Generation of a synthetic memory trace

Abstract

We investigated the effect of activating a competing, artificially generated, neural representation on encoding of contextual fear memory in mice. We used a c-fos-based transgenic approach to introduce the hM(3)D(q) DREADD receptor (designer receptor exclusively activated by designer drug) into neurons naturally activated by sensory experience. Neural activity could then be specifically and inducibly increased in the hM(3)D(q)-expressing neurons by an exogenous ligand. When an ensemble of neurons for one context (ctxA) was artificially activated during conditioning in a distinct second context (ctxB), mice formed a hybrid memory representation. Reactivation of the artificially stimulated network within the conditioning context was required for retrieval of the memory, and the memory was specific for the spatial pattern of neurons artificially activated during learning. Similar stimulation impaired recall when not part of the initial conditioning.

Figure 1. Expression and activation of the hM₃D_q transgene

A) Transgenic mice used in this study carry the 2 transgenes shown allowing Dox regulated and neural activity dependent expression of the hM₃D_q receptor. B) Overall spatial expression profile of the hM₃D_q transgene in mice off dox maintained in the homecage. Immunofluorescence was strong in hippocampus, basalateral amygdala, and throughout the cortex. Fluorescence was also observed to a small extent in the pontine nucleus and in brainstem. C) Expression in the CA1 region of the hippocampus showing sparse and distributed expression of the hM₃D_q transgene. D) CNO injection causes increased neural activity in hM₃D_q^fos mice. Red curve shows multi unit activity (MUA) recorded from dorsal CA1of an anesthetized hM₃D_q^fos mouse over time. Inset gives fold increase in MUA (4.76 for hM₃D_q^fos vs. .9 for WT, mean 30-40 minutes post-injection/mean pre-injection baseline. n=6 and 6, *=Wilcoxon signed-rank: P<0.01). E & F) cfos induction 1.5 hours after CNO administration in a control (left) and hM₃D_q^fos (right) mouse. hM₃D_q^fos mice showed on average a 2.5-fold increase in cfos expression in the hippocampal CA1 region compared to control mice (see supplementary table 1 hM₃D_q^fos n = 10, control, n = 10, T-test p <.02).

Figure 2. Incorporation of Synthetic Neural Activity into a 24-hr Memory Representation

A) Freezing in ctxA 24-hours after conditioning in ctxB. hM₃D_q^fos n = 14, control n = 13. hM₃D_q^fos mice freeze significantly less than control mice in ctxA in the absence and presence of CNO. Repeated measures ANOVA main effect of genotype F(1,26) = 10.96, p <.005. CNO has no significant effect on freezing in either group. Post hoc Bonferroni hM3D^fos p = 0.192, control p = 1.00. B) Transgenic hM₃D_q^fos mice show impaired 24-hour memory for ctxB that is rescued by injection of CNO. Repeated measures ANOVA genotype x CNO interaction F(1.25) = 10.15, p <.005. Post hoc Fisher’s LSD found that hM₃D_q^fos mice were freezing significantly less than control mice in ctxB in the absence of CNO, p < 0.001, but were statistically similar in ctxB in the presence of CNO, p = 0.117, and showed a significant increase in freezing in ctxB with CNO compared to ctxB alone, p < 0.001. C and D) Correlation between the difference in freezing scores in the presence and absence of CNO and endogenous cfos expression 1 hour after memory testing in hippocampal area CA1, D, r = 0.8276, p <.005 and CA3, E, r = 0.6742, p <.05.

Figure 3. Disruption of Memory Retrieval by Synthetic Neural Activation

Transgenic hM3D^fos mice develop a normal 24-hr context memory when conditioned in the absence of CNO. This memory is disrupted by CNO injection to activate the competing ctxA representation. hM₃D_q^fos n = 12, control n = 12. Repeated measures ANOVA main effect of genotype F(1, 22) = 5.3, p <.05, CNO F(1, 22) = 28.6, p < 0.001, and genotype × CNO interaction F(1, 22) = 13.5, p = 0.001. Post-hoc Fisher LSD revealed that hM₃D^fos mice were freezing significantly less in the presence of CNO compared to before CNO administration p < 0.001, and were freezing significantly less that control mice in the presence of CNO p < 0.001.

Figure 4. Memory performance during synthetic reactivation is network specific

A) When CNO induced synthetic activation does not occur in identical neural populations during memory formation and memory retrieval, a memory deficit is observed. hM3D^fos mice show significantly less freezing than control mice in ctxB both in the absence and presence of CNO. hM3D^fos n = 14, control = 17. Repeated measures ANOVA main effect of genotype F(1, 23) = 51.15, p < 0.001. B) When hM₃D_q^fos mice are exposed to ctxB off of dox to induce hM₃D_q expression and then fear conditioned on dox after CNO injection in ctxB, synthetic activation by CNO is not necessary for memory recall in ctxB. ctxB: hM₃D_q^fos n = 9, control n = 10, ctxBcno: hM₃D_q^fos n = 5, control n = 6. Repeated measures ANOVA F(2, 18) = 0.0474, p = 0.954.