented at least two different aspects of fictive feeding (i.e., ingestion-like and rejection-like motor patterns) were elicited by tonic stimulation of a peripheral buccal nerve (n.2,3). Three groups of preparations were examined. In a contingent-reinforcement group, stimulation of E n. was contingent on the expression of a specific type of motor pattern (i.e., either ingestion-like or rejection-like). In a yoke-control group, stimulation of E n. was not contingent on any specific pattern. In a control group, E n. was not stimulated. The frequency of the reinforced pattern increased significantly only in the contingent-reinforcement group. No changes were observed in nonreinforced patterns or in the motor patterns of the control and yoke-control groups. Contingent reinforcement of the ingestion-like pattern was associated with an enhancement of activity in motor neuron B8, and this enhancement was specific to the reinforced pattern. These results suggest that the isolated buccal ganglia expressed an essential feature of operant conditioning (i.e., contingent reinforcement modified a designated operant) and that this analog of operant conditioning is accessible to cellular analysis.

Fig. 1.

The buccal ganglia and its peripheral nerves. A, Schematic representation of the buccal mass and the location of the buccal ganglia and its peripheral nerves. B, Schematic representation of in vitro buccal ganglia preparation showing the position of the recording electrodes (white triangles) on I2 n., n.2,1, and R n.1 and the stimulating electrodes (black triangles) on E n.2 and n.2,3. This schematic illustrates the placement of electrodes that was used in nondesheathed preparations (i.e., the E n.2 that was stimulated was ipsilateral to the nerves from which recordings were made). In desheathed preparations the E n.2 that was contralateral to the ganglion from which recordings were made was stimulated (data not shown). C-B conn., Cerebrobuccal connectives; E n., esophageal nerve; I2 n., nerve to intrinsic buccal muscle 2; n., buccal nerve; R n., radular nerve.

Fig. 3.

Identification of neurons contributing to activity in I2 n., R n.1, and n.2,1. A, B, Simultaneous extra- and intracellular recordings during patterned activity elicited by stimulation of n.2,3. Cell B31/32 fires in phase with I2 n., cell B9 fires in phase with activity in n.2,1 (A), and cell B8 fires in phase with the large unit activity in R n.1 (B).C, A one-for-one relationship between intracellularly recorded action potentials from B31/32 and extracellular activity recorded from I2 n. (circle, C1) and antidromically activated action potentials in B31/32 elicited by stimulation of I2 n. C2 demonstrated thatB31/32 projects through I2 n. Four oscilloscope traces triggered by intrasomatic action potentials recorded fromB31/32 (C1) and six traces triggered by nerve stimulation (C2) were superimposed.D, A similar study as in C indicated that neuronB9 sends axons in both ipsilateral (i) and contralateral (c) n.2,1 (n.2,1i, n.2,1c;circles in D1 indicate the time-locked extracellular action potentials). Six oscilloscope traces triggered by intrasomatic action potentials recorded from B9(D1) and five traces triggered by nerve stimulation (D2) were superimposed. E, CellB8 projects bilaterally through the ipsilateral and contralateral R n.1 (R n.1i, R n.1c;circles in E1 indicate the time-locked extracellular action potentials). Five oscilloscope traces triggered by intrasomatic action potentials recorded from B8(E1) and four traces triggered by nerve stimulation (E2) were superimposed.

Fig. 2.

Both patterns I and II were elicited by tonic stimulation of n.2,3. A, In both patterns I and II, a burst of spikes in I2 n. preceded a burst of spikes in n.2,1. Pattern I was defined as one in which 50% or more of the large-amplitude activity in the R n.1 occurred after the end of the I2 n. burst (dashed line). In pattern II, large-amplitude activity in R n.1 (black arrow), which can be distinguished from small-amplitude activity (white arrow), does not extend beyond the burst in I2 n. (dashed line). These examples of pattern I and II were recorded from the same preparation. An artifact of the tonic stimulation of n.2,3 appears in I2 n. and n.2,1 traces. B, The average phase relationship of activity in I2 n. (black), n.2,1 (gray), and the large-amplitude R n.1 activity (white) in pattern I (n = 46) and pattern II (n = 8) recorded during the test period in the 10 nondesheathed control preparations (see Fig. 4). The key distinguishing feature of patterns I and II was the duration of large-amplitude activity in R n.1 that extended beyond the termination of the I2 n. phase. In this and subsequent figures, the bars indicate the mean values ± SEM.

Fig. 4.

Experimental paradigms for neural analog of operant conditioning. A, In all paradigms, tonic stimulation of n.2,3 (n.2,3 stim.) was delivered throughout the experiment. The type of patterned activity induced by stimulation of n.2,3 was represented by black circles(pattern I) or by white circles (pattern II and intermediate patterns). Experiments were divided into three periods: a pretraining period (Pre-Training), a 10 min training period (Training), and a 10 min test period (Test), which immediately followed the training period. In a single block of three matched preparations, each preparation received one of the different stimulus paradigms (i.e.,Contingent Reinforcement, B; Yoke Control, C; Control, D). B, Contingent reinforcement. During the training period phasic (10 Hz, 6 sec) stimulation of E n.2 (black squares on E n.2 stim.) was delivered immediately after expression of each pattern I (black circles). In an experimental block the beginning of the training period was determined by the first occurrence of a pattern I and the contingent stimulation of E n.2.C, Yoke control. Stimulation of E n.2 (black squares in E n.2 stim.) was applied with the same parameters and the same timing as that in the contingent stimulation paradigm (compare E n.2 stim. with that inB). In this paradigm, however, E n.2 stimulation was not contingent with any specific pattern; rather, it was “yoked” to the previous contingent-stimulation preparation in the block.D, Control. In this paradigm, no stimulation of E n.2 was delivered.

Fig. 5.

Representative recordings of rhythmic activity during the test period. The patterned activity (•, pattern I; ○, other patterns) recorded from I2 n., R n.1, and n.2,1 in nondesheathed preparations during a 10 min test period in a control (A), a contingent-reinforcement (B), and a yoke-control (C) preparation. The frequency of patterned activity was enhanced after contingent reinforcement, as compared with the control and yoke-control preparations.

Fig. 6.

Contingent reinforcement increased the frequency of the rhythmic activity. Statistical comparison of the number of patterns expressed during the 10 min test period in the control (white bar), in the contingent-reinforcement (black bar), and in the yoke-control (gray bar) groups from both nondesheathed and desheathed preparations (n = 20 in each group). A significantly higher frequency of rhythmic activity was expressed in the contingent-reinforcement group, as compared with the control (p < 0.001) or yoke-control groups (p < 0.001). This effect resulted from the contingency of the reinforcement because no significant difference (N.S.) was observed between the yoke-control and the control groups.

Fig. 7.

Only the reinforced pattern of activity was increased. A, Selective increase of pattern I. In both nondesheathed and desheathed preparations during a 10 min test period immediately after the training session in which stimulation of E n.2 was contingent on pattern I, the number of occurrences of pattern I was increased significantly in the contingent-reinforcement group (black bar), as compared with the control (white bar; p < 0.001) or the yoke-control groups (gray bar;p < 0.001), and no significant difference (N.S.) was observed between the control and yoke-control groups (A1). In contrast, in the same preparations and during the same test period the number of occurrences of other patterns (i.e., the nonreinforced patterns: pattern II and intermediate patterns) was not significantly different (N.S.) among the groups (A2). B, Selective increase of pattern II. In nondesheathed preparations contingent reinforcement of pattern II increased the number of occurrences of pattern II during the 10 min test period in the contingent-reinforcement group (black bar), as compared with the control (white bar; p < 0.05) or the yoke-control groups (gray bar; p< 0.005). No significant difference (N.S.) was observed between the control and yoke-control groups (B1), but in the same preparations and during the same test period the number of occurrences of the other patterns (i.e., the nonreinforced patterns: pattern I and intermediate patterns) was not significantly different among the groups (B2).

Fig. 8.

Long-lasting effects of the contingent reinforcement. During a 10 min test period that began 1 hr after the training session, the contingent-reinforcement group (black bar) expressed a significantly greater number of occurrences of the reinforced pattern (pattern I, A), but not of the nonreinforced patterns (pattern II and intermediate, B), than in the control (white bar;p < 0.001) and the yoke-control groups (gray bar; p < 0.05). The effects of the contingent reinforcement persisted for >1 hr. Five nondesheathed preparations in each group were used.

Fig. 9.

Neural modification induced by contingent reinforcement. A, The phase relationship of the discharge in the closure motor neuron B8 relative to the burst of activity in I2 n. can vary from one pattern to another. In some B8 bursts at least 50% of the activity occurred after termination of burst in I2 n. In this case the majority of activity in B8 was out of phase with activity in I2 n. B, The number of occurrences of bursts in B8 characterized in A and that were associated with expression of pattern I significantly increased in the contingent-reinforcement group (black bar), as compared with the control (white bar;p < 0.001) and yoke-control groups (gray bar; p < 0.05). No significant difference (N.S.) was observed between the control and the yoke-control groups. The contingent reinforcement specifically enhanced the discharge of B8 during the retraction phase (i.e., after the burst in I2 n. terminated).