Intermediate-term memory in Aplysia involves neurotrophin signaling, transcription, and DNA methylation

Abstract

Long-term but not short-term memory and synaptic plasticity in many brain areas require neurotrophin signaling, transcription, and epigenetic mechanisms including DNA methylation. However, it has been difficult to relate these cellular mechanisms directly to behavior because of the immense complexity of the mammalian brain. To address that problem, we and others have examined numerically simpler systems such as the hermaphroditic marine mollusk Aplysia californica. As a further simplification, we have used a semi-intact preparation of the Aplysia siphon withdrawal reflex in which it is possible to relate cellular plasticity directly to behavioral learning. We find that inhibitors of neurotrophin signaling, transcription, and DNA methylation block sensitization and classical conditioning beginning ∼1 h after the start of training, which is in the time range of an intermediate-term stage of plasticity that combines elements of short- and long-term plasticity and may form a bridge between them. Injection of decitabine (an inhibitor of DNA methylation that may have other actions in these experiments) into an LE sensory neuron blocks the neural correlates of conditioning in the same time range. In addition, we found that both DNA and RNA methylation in the abdominal ganglion are correlated with learning in the same preparations. These results begin to suggest the functions and integration of these different molecular mechanisms during behavioral learning.

Figure 1.

The preparation (A) and behavioral protocols (B) for sensitization and conditioning. See Materials and Methods for details. The large arrow indicates a train of four shocks, the small arrows indicate single shocks, and the vertical bars indicate siphon taps.

Figure 2.

Inhibitors of neurotrophin signaling, transcription, and DNA methylation block conditioning in the siphon-withdrawal preparation. (A) Examples of siphon withdrawal (SWR) before (PreTest) and 45 min after (PostTest) conditioning. (B) The average amplitude of siphon withdrawal on each test in groups that received paired or unpaired training with the abdominal ganglion bathed in either IgG (n = 8 paired and 6 unpaired) or Trk-Fc (n = 6 and 8). There was a significant overall drug × pairing × test interaction (F_(3,72) = 3.47, P < 0.05). Responses have been normalized to the value on the pretest in each experiment. The average pretest value was 2.0 ± 0.1 mm for IgG and 1.8 ± 0.1 mm for Trk-Fc and the average response to the first tail shock US was 6.4 ± 0.4 mm for IgG and 7.1 ± 0.3 mm for Trk-Fc, not significantly different. (C) Average siphon withdrawal on each test in groups that received paired or unpaired training with the abdominal ganglion bathed in either normal seawater or DMSO (Control, n = 37 paired and 29 unpaired), DRB (n = 9 and 8), Actinomycin D (n = 10 and 7, not shown), RG108 (n = 10 and 8), or decitabine (n = 7 and 6, not shown). There was a significant overall drug × pairing × test interaction (F_(12,363) = 2.57, P < 0.01). The average pretest value was 3.1 ± 0.1 mm for control, 3.0 ± 0.3 mm for DRB, 3.5 ± 0.2 mm for Actinomycin D, 3.4 ± 0.2 mm for RG108, and 2.9 ± 0.3 mm for decitabine, and the average response to the first tail shock US was 5.1 ± 0.5 mm, 5.7 ± 0.3 mm, 6.1 ± 0.4 mm, 5.8 ± 0.8 mm, and 5.6 ± 0.6 mm, not significantly different in one-way ANOVAs. In this and subsequent figures error bars indicate SEMs, x = P < 0.05 one-tail, (*) P < 0.05, (**) P < 0.01 for the difference between the trained and control groups, and + = P < 0.05 one-tail, (#) P < 0.05, (##) P < 0.01 for the drug × training interaction.

Figure 3.

Intracellular injection of decitabine into the LE SN blocks neural correlates of conditioning. (A) Examples of siphon withdrawal (SW), evoked firing of an LFS siphon MN and an LE siphon SN, the membrane resistance of the neurons, and the monosynaptic EPSP from the LE neuron to the LFS neuron on the pretest, the post-test, and a 24 h test following paired or unpaired training in a control experiment. (B) Average results on each test from experiments like the one shown in A with paired or unpaired training following injection of vehicle or decitabine into the LE neuron (n = 3 per group). There were significant overall effects of pairing for siphon withdrawal (F_(1,8) = 14.72, P < 0.01), LE spikes (F = 55.71, P < 0.001), and the EPSP (F = 12.25, P < 0.01) and a significant overall drug × pairing interaction for LE spikes (F = 42.57, P < 0.001). The data have been normalized to the value on the pretest in each experiment. The average pretest value for siphon withdrawal was 2.5 ± 0.8 mm for control and 1.9 ± 0.5 mm for decitabine, for evoked LFS firing 6.0 ± 0.3 spikes for control and 5.7 ± 0.8 spikes for decitabine, for evoked LE firing 3.3 ± 0.5 spikes for control and 4.0 ± 0.6 spikes for decitabine, and for the amplitude of the EPSP 8.4 ± 0.9 mV for control and 5.6 ± 0.9 mV for decitabine, and the average siphon withdrawal in response to the first tail shock was 4.9 ± 0.3 mm for control and 4.2 ± 0.3 mm for decitabine, not significantly different.

Figure 4.

Trk-Fc, DRB, and cytosine analogs block intermediate-term sensitization in the siphon-withdrawal preparation. (A) Examples of siphon withdrawal before (PreTest) and after (PostTest) tail shock (sensitization). (B) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either IgG (n = 8 shock and 12 no-shock) or Trk-Fc (n = 13 and 10). The average pretest value was 2.2 ± 0.1 mm for IgG and 2.3 ± 0.2 mm for Trk-Fc and the average response to the tail shock was 7.8 ± 0.5 mm for IgG and 8.1 ± 0.2 mm for Trk-Fc, not significantly different. (C) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either ASW or DMSO (Control, n = 12 shock and 11 no-shock), DRB (n = 9 and 8), or decitabine (n = 9 and 8). There was a significant overall drug × shock interaction (F_(2,51) = 5.02, P < 0.01). Responses have been normalized to the average value on the three pretests in each experiment. The average pretest value was 2.6 ± 0.2 mm for control, 2.9 ± 0.1 mm for DRB, and 3.1 ± 0.2 mm for decitabine, and the average response to the tail shock was 7.2 ± 0.5 mm for control, 7.0 ± 0.6 mm for DRB, and 8.3 ± 0.9 mm for decitabine, not significantly different. We also obtained similar results with another cytosine analog, 5-aza-cytidine (control n = 11 shock and 8 no-shock, 5-aza-cytidine n = 3 and 3, not shown). There was a significant overall drug × shock interaction (F_(1,21) = 3.04, P < 0.05 one-tail). The average pretest value was 2.7 ± 0.2 mm for control and 2.4 ± 0.1 mm for 5-aza-cytidine and the average response to the tail shock was 8.3 ± 0.6 mm for control and 6.8 ± 0.6 mm for 5-aza-cytidine, not significantly different.

Figure 5.

Behavioral learning is correlated with DNA and RNA methylation in the abdominal ganglion. (A) Average siphon withdrawal on each test in groups that received tail shock or no-shock control with the abdominal ganglion bathed in either normal seawater (control, n = 11 shock and 10 no-shock) or decitabine (n = 10 and 10). There was a significant overall effect of shock (F_(1,37) = 16.58, P < 0.001). Responses have been normalized to the average value on the three pretests in each experiment. The average pretest value was 2.3 ± 0.2 mm for control and 2.7 ± 0.2 mm for decitabine and the average response to the tail shock was 7.8 ± 0.4 mm for control and 7.4 ± 0.5 mm for decitabine, not significantly different. (B) DNA methylation in the abdominal ganglion assayed immediately after the last test in some of the experiments shown in A. (B1) Average 5mC DNA as a percent of total DNA in the different groups (control, n = 2 shock and 2 no-shock, decitabine, n = 2 shock and 2 no-shock). (B2) Within-groups correlation between the change in siphon withdrawal and 5mC DNA as a percent of total DNA in the abdominal ganglion assayed immediately after the last test in some of the experiments shown in A. There was a negative linear regression in each group and overall (r = −0.88, P < 0.05) in an ANCOVA. To illustrate the within-groups correlation graphically the mean of each group is set to zero and the groups are pooled, so the correlation reflects the relation independent of group. (C) RNA methylation in experiments similar to those shown in B. (C1) Average 5mC RNA as a percent of total RNA in the different groups (control, n = 9 shock and 8 no-shock, decitabine, n = 8 shock and 8 no-shock). (C2) Within-groups correlation between the change in siphon withdrawal and 5mC RNA as a percent of total RNA in the abdominal ganglion assayed immediately after the last test in some of the experiments shown in A. There was a positive linear regression in each group (except decitabine, control) and overall (r = 0.36, P < 0.05).