A ventral CA1 to nucleus accumbens core engram circuit mediates conditioned place preference for cocaine

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Abstract

The importance of neuronal ensembles, termed engram cells, in storing and retrieving memory is increasingly being appreciated, but less is known about how these engram cells operate within neural circuits. Here we tagged engram cells in the ventral CA1 region of the hippocampus (vCA1) and the core of the nucleus accumbens (AcbC) during cocaine conditioned place preference (CPP) training and show that the vCA1 engram projects preferentially to the AcbC and that the engram circuit from the vCA1 to the AcbC mediates memory recall. Direct activation of the AcbC engram while suppressing the vCA1 engram is sufficient for cocaine CPP. The AcbC engram primarily consists of D1 medium spiny neurons, but not D2 medium spiny neurons. The preferential synaptic strengthening of the vCA1→AcbC engram circuit evoked by cocaine conditioning mediates the retrieval of cocaine CPP memory. Our data suggest that the vCA1 engram stores specific contextual information, while the AcbC D1 engram and its downstream network store both cocaine reward and associated contextual information, providing a potential mechanism by which cocaine CPP memory is stored.

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Fig. 1: Activation of the AcbC-projecting vCA1 engram is required for the retrieval of cocaine memory.
Fig. 2: The vCA1→AcbC engram circuits are required for cocaine memory storage.
Fig. 3: Activation of the AcbC engram retrieves cocaine CPP memory independent of the vCA1 engram.
Fig. 4: Cocaine CPP training induces synaptic potentiation of AcbC D1 engram cells.
Fig. 5: Cocaine CPP training preferentially increases the synaptic connectivity from the vCA1 engram to AcbC D1 engram cells.
Fig. 6: LFS of vCA1→AcbC engram circuits inhibits and HFS promotes the retrieval of cocaine CPP memory.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Custom Matlab code is available on reasonable request from L.M.

References

  1. 1.

    Tonegawa, S., Liu, X., Ramirez, S. & Redondo, R. Memory engram cells have come of age. Neuron 87, 918–931 (2015).

  2. 2.

    Josselyn, S. A. Continuing the search for the engram: examining the mechanism of fear memories. J. Psychiatry Neurosci. 35, 221–228 (2010).

  3. 3.

    Reijmers, L. G., Perkins, B. L., Matsuo, N. & Mayford, M. Localization of a stable neural correlate of associative memory. Science 317, 1230–1233 (2007).

  4. 4.

    Tonegawa, S., Morrissey, M. D. & Kitamura, T. The role of engram cells in the systems consolidation of memory. Nat. Rev. Neurosci. 19, 485–498 (2018).

  5. 5.

    Denny, C. A. et al. Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis. Neuron 83, 189–201 (2014).

  6. 6.

    Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).

  7. 7.

    Zhou, Y. et al. CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala. Nat. Neurosci. 12, 1438–1443 (2009).

  8. 8.

    Han, J. H. et al. Selective erasure of a fear memory. Science 323, 1492–1496 (2009).

  9. 9.

    Ryan, T. J., Roy, D. S., Pignatelli, M., Arons, A. & Tonegawa, S. Memory. Engram cells retain memory under retrograde amnesia. Science 348, 1007–1013 (2015).

  10. 10.

    Josselyn, S. A., Kohler, S. & Frankland, P. W. Finding the engram. Nat. Rev. Neurosci. 16, 521–534 (2015).

  11. 11.

    Tonegawa, S., Pignatelli, M., Roy, D. S. & Ryan, T. J. Memory engram storage and retrieval. Curr. Opin. Neurobiol. 35, 101–109 (2015).

  12. 12.

    Lowel, S. & Singer, W. Selection of intrinsic horizontal connections in the visual cortex by correlated neuronal activity. Science 255, 209–212 (1992).

  13. 13.

    Munakata, Y. & Pfaffly, J. Hebbian learning and development. Dev. Sci. 7, 141–148 (2004).

  14. 14.

    Roy, D. S., Muralidhar, S., Smith, L. M. & Tonegawa, S. Silent memory engrams as the basis for retrograde amnesia. Proc. Natl Acad. Sci. USA 114, E9972–E9979 (2017).

  15. 15.

    Choi, J. H. et al. Interregional synaptic maps among engram cells underlie memory formation. Science 360, 430–435 (2018).

  16. 16.

    Kim, W. B. & Cho, J. H. Encoding of discriminative fear memory by input-specific LTP in the amygdala. Neuron 95, 1129–1146 e1125 (2017).

  17. 17.

    Tanaka, K. Z. et al. Cortical representations are reinstated by the hippocampus during memory retrieval. Neuron 84, 347–354 (2014).

  18. 18.

    Huganir, R. L. & Nicoll, R. A. AMPARs and synaptic plasticity: the last 25 years. Neuron 80, 704–717 (2013).

  19. 19.

    Diering, G. H., Gustina, A. S. & Huganir, R. L. PKA-GluA1 coupling via AKAP5 controls AMPA receptor phosphorylation and cell-surface targeting during bidirectional homeostatic plasticity. Neuron 84, 790–805 (2014).

  20. 20.

    Kessels, H. W. & Malinow, R. Synaptic AMPA receptor plasticity and behavior. Neuron 61, 340–350 (2009).

  21. 21.

    Bagot, R. C. et al. Ventral hippocampal afferents to the nucleus accumbens regulate susceptibility to depression. Nat. Commun. 6, 7062 (2015).

  22. 22.

    Nicola, S. M. The nucleus accumbens as part of a basal ganglia action selection circuit. Psychopharmacology 191, 521–550 (2007).

  23. 23.

    Pignatelli, M. & Bonci, A. Role of dopamine neurons in reward and aversion: a synaptic plasticity perspective. Neuron 86, 1145–1157 (2015).

  24. 24.

    Joseph, M. H., Datla, K. & Young, A. M. The interpretation of the measurement of nucleus accumbens dopamine by in vivo dialysis: the kick, the craving or the cognition? Neurosci. Biobehav. Rev. 27, 527–541 (2003).

  25. 25.

    Miller, C. A. & Marshall, J. F. Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory. Neuron 47, 873–884 (2005).

  26. 26.

    Otis, J. M., Fitzgerald, M. K. & Mueller, D. Inhibition of hippocampal beta-adrenergic receptors impairs retrieval but not reconsolidation of cocaine-associated memory and prevents subsequent reinstatement. Neuropsychopharmacology 39, 303–310 (2014).

  27. 27.

    Degoulet, M., Stelly, C. E., Ahn, K. C. & Morikawa, H. L-type Ca2+ channel blockade with antihypertensive medication disrupts VTA synaptic plasticity and drug-associated contextual memory. Mol. Psychiatry 21, 394–402 (2016).

  28. 28.

    Calipari, E. S. et al. In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward. Proc. Natl Acad. Sci. USA 113, 2726–2731 (2016).

  29. 29.

    Bertran-Gonzalez, J. et al. Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol. J. Neurosci. 28, 5671–5685 (2008).

  30. 30.

    Kim, J., Park, B. H., Lee, J. H., Park, S. K. & Kim, J. H. Cell type-specific alterations in the nucleus accumbens by repeated exposures to cocaine. Biol. Psychiatry 69, 1026–1034 (2011).

  31. 31.

    Sjulson, L., Peyrache, A., Cumpelik, A., Cassataro, D. & Buzsaki, G. Cocaine place conditioning strengthens location-specific hippocampal coupling to the nucleus accumbens. Neuron 98, 926–934.e5 (2018).

  32. 32.

    Lobo, M. K. et al. Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward. Science 330, 385–390 (2010).

  33. 33.

    Graziane, N. M. et al. Opposing mechanisms mediate morphine- and cocaine-induced generation of silent synapses. Nat. Neurosci. 19, 915–925 (2016).

  34. 34.

    Tzschentke, T. M. Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade. Addict. Biol. 12, 227–462 (2007).

  35. 35.

    Sun, Y., Chen, G., Zhou, K. & Zhu, Y. A conditioned place preference protocol for measuring incubation of craving in rats. J. Vis. Exp. https://doi.org/10.3791/58384 (2018).

  36. 36.

    Dong, Y. et al. Cocaine-induced potentiation of synaptic strength in dopamine neurons: behavioral correlates in GluRA −/− mice. Proc. Natl Acad. Sci. USA 101, 14282–14287 (2004).

  37. 37.

    Di Chiara, G. & Imperato, A. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Natl Acad. Sci. USA 85, 5274–5278 (1988).

  38. 38.

    Cowansage, K. K. et al. Direct reactivation of a coherent neocortical memory of context. Neuron 84, 432–441 (2014).

  39. 39.

    Jones, E. G. Santiago Ramon y Cajal and the Croonian lecture, March 1894. Trends Neurosci. 17, 190–192 (1994).

  40. 40.

    Morris, R.G. D.O. Hebb: The Organization of Behavior, Wiley: New York; 1949. Brain Res. Bull. 50, 437 (1999).

  41. 41.

    Konorski, J. Mechanisms of learning. Sym. Soc. Exp. Biol. 4, 408–431 (1950).

  42. 42.

    Liu, X. et al. β-Arrestin-biased signaling mediates memory reconsolidation. Proc. Natl Acad. Sci. USA 112, 4483–4488 (2015).

  43. 43.

    Mondello, S. E., Jefferson, S. C., O’Steen, W. A. & Howland, D. R. Enhancing Fluorogold-based neural tract tracing. J. Neurosci. Methods 270, 85–91 (2016).

  44. 44.

    Ting, J. T., Daigle, T. L., Chen, Q. & Feng, G. Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol. Biol. 1183, 221–242 (2014).

  45. 45.

    Zhao, S. et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8, 745–752 (2011).

  46. 46.

    Redondo, R. L. et al. Bidirectional switch of the valence associated with a hippocampal contextual memory engram. Nature 513, 426–430 (2014).

  47. 47.

    Lee, J., Finkelstein, J., Choi, J. Y. & Witten, I. B. Linking cholinergic interneurons, synaptic plasticity, and behavior during the extinction of a cocaine-context association. Neuron 90, 1071–1085 (2016).

  48. 48.

    Huang, B. et al. β-Arrestin-biased beta-adrenergic signaling promotes extinction learning of cocaine reward memory. Sci. Signal. 11, eaam5402 (2018).

  49. 49.

    Kitamura, T. et al. Engrams and circuits crucial for systems consolidation of a memory. Science 356, 73–78 (2017).

  50. 50.

    Dobi, A., Margolis, E. B., Wang, H. L., Harvey, B. K. & Morales, M. Glutamatergic and nonglutamatergic neurons of the ventral tegmental area establish local synaptic contacts with dopaminergic and nondopaminergic neurons. J. Neurosci. 30, 218–229 (2010).

  51. 51.

    Franklin, K. B. J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates (Academic Press, 2013).

  52. 52.

    Dumitriu, D., Rodriguez, A. & Morrison, J. H. High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy. Nat. Protoc. 6, 1391–1411 (2011).

  53. 53.

    Yuste, R. & Bonhoeffer, T. Genesis of dendritic spines: insights from ultrastructural and imaging studies. Nat. Rev. Neurosci. 5, 24–34 (2004).

  54. 54.

    Rodriguez, A., Ehlenberger, D. B., Dickstein, D. L., Hof, P. R. & Wearne, S. L. Automated three-dimensional detection and shape classification of dendritic spines from fluorescence microscopy images. PLoS One 3, e1997 (2008).

  55. 55.

    Rodriguez, A., Ehlenberger, D. B., Hof, P. R. & Wearne, S. L. Rayburst sampling, an algorithm for automated three-dimensional shape analysis from laser scanning microscopy images. Nat. Protoc. 1, 2152–2161 (2006).

  56. 56.

    Li, Y. et al. Serotonin neurons in the dorsal raphe nucleus encode reward signals. Nat. Commun. 7, 10503 (2016).

  57. 57.

    Guo, Q. et al. Multi-channel fiber photometry for population neuronal activity recording. Biomed. Opt. Express 6, 3919–3931 (2015).

  58. 58.

    Gunaydin, L. A. et al. Natural neural projection dynamics underlying social behavior. Cell 157, 1535–1551 (2014).

  59. 59.

    Martin-Fernandez, M. et al. Synapse-specific astrocyte gating of amygdala-related behavior. Nat. Neurosci. 20, 1540–1548 (2017).

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Acknowledgements

This research was supported by grants from the National Natural Science Foundation of China (31430033, 31930046 and 91632307 to L.M., 31771176 and 31571036 to X.L.), the Ministry of Science and Technology (2015CB553501 to L.M.), the Shanghai Municipal Science and Technology Major Project (2018SHZDZX01 to L.M.) and the ZJLab.

Author information

L.M., X.L., Y.Z. and H.Z designed the experiments. H.Z., Y.Z., X.C. and Z.L. performed the behavioral experiments. Y.Z. and X.L. designed the electrophysiology experiments and Y.Z. performed most of the electrophysiological experiments. H.Z., Z.L. and X.C. assisted with the electrophysiology experiments. Figure 1b and Supplementary Fig. 8j,k were prepared by Z.L.. H.Z., C.M. and X.C. processed and analyzed the electrophysiological data. H.Z., Y.Z. and X.L. performed viral injections, brain preparation, imaging and analysis. Z.L., X.S., C.M., Z.T. and E.Y. assisted with viral injections and cocaine conditioning. B.H. performed molecular cloning. L.M. and X.L. supervised the project. L.M., X.L. and Y.Z. wrote the paper.

Correspondence to Xing Liu or Lan Ma.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Denise Cai, Yan Dong, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Retrieval of cocaine-CPP memory induces engram cells activation in the vCA1, AcbC, and PrL.

No-doxycycline (Dox) diet was provided for c-fos-tTA × tetO-Cre × Ai14 mice during cocaine- conditioning (days 2 and 3). Mice were returned to the CPP apparatus on Day 5 for a CPP test. 1 hr after the test, mice were sacrificed for immunofluorescent analysis. (a) The c-fos-driven expression of tetracycline-controlled transactivator (tTA) induces Cre expression and Cre-dependent expression of tdTomato, which can be blocked by Dox, an inhibitor of tTA. (b) Experiment scheme. (c) TdTomato+ cell counts in the indicated brain areas of mice experienced cocaine-conditioning or control mice that stayed in home cage. [Mann-Whitney Rank Sum Test, AcbC: control n = 6, Cocaine n = 13, Z = −3.421, p = 0.000625; BLA: Control n = 6, Cocaine n = 13, Z = −3.454, p = 0.000552; Two-tailed Student’s t-test, vCA1: Control n = 3, Cocaine n = 13, t (14) = −5.472, p = 0.000082; PrL: Control n = 3, Cocaine n = 9, t (12) = −3.127, p = 0.009]. **p < 0.01, ***p <0.001. (d,e) Representative images (d) and summary graphs (e) of tdTomato and c-Fos fluorescence in indicated brain areas. Scale bar: 100 µm. [One Way ANOVA or Kruskal-Wallis One-way ANOVA on ranks, No Retrieval n = 5, Retrieval n = 8, AcbC: tdTomato+, F (1, 11) = 0.978, p = 0.344, c-Fos+, H = 8.571 with 1 degrees of freedom, p = 0.003, c-Fos+ tdTomato+: F (1, 11) = 10813, p = 0.007, c-Fos+ tdTomato+ / tdTomato+, F (1, 11) = 14.063, p = 0.003; vCA1: tdTomato+, F (1, 11) = 0.047, p = 0.833, c-Fos+, H = 8.571 with 1 degrees of freedom, p = 0.003, c-Fos+ tdTomato+: F (1, 11) = 15.178, p = 0.002, c-Fos+ tdTomato+ / tdTomato+: H = 8.571 with 1 degrees of freedom, p = 0.003; No Retrieval n = 5, Retrieval n = 6, PrL: tdTomato+: F (1, 9) = 0.039, p = 0.847, c-Fos+: F (1, 9) = 8.757, p = 0.016, c-Fos+ tdTomato+: F (1,9) = 5.475, p = 0.044, c-Fos+ tdTomato+ / tdTomato+: H = 1.205 with 1 degrees of freedom, p = 0.329; IL: tdTomato+: H = 0.133 with 1 degrees of freedom, p = 0.792, c-Fos+: H = 0.833 with 1 degrees of freedom. p = 0.429, c-Fos+ tdTomato+: H = 0.837 with 1 degrees of freedom, p = 0.429, c-Fos+ tdTomato+/ tdTomato+: H = 1.633 with 1 degrees of freedom, p = 0.247 ; No Retrieval n = 5, Retrieval n = 8, BLA: tdTomato+: F (1, 11) = 0.234, p = 0.638, c-Fos+: F (1, 11) = 6.997, p = 0.023, c-Fos+ tdTomato+: H = 0.095 with 1 degrees of freedom, p = 0.833, c-Fos+ tdTomato+ / tdTomato+: H = 0.024 with 1 degrees of freedom, p = 0.943.] *p < 0.05, **p < 0.01, ***p <0.001. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 2 Inhibition of cocaine-engram in the AcbC and vCA1 does not affect locomotion.

C57BL/6 mice were trained to acquire cocaine-CPP, and tested for CPP on Day 4 (Test). CNO (1 mg/kg, i.p.) or saline was injected 30 min before Test. (a) Experiment scheme. (b) Summary bar graph of CPP scores. [Saline, n = 10, CNO, n = 11, Friedman’s two-way RM ANOVA by ranks, Ftreatment × session (1, 19) = 0.11, p = 0.748]. (c-g) AAV TRE-tight-hM4D-mCherry was infused into the AcbC or vCA1 of c-fos-tTA mice 2 weeks before cocaine-CPP training. Clozapine N-oxide (CNO, 1 mg/ kg, i.p.) or saline was injected 30 min before open field test. (c) Experiment scheme. (d, f) Schematics of chemogenetic stimulation (left) and representative fluorescence images of hM4D-mCherry (right). The broken white line shows the position of AC. The experiment was independently repeated with similar results in the experimental mice in panel e or g. Scale bar: 100 μm. (e, g) Summary bar graphs of distance traveled in the open field. [AcbC: Saline, n = 11, CNO, n = 12, One-way ANOVA, F (1, 21) = 0.632, p = 0.435; vCA1: Saline, n = 12, CNO, n = 11, One-way ANOVA, F (1, 21) = 1.574, p = 0.223]. (h, i) Retrograde tracing of AcbC. The experiment was independently repeated with similar results in n = 5 mice. (h) Schematic and representative image illustrating retrograde tracer Fluorogold injection in the AcbC of C57BL/6 mice. The broken white line shows the position of AC. (i) Representative images of Fluorogold fluorescence in the BLA, PrL, VTA, and vCA1. Scale bar: 100 µm. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 3 Cocaine-engram cells, but not non-cocaine control-engram cells in the vCA1 are activated by memory retrieval of cocaine-CPP.

(a, b) AAV TRE-tight-hM4D-mCherry was infused into the vCA1 of c-fos-tTA mice 2 weeks before the beginning of behavioral experiments. To label control-engram cells, mice were exposed to a novel context in the absence of Dox before cocaine-CPP training. Alternatively, to label cocaine-engram cells, mice were conditioned with cocaine in the absence of Dox. (a) Experiment scheme. (b) Left: strategy for engram labeling. Right: plot of cell counts and fluorescence images of cocaine-engram and non-cocaine control-engram in the vCA1. Scale bar: 100 µm. [One-way ANOVA, Non-cocaine control engram, n = 8, Cocaine engram, n = 10, F (1, 16) = 0.118, p = 0.735]. (c-f) AAV TRE-tight-mCherry was infused into the vCA1 of c-fos-tTA mice 2 weeks before behavioral experiments. To label control-engram cells, mice were exposed to a novel context in the absence of Dox before cocaine-CPP training. To label cocaine-engram cells, mice were conditioned with cocaine in the absence of Dox. One hour after memory retrieval, mice were sacrificed for c-Fos immunostaining. (c) Experiment scheme. (d) Strategy for engram labeling (left) and representative images of c-Fos expression in cocaine engram and non-cocaine control engram cells (right). The experiment was independently repeated with similar results in the experimental mice in panel e and f. Scale bar: 100 µm. (e) Venn diagram and bar graphs showed activation of non-cocaine control engram and cocaine engram (c-Fos+ mCherry+) induced by reexposure to CPP apparatus. [n = 6 of each group, Kruskal-Wallis One-way ANOVA on ranks or One-way ANOVA, H = 8.337 with 1 degrees of freedom, p = 0.004]. (f) Summary graphs of percentage of control engram and cocaine engram activated after memory retrieval. [n = 6 of each group, One-way ANOVA, c-Fos+ mCherry+ / c-Fos+: F (1, 10) = 45.808, p = 0.000049; c-Fos+ mCherry+ / mCherry+: F (1, 10) = 71.800, p = 0.000007]. **p < 0.01, ***p < 0.001. Bar graphs show mean (bar) ± s.e.m. and individual data (circles).

Supplementary Figure 4 vCA1 cocaine-engram collateralization is preferentially recruited in the Acb.

(a-c) AAVs TRE-tight-eNpHR3.0-eYFP and CaMKIIα:ChR2-mCherry were infused into the vCA1 of c-fos-tTA mice 2 weeks before behavioral experiments. No-Dox diet was provided either during a novel context exposure (days −2 and −1) or cocaine conditioning (days 2 and 3) to allow c-fos driven eYFP expression. 21 days after labeling, the quantification of eYFP and mCherry fluorescence in Acb, PrL, and BLA were performed. (a) Experiment scheme. (b) Strategy for terminal labeling of vCA1 engram (left) and representative images of projections from cocaine-engram and non-cocaine control-engram of vCA1. The broken white lines show the position of AC. The experiment was independently repeated with similar results in the experimental mice in panel c. (c) Summary graphs of eYFP / mCherry fluorescence ratios. Scale bar: 100 µm. [Non-cocaine control-engram, n = 8, Cocaine-engram, n = 9, Kruskal-Wallis One-way ANOVA on ranks or One-way ANOVA, vCA1: H = 0.454 with 1 degrees of freedom, p = 0.501; AcbC: H = 8.333 with 1 degrees of freedom, p = 0.004; PrL: H = 0.333 with 1 degrees of freedom, p = 0.564; Amygdala: F (1, 15) = 0.009, p = 0.924.] **p < 0.01. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 5 Photoactivation of vCA1-engram increases GCaMP fluorescence of AcbC-engram simultaneously.

(a-c) AAV TRE-ChR2-mCherry was infused into the vCA1, and AAVs TRE3g-Cre, DIO-GCaMP6s, and hSyn-mCherry were infused into the AcbC of c-fos-tTA mice. Optical fibers were implanted in the vCA1 and AcbC, and GCaMP6s response to photostimulation of vCA1 were recorded in AcbC on Day 5. (a) Behavioral scheme. (b) Schematic of virus injection, photostimulation and fluorescence recordings. (c,d) Heatmaps illustrate the mCherry or GCaMP fluorescence (ΔF/F %) of 90 trials per mice from -2 s to 10 s in response to photostimuation (473 nm laser, a train of ten 15-ms light pulses at 20 Hz every 10 s for 15 min, blue vertical bars). Peri-event plots illustrate the averaged fluorescence of each mice. The curves and shaded regions indicate the mean ± s.e.m. [n = 7 mice].

Supplementary Figure 6 Chemogenetic stimulation of vCA1-engram induces AcbC-engram activation, while suppression of vCA1-engram inhibits AcbC-engram activation induced by memory retrieval.

(a-c) AAV TRE-tight-hM3D-mCherry was infused into the vCA1 of c-fos-tTA×tetO-Cre×Ai14 mice 2 weeks before behavioral experiments. No-Dox diet was provided during cocaine conditioning. On Day 5, 90 min after CNO (1 mg/kg, i.p.) or saline injection mice were sacrificed for immunohistochemical analysis. (a) Experiment scheme and schematic of virus injection. (b, c) Representative images (b) and summary graphs (c) of tdTomato and c-Fos fluorescence in the AcbC. The broken white lines show the position of AC. Scale bar: 100 µm. [Saline n = 4, CNO n = 4. One-way ANOVA, tdTomato+ cell counts: F (1, 6) = 0.561, p = 0.482, c-Fos+ cell counts: F (1, 6) = 15.526, p = 0.008; Kruskal-Wallis One Way Analysis of Variance on ranks, tdTomato+ c-Fos+ cell counts: H = 5.600 with 1 degrees of freedom. p = 0.020, tdTomato+ c-Fos+ / tdTomato+: F (1, 6) = 15.672, p = 0.007]. *p <0.05, **p < 0.01. (d-f) AAV TRE-tight-hM4D-mCherry was infused into the vCA1 of c-fos-tTA × tetO-Cre × Ai14 mice 2 weeks before behavioral experiments. No-Dox diet was provided during cocaine-paired conditioning. CNO treatment (1 mg/kg, i.p.) was given 30 min before Test 1 and mice were sacrificed for immunohistochemical analysis of tdTomato and c-Fos expression 60 min after Test 1. (d) Experiment scheme and schematic of virus injection. (e, f) Representative images (e) and summary graphs (f) of tdTomato and c-Fos fluorescence in the AcbC. The broken white lines show the position of AC. Scale bar: 100 µm. [One-way ANOVA, Saline n = 3, CNO n = 6. tdTomato+ cell counts: F (1, 7) = 3.731, p = 0.095, c-Fos+ cell counts: F (1, 7) = 113.655, p = 0.000014; tdTomato+ c-fos+ cell counts: F (1, 7) = 43.839, p = 0.000298, tdTomato+ c-fos+ / tdTomato+: F (1, 7) = 112.664, p = 0.000017]. ***p < 0.001. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 7 Chemogenetic activation of vCA1-engram does not change memory retrieval of cocaine-CPP or locomotion.

(a-c) AAV TRE-tight-hM3D-mCherry was infused into the vCA1 of c-fos-tTA mice. No-Dox food was provided during cocaine conditioning to allow the c-fos-driven expression of hM3D-mCherry. On Day 5, CNO (1 mg/ kg, i.p.) or saline was injected 30 min before Test 1, and memory retention was tested on Day 6 (Test 2). In another cohort of mice, on Day 5, CNO (1 mg/ kg, i.p.) or saline was injected 30 min before open field test. (a) Experiment scheme. (b) Schematic of chemogenetic stimulation (left) and expression of hM3D-mCherry in vCA1-engram (right). The experiment was independently repeated with similar results in the experimental mice in panel c or d. Scale bar: 100 μm. (c) Summary bar graphs of CPP scores. [Saline n = 12, CNO n = 11, Two-way RM ANOVA, Ftreatment × block (2, 42) = 0.176, p = 0.839.] (d) Summary bar graphs of distance traveled in the open field test. [Saline n = 18, CNO n = 18, One-way ANOVA, F (1, 34) = 0.752, p = 0.392]. Bar graphs show mean (bar) ± s.e.m. and individual data (circles).

Supplementary Figure 8 AcbC neurons activated by the retrieval of cocaine-CPP memory are mostly D1-MSNs.

(a-c) D1-tdTomato or D2-eGFP mice were injected with AAV c-fos-tTA and TRE-tight-eYFP or TRE-tight-mCherry in the AcbC 2 weeks before cocaine conditioning. No-Dox food was provided during cocaine-paired conditioning to allow the c-fos-driven expression of eYFP or mCherry. Mice were sacrificed one day after cocaine-conditioning for analysis of c-Fos+ D1+ and c-Fos+ D2+ fluorescence in the AcbC. (a) Experiment scheme. (b) Strategy for engram labeling during training and representative images. The broken white lines show the position of AC. (c) Summary bar graphs of c-Fos+ D1+ and c-Fos+ D2+ fluorescence. [D1-tdTomato: n = 5, D2-eGFP: n = 4, Kruskal-Wallis One-way ANOVA on ranks or One-way ANOVA, left: H = 6.000 with 1 degrees of freedom, p = 0.014; right: F (1, 7) = 569.459, p < 0.000001]. *p < 0.05, ***p < 0.001. (d-h) D1-tdTomato and D2-eGFP mice were conditioned for cocaine-CPP from days 1 to 3. On Day 4, 1 hr after memory retrieval, mice were used for histochemical analysis of tdTomato, eGFP, and c-Fos fluorescence in the AcbC. (d) Experiment scheme. (e-h) Representative images (e, f) and summary bar graphs (g, h) of tdTomato, eGFP, and c-Fos fluorescence. The broken white lines show the position of AC. Scale bar: 100 µm. [One-way ANOVA, g: D1-tdTomato n = 4, D2-eGFP n = 7, double positive cell counts: F (1, 9) = 32.749, p = 0.000286; double positive / c-fos+: F (1, 9) = 77.588, p = 0.00001; h: D1-tdTomato: Non-Retrieval, n = 4, Retrieval, n = 4, F (1, 6) = 150.821, p = 0.000018, D2-eGFP: Non-Retrieval, n = 4, Retrieval, n = 7, F (1, 9) = 7.128, p = 0.026]. *p < 0.05, ***p < 0.001. (i-k) AAV DIO-hM4D-mCherry or DIO-hM3D-mCherry was infused in the AcbC of D1-Cre mice. (i) Schematic of chemogenetic stimulation and electrophysiological recordings. (j) Action potentials recorded from hM4D or hM3D-expressing D1-MSNs in response to progressively higher depolarizing currents before (baseline) and 15 min after CNO application (5 μM). (k) Rheobase increased in hM4D expressing D1-MSNs and decreased in hM3D expressing D1-MSNs after CNO application [n = 8 neurons from 3 mice of each group, Kruskal-Wallis One-way ANOVA on ranks or One-way ANOVA, top: H = 0.011 with 1 degrees of freedom, p = 0.959; middle: F (1, 14) = 30.096, p = 0.000080; bottom: F (1, 14) = 7.559, p = 0.016]. *p < 0.05, ***p < 0.001. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 9 Anatomic engram connection from vCA1 to AcbC.

(a-c) AAV DIO-WGA was infused into the AcbC of D1-Cre (a) or D2-Cre (b) mice. (a, b) Strategy for retrograde labeling and representative images. The broken white lines show the position of AC. (c) Summary bar graph of WGA+ cells counts in the vCA1. [D1-Cre n = 10, D2-Cre n = 12, Kruskal-Wallis One-way ANOVA on ranks, H = 15.110 with 1 degrees of freedom, p = 0.000101] ***p < 0.001. (d-f) Retrograde labelling with rabies-virus system. AAV EF1α-DIO-his-EGFP-2a-TVA and AAV-EF1α-DIO-RVG were injected in the AcbC of D1-Cre or D2-Cre mice, followed by RV-ENVA-deltaG-dsRed injection 2 weeks later. (d, e) Strategy for retrograde labeling and representative images of starter cells in the AcbC and RVdG-dsRed+ cells in the vCA1 that provide direct monosynaptic inputs on D1-MSNs (d) and D2-MSNs (e). The broken white lines show the position of AC. (f) Summary bar graph of input connection strength index (numbers of labeled presynaptic neurons / numbers of starter neurons) in the vCA1. [D1-Cre n = 5, D2-Cre n = 4, Kruskal-Wallis One-way ANOVA on ranks, H = 6.000 with 1 degrees of freedom, p = 0.014] *p < 0.05. (g, h) Anterograde labelling with scAAV system. scAAV2/1-hSyn-Cre was injected in the vCA1 of Ai14 mice and smFISH was performed with probes of drd1, drd2, and tdTomato. (g) Strategy for anterograde labeling and representative image. (h) Summary bar graph of Drd1+ tdTomato+ and Drd2+ tdTomato+ cell counts in the AcbC. [n = 5, One-way ANOVA, F (1, 8) = 158.833, p = 0.000001] ***p < 0.001. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 10 Enhanced synaptic potentiation of AcbC-engram cells over non-engram cells.

(a-d) Whole-cell patch clamp was performed on AcbC-containing slices of D1-tdTomato and D2-eGFP mice one day after saline or cocaine conditioning. (a) Experiment scheme. (b) Representative traces of mEPSC. (c,d) Summary plots of mEPSC amplitude (c) and frequency (d) [Two-way ANOVA, Amplitude: D1-MSN Saline n = 18 from 5 mice, Cocaine: n = 22 from 6 mice, D2-MSN Saline n = 15 from 4 mice, Cocaine n = 20 from 5 mice, Ftreatment×cell type (1, 71) = 2.318, p = 0.132; Frequency: D1-MSN Saline n = 18 from 5 mice, Cocaine n = 22 from 6 mice; D2-MSN Saline n = 15 from 4 mice, Cocaine n = 20 from 6 mice, Ftreatment×cell type (1, 71) = 8.245, p = 0.005]. *p < 0.05, ***p <0.001. (e-h) No-Dox diet was provided during cocaine- conditioning in c-fos-tTA × tetO-H2B-GFP mice. Electrophysiological recordings were carried out on Day 5 on brain slices containing AcbC. (e) Experiment scheme. (f) Representative mEPSC traces of engram and non-engram cells. (g, h) Summary plots of mEPSC amplitude (g) and frequency (h) of engram and non-engram cells. [Non-engram: 16 neurons from 5 mice, Engram: 22 neurons from 5 mice, Kruskal-Wallis One-way ANOVA on ranks, Amplitude: H = 16.889 with 1 degrees of freedom, p = 0.00004; Frequency: H = 9.094 with 1 degrees of freedom, p = 0.003. Kolmogorov-Smirnov test, Cumulative probability of the amplitude: p < 0.000001; Cumulative probability of the interevent intervals: p < 0.000001]. **p < 0.01, ***p < 0.001. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 11 Cocaine conditioning does not strengthen the synaptic connectivity of vCA1-AcbC circuits of control-engram or D2-engram.

(a-c) AAVs c-fos-tTA, TRE-tight-mCherry, and DIO-H2B-GFP were infused into the AcbC, and AAVs c-fos-tTA and TRE3g-ChR2-mCherry were infused into the vCA1 of D1-Cre mice. No-Dox food was provided during neutral context exposure, followed by cocaine-CPP conditioning. Electrophysiological recording on AcbC control-engram were performed on slice on Day 5. (a) Experiment scheme and schematic of photostimulation and electrophysiological recordings. (b, c) Representative traces, summary bar graphs of PPR (b), and A/N ratio (c) of AcbC control-engram cells recorded upon photostimulation (473 nm laser, 5 ms pulse width, blue vertical bar) of ChR2-expressing axon terminals of vCA1control-engram cells in the AcbC. [One-way ANOVA, PPR (50 ms): 16 D1-Non-engram neurons from 5 mice, 13 D1-Engram neurons from 5 mice, F (1, 27) = 0.056, p = 0.815; PPR (100 ms): 15 D1-Non-engram neurons from 5 mice, 13 D1-Engram neurons from 5 mice, F (1, 26) = 0.287, p = 0.597; A/N: 14 D1-Non-engram neurons from 5 mice, 15 D1-Engram neurons from 5 mice, F (1, 27) = 1.287, p = 0.266]. (d-f) AAVs c-fos-tTA, TRE-tight-mCherry, and DIO-H2B-GFP were infused into the AcbC, and AAVs c-fos-tTA and TRE3g-ChR2-mCherry were infused into the vCA1 of D2-Cre mice. No-Dox food was provided during cocaine-conditioning. On Day 5, electrophysiological recording of AcbC D2-engram (H2B-GFP+ mCherry+) and D2-non-engram (H2B-GFP+ mCherry—) cells were performed on slice. (d) Experiment scheme and schematic of photostimulation and electrophysiological recordings. (e, f) Representative traces, summary bar graphs of PPR (e), and A/N ratio (f) of AcbC D2-non-engram and D2-engram cells recorded upon photostimulation (473 nm laser, 5 ms pulse width, blue vertical bar) of ChR2-expressing axon terminals of vCA1-engram cells in the AcbC. [One-way ANOVA, PPR (50 ms): 11 D2-Non-engram neurons from 7 mice, 10 D2-Engram neurons from 7 mice, F (1, 19) = 0.288, p = 0.598; PPR (100 ms): 11 D2-Non-engram neurons from 7 mice, 10 D2-Engram neurons from 7 mice, F (1, 19) = 0.44, p = 0.515; A/N: 12 D1-Non-engram neurons from 7 mice, 11 D1-Engram neurons from 7 mice, F (1, 21) = 0.008, p = 0.929]. Bar graphs show mean ± s.e.m. and individual data (circles).

Supplementary Figure 12 LTD induction in vCA1-AcbC engram projection does not affect locomotion.

(a-d) AAV TRE-ChR2-mCherry was infused into the vCA1 of c-fos-tTA mice 2 weeks before behavioral experiments. No-Dox diet was provided during cocaine-paired conditioning. On days 5–7, Open-field, light/dark box, and elevated O maze tasks were performed, and in vivo photostimulation (LFS: 473 nm laser, 4 ms pulse width, 1 Hz, 10 min) in the AcbC was given 40 min before each task. (a) Experiment scheme and schematic of photostimulation. (b) Open-field test. (c) Light/dark box task. (d) O-maze task. [mCherry: n = 10, ChR2-mCherry: n = 11. b, Left, Kruskal-Wallis One Way Analysis of Variance on ranks, H = 0.600 with 1 degrees of freedom, p = 0.439; b, Middle, Friedman’s two way RM ANOVA by ranks, F (5, 95) = 0.75, p = 0.588; b, Right, One way ANOVA, F (1, 19) = 0.295, p = 0.594. c, One way ANOVA, F (1, 19) = 2.923, p = 0.104. d, One way ANOVA, F (1, 19) = 1.750, p = 0.202]. Bar graphs show mean ± s.e.m. and individual data (circles).

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Zhou, Y., Zhu, H., Liu, Z. et al. A ventral CA1 to nucleus accumbens core engram circuit mediates conditioned place preference for cocaine. Nat Neurosci 22, 1986–1999 (2019) doi:10.1038/s41593-019-0524-y

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