Successful and unsuccessful attempts to swallow in a reduced Aplysia preparation regulate feeding responses and produce memory at different neural sites

Abstract

Sensory feedback shapes ongoing behavior and may produce learning and memory. Motor responses to edible or inedible food in a reduced Aplysia preparation were examined to test how sensory feedback affects behavior and memory. Feeding patterns were initiated by applying a cholinomimetic onto the cerebral ganglion. Feedback from buccal muscles increased the response variability and response rate. Repeated application of the cholinomimetic caused decreased responses, expressed in part by lengthening protractions. Swallowing strips of "edible" food, which in intact animals induces learning that enhances ingestion, increased the response rate, and shortened the protraction length, reflecting more swallowing. Testing memory by repeating the procedure prevented the decrease in response rate observed with the cholinomimetic alone, and shortened protractions. Training with "inedible" food that in intact animals produces learning expressed by decreased responses caused lengthened protractions. Testing memory by repeating the procedure did not cause decreased responses or lengthened protractions. After training and testing with edible or inedible food, all preparations were exposed to the cholinomimetic alone. Preparations previously trained with edible food displayed memory expressed as decreased protraction length. Preparations previously trained with inedible food showed decreases in many response parameters. Memory for inedible food may arise in part via a postsynaptic decrease in response to acetylcholine released by afferents sensing food. The lack of change in response number, and in the time that responses are maintained during the two training sessions preceding application of the cholinomimetic alone suggests that memory expression may differ from behavioral changes during training.

Figure 1.

Changes in patterning of feeding responses as a result of the buccal mass remaining attached to the buccal and cerebral ganglia. Examples of fictive feeding induced by CCh applied to the cerebral ganglion in: (A) a preparation in which the buccal muscles were not present, and (B) a preparation in which the buccal muscles remained attached to the buccal ganglia. The records shown are portions of longer recordings, and were chosen to display the patterning and rate of responses during a 200 sec interval at the peak of responses to CCh (A—200–400 sec after application of CCh; B—270–470 sec after application of CCh). (I2) EMG recordings from the I2 muscle, (Rad N) recording from the Radula Nerve, (BN2) recording from the right Buccal Nerve 2, (BN3) recording from the right Buccal Nerve 3. In addition to an increase in the rate at which CCh generates fictive feeding, attachment of the muscle also increases the variability of the feeding bursts that are elicited. In the isolated ganglia, note the relatively fixed lengths of I2 and BN2 bursts, which mark protraction and retraction phases, respectively, and the overlap between Rad activity and BN2, which has been used as an indicator of ingestion activity. By contrast, when the muscle is attached, note the variability in the lengths of I2 and BN2 activities, and the change in the position of Rad activity from an overlap with I2 in the first half of the record to an overlap with BN2 in the latter half.

Figure 2.

Parameters of motor responses induced by CCh with and without the buccal musculature. (A–C) Summary data comparing response parameters in the presence and absence of the buccal musculature. Asterisks mark significant differences. Data on bursting in the absence of the buccal muscles are from the first of five repetitions with CCh applied to the cerebral ganglion that were reported in Susswein et al. (1996) (N = 10). Data on bursting in the presence of the buccal muscles is from the first of three repetitions with CCh applied to the cerebral ganglion reported in the present paper (N = 7). There were no significant differences between preparations with and without the buccal musculature for the total time that bursting was maintained (P = 0.17, t₍₁₄₎ = 1.44), or for the number of responses recorded during this period (P = 0.36, t₍₁₄₎ = 0.94). In contrast, the maximum response rate was higher when the musculature was attached (P = 0.01, t₍₁₃₎ = 3.00; all test are two-tailed t-tests), presumably as a result of proprioceptive feedback. (D–F) Latency and pattern of responses to CCh in a suspended buccal mass preparation. (D) The CCh was applied 20–30 sec after the start of the recording in a preparation in which the buccal and cerebral ganglia remained attached to the buccal mass. Regular motor programs were initiated ∼5 min after the start of the recording. (I2) EMG recordings from the I2 muscle, which is active during protraction, (RN) recording from the radular nerve, which is a monitor of radular closing, (BN2-R) Recording from the right Buccal Nerve 2, which is active during retraction, (BN3-R) Recording from the right Buccal Nerve 3, in which the largest units are B4/B5, which are active at the start of retraction. (E) The number of responses per minute, from the application of CCh onto the cerebral ganglion. Note that data are shown only from the first of three exposures to CCh (see below). The figure shows the mean and standard error of each minute for six of the seven preparations exposed to CCh alone. One preparation stopped responding within 6 min after the CCh was applied, and for this reason data from this preparation were are not included. Preparations differed in the length of time that they continued to respond. In the period shown in the figure, all six preparations continued to respond. Beyond this point, progressively fewer preparations continued to respond, and so these data are not shown. Note that the time at which responses began, and their rate of increase, differed somewhat for each preparation. The figure shows that after a delay, the rate of responses increased, and then slowly decreased. (F) There was no significant difference in the latency from application of CCh to begin bursting between preparations with and without the buccal muscles (P = 0.91, t₍₁₅₎ = 0.11; two-tailed t-test).

Figure 3.

Changes in protraction length during the first exposure to CCh. (A) The mean protraction length during the first and last 10 feeding responses in six preparations exposed to CCh alone. Standard errors are shown. During the first few feeding responses, when response rate is low, protractions are relatively long. A one-way analysis of variance showed significant differences in protraction length among the first 10 protractions (P = 0.0007, F_(9,53) = 3.91). To be certain that protraction length had reached baseline values, we elected to analyze protraction length from after the fifth response. During the last 10 responses, the protractions are similarly long. (B) The time from the start of regular motor programs until the criterion for cessation was divided in halves, and the distribution of protraction lengths during each half was plotted. Bins are 1 sec each. Since response rate is higher during the first half than during the second half, there are more protractions in the first half (N = 310) than in the second half (N = 154). To provide a common scale of frequencies, the frequency was expressed as a percentage of the total number of responses elicited by CCh. A Kolmogorov–Smirnov test showed that there was a significant difference in the distribution of the protraction lengths between the first and second halves (D = 0.2164, P < 0.0001), with a more prominent tail of long protractions found in the second half.

Figure 4.

Parameters of feeding responses during the first exposure to CCh alone, and when either edible or inedible foods were also present. Asterisks mark significant differences. (A) Time from the start of active bursting to the 60 sec criterion for cessation of bursting. There was no significant difference between the three treatments (P = 0.48, F_(2,18) = 0.77, one-way analysis of variance). (B) The total number of feeding responses elicited from the application of CCh until the criterion for cessation of response was reached. There was no significant difference between the three treatments (P = 0.44, F_(2,18) = 0.87, one-way analysis of variance). (C) The mean response rate (defined as total number of responses/total response time (in minutes)). There was no significant difference between the three treatments (P = 0.52, F_(2,18) = 0.67, one-way analysis of variance). (D) The peak response rate. There was a significant difference between the three treatments (P = 0.02, F_(2,18) = 4.78, one-way analysis of variance). A Tukey HSD post-hoc test showed no significant difference between preparations treated with CCh alone and those treated with CCh + inedible food (P = 0.80). The difference between preparations treated with CCh alone and those treated with CCh + edible food approached significance (P = 0.07). There was a significant increase in the maximum response rate in animals treated with CCh + edible food with respect to those treated with CCh + inedible food (P = 0.01). (E) Mean protraction lengths during the first half of the CCh exposure, with the first five feeding responses (when the preparation is not maximally aroused) removed (one of the seven preparation exposed to CCh alone had fewer than 20 responses, and so was not included in the analysis, since there were not enough responses to provide estimates of protraction length after the first five responses were subtracted). Edible food (N = 215 protractions) showed significantly shortened protraction (P < 0.0002) compared to CCh alone (N = 277 protractions), whereas inedible food (N = 202 protractions) showed significantly lengthened protraction (P = 0.0452, Mann–Whitney U-test, which was used because of the clear nonnormal distribution of protraction length—see Fig. 4).

Figure 5.

Distributions of protraction lengths in preparations treated with CCh alone, and in preparations treated with CCh and edible or inedible foods. As in Figure 3B, bins of the protraction lengths are 1 sec each. To provide a common scale of frequencies, the frequency was expressed as a percentage of the total number of responses from the application of CCh until the criterion for cessation of responses was reached. (A) First treatment with CCh. Kolmogorov–Smirnov tests showed that there were significant differences in the distribution of the protraction lengths between treatment with CCh alone (N = 461) and with CCh + edible food (N = 323) (P < 0.0001, D = 0.3189), and between CCh alone and CCh + inedible food (N = 379) (P = 0.002, D = 0.1265). In addition, Mann–Whitney U-tests were performed to test whether the populations were ranked differently. There was a significant difference between CCh alone and CCh + edible food (P = 0.002, Mann–Whitney U-test with Bonferroni correction), but not between CCh alone and CCh + inedible food (P = 0.50, Mann–Whitney U-test). A comparison of protraction lengths in response to edible and inedible foods showed that protraction length in response to edible foods was significantly shorter than in response to inedible food (P = 0.018, Mann–Whitney U-test with Bonferroni correction). The shortened protraction in response to edible food is likely to be because they elicited more swallowing responses, which are characterized by weak, short protractions. (B) The second treatment with CCh. Protraction lengths during the second exposure were compared to those during the first exposure, for the same treatments. Kolmogoroff- Smirnov tests were significant for CCh alone (N = 179) (P < 0.001, D = 0.2866) and for CCh + edible food (N = 221) (P < 0.001, D = 0.2012), but not for CCh + inedible food (N = 171) (P = 0.471, D = 0.0770). Mann–Whitney U-tests with Bonferroni corrections (used because the data are not normally distributed) showed a significant increase in protraction length for preparations treated with CCh alone (P < 0.0001), a significant decrease in protraction length for preparations treated with CCh + edible food (P = 0.009), and no significant change in protraction length in preparations treated with CCh + inedible food (P = 0.704).

Figure 6.

One hour after the start of the three treatments whose results are shown in Figures 4 and 5A, the treatments were repeated. Asterisks mark significant differences. (A) There were no significant differences in the time to stop responding between the three treatments (P = 0.35, F_(2,18) = 1.13, one-way analysis of variance). (B) There were no significant differences in the number of responses between the three treatments (P = 0.12, F_(2,18) = 2.41, one-way analysis of variance). (C) There was a significant difference in the mean response rate between the three treatments (P = 0.046, F_(2,18) = 3.66, one-way analysis of variance). A Tukey HSD post-hoc test showed that there was no significant difference between preparations treated with CCh alone and those treated with CCh + inedible food (P = 0.90). By contrast, there was a significant difference between preparations treated with CCh + edible and CCh + inedible food (P = 0.039), and the difference between preparations treated with CCh alone and those treated with CCh + edible food approached significance (P = 0.088). (D) There was a significant difference in the maximum response rate between the three treatments (P = 0.008, F_(2,18) = 6.38). A Tukey HSD post-hoc test showed that there was no significant difference between preparations treated with CCh alone and those treated with CCh + inedible food (P = 0.90). By contrast, there were significant differences between preparations treated with CCh + edible and CCh + inedible food (P = 0.014), and between preparations treated with CCh alone and those treated with CCh + edible food (P = 0.012). (E) Mean protraction lengths during the first half of the CCh exposure, with the first five feeding responses (when the preparation is not maximally aroused) removed. Edible food (N = 113 protractions) significantly shortened protraction (P < 0.0001), with respect to CCh alone (N = 79 protractions), whereas inedible food (N = 81 protractions) had no significant effect on protraction (P = 0.023; Mann–Whitney U-test).

Figure 7.

Comparison between parameters of feeding responses during the first and second test with CCh. Asterisks mark significant differences. (A) Because there were no significant differences in the time to stop responding among the three groups tested in either the first or the second exposure to CCh (see Fig. 6A,B), data from the three treatments were combined for the first exposure to CCh, and again for the second exposure to CCh. The time to stop responding during the second exposure was significantly less than the time to stop during the first exposure to CCh (P = 0.002, t = 3.64, df = 20, two-tailed paired t-test, comparing all preparations from the first to the second CCh exposure). (B) There was also no significant difference in number of responses between the three treatments during either of the exposures to CCh, and therefore data were combined for each exposure to CCh. The number of feeding responses during the second exposure was significantly less than the number of responses during the first exposure to CCh (P = 0.003, t = 3.45, df = 20, two-tailed paired t-test, comparing all preparations from the first to the second CCh exposure). (C) Because there were significant differences between the three treatments during the second exposure to CCh, the mean response rate between the first and second exposures to CCh for the treatment that was significantly different from the other two (CCh + edible food) was analyzed separately from the mean response rate for CCh alone and for inedible food, which were combined. There was a significant reduction in mean response rate for preparations treated with CCh alone and with CCh + inedible food (P = 0.006, t = 3.23, df = 15, two-tailed paired t-test), with no significant difference for preparations treated with edible food (P = 0.95, t = 0.07, df = 4, two-tailed paired t-test). (D) Because there were significant differences between the three treatments during both the first and second exposures to CCh for the maximal response rate, the values between the first and second exposures to CCh for the group that differed from the others (CCh + edible food) were analyzed separately, whereas data from the two groups that were not significantly different (CCh alone and CCh + inedible food) were combined. There was a significant reduction in mean response rate for preparations treated with CCh alone and with CCh + inedible food (P = 0.0003, t = 4.75, df = 15, two-tailed paired t-test), but not for preparations treated with CCh + edible food (P = 0.12, t = 1.92, df = 4, two-tailed paired t-test). Note that the data for the first and second exposures to CCh are plotted separately for each of the three procedures (i.e., no combining of data from different procedures) are presented in Supplemental Figure 1.

Figure 8.

One hour after the start of the treatments whose results are shown in Figures 6 and 7, a second test of memory examined the response to CCh alone. (A) There were significant differences in the time to stop responding based on which of the three treatments preceded the CCh alone (P = 0.013, F_(2,18) = 5.55, one-way analysis of variance). The difference arose because of a decrease in response time of preparations that were previously treated with CCh + inedible food with respect to preparations previously treated with CCh alone (P = 0.010, Tukey HSD post-hoc test), with no significant difference between preparations previously treated with CCh + edible food and CCh alone (P = 0.46, Tukey HSD post-hoc test). (B) There were significant differences in the number of responses to CCh alone based on which of the three treatments preceded the CCh alone (P = 0.003, F_(2,18) = 8.31, one-way analysis of variance). The difference arose because of a decrease in response time of preparations that were previously treated with CCh + inedible food with respect to preparations previously treated with CCh alone (P = 0.002, Tukey HSD post-hoc test), with no significant difference between preparations previously treated with CCh + edible food and CCh alone (P = 0.44, Tukey HSD post-hoc test). (C) There were significant differences in the mean response rate to CCh alone, based on which of the three prior treatments was applied previously (P = 0.009, F_(2,18) = 6.28, one-way analysis of variance). The difference arose because of a decrease in response time of preparations that were previously treated with CCh + inedible food with respect to preparations previously treated with CCh alone (P = 0.011, Tukey HSD post-hoc test), with no significant difference between preparations previously treated with CCh + edible food and CCh alone (P = 0.49, Tukey HSD post-hoc test). (D) There were no significant differences in the peak response rate to CCh after the three preceding treatments (P = 0.08, F_(2,18) = 2.85, one-way analysis of variance). However, when the data from preparations that were exposed previously to CCh alone and to CCh + edible food were combined, and were compared to data from preparations that had been previously exposed to CCh + inedible food, there was a significant difference (P = 0.026, t₍₁₉₎ = 2.41). In addition, there was a significant difference between preparations previously tested with CCh alone and those previously tested with CCh + inedible food (P = 0.03, t₍₁₄₎ = 2.36). (E) Distribution of protraction lengths for preparations treated previously with (1) CCh alone (N = 392), and (2) CCh + edible food (N = 155). There were too few responses in seven of the nine preparations trained with inedible food to meaningfully compare preparations previously treated with CCh + inedible food to the other two groups. There were no significant differences in protraction length between the two groups shown (Kolmogorov–Smirnov test: D = 0.1124, P = 0.112; Mann–Whitney U-test: U = 27211, P = 0.057). (F) Comparison of protraction lengths during the first half of the exposure to CCh alone in preparations treated previously with CCh alone or with CCh + edible food. There was a significant decrease in protraction length in preparations previously treated with CCh + edible food (P = 0.00022, Mann–Whitney U-test) during the first half of exposure to CCh. Note that data on protraction lengths during the second half of all three treatments with CCh are shown in Supplemental Figure 2.

Figure 9.

Summary of the findings. The cholinergic agonist CCh applied to the cerebral ganglion induces repetitive feeding motor programs. When the buccal muscles remain attached to the buccal ganglia, there is an increase in peak frequency, and an increase in the variability of the motor patterns. Repetition of this procedure leads to a decrease in responses, as measured by a shorter time that the preparation responds, fewer responses, and a lengthening of protractions. Challenging the preparation with edible food leads to an increase in mean and peak response rates, and a shortening of the protractions. Repetition of this procedure does not lead to the decrease in mean or peak response rates seen when the preparation is exposed to CCh alone, and leads to shorter protractions than during the initial training with edible food. Challenging the preparation with inedible foods causes responses that are similar to those to CCh alone during the training and during the repetition, except that the increased protraction length during the repetition does not occur, because protraction length is paradoxically decreased during the second half of the CCh exposure. When the preparations are again challenged with CCh alone, there are shorter protractions in preparations previously treated with CCh and edible food relative to preparations previously treated with CCh alone, with no other differences in other parameters of feeding. However, preparations previously treated with inedible food show reductions in many response parameters, showing memory similar to that in intact animals.

Figure 10.

Hypothesis of mechanism of learning that food is inedible in the cerebral ganglion. Taste receptors respond to different tastes, but all release ACh onto different neurites of command-like CBI neurons. These excite the CPG within the buccal ganglia. Pairing activation of a specific taste with unrewarded effort, signaled by the release of Nitric Oxide (NO) and histamine (Susswein and Chiel 2012), causes a decrease in responsivity to ACh at the specific neurite (or combination of neurites) that were activated, while leaving intact the responses at neurites that were not paired with NO and histamine.