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Memory formation in the absence of experience

Abstract

Memory is coded by patterns of neural activity in distinct circuits. Therefore, it should be possible to reverse engineer a memory by artificially creating these patterns of activity in the absence of a sensory experience. In olfactory conditioning, an odor conditioned stimulus (CS) is paired with an unconditioned stimulus (US; for example, a footshock), and the resulting CS–US association guides future behavior. Here we replaced the odor CS with optogenetic stimulation of a specific olfactory glomerulus and the US with optogenetic stimulation of distinct inputs into the ventral tegmental area that mediate either aversion or reward. In doing so, we created a fully artificial memory in mice. Similarly to a natural memory, this artificial memory depended on CS–US contingency during training, and the conditioned response was specific to the CS and reflected the US valence. Moreover, both real and implanted memories engaged overlapping brain circuits and depended on basolateral amygdala activity for expression.

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Fig. 1: Pairing either acetophenone or M72 photostimulation with footshock produces conditioned odor aversion.
Fig. 2: Generation of artificial memories by pairing M72 photostimulation with photostimulation of distinct LHb inputs into the VTA.
Fig. 3: Real and artificial memories engage similar neural circuits.
Fig. 4: Silencing the BLA prevents expression of real and artificial odor memories.

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Data availability

All data supporting the findings of this study are available from the corresponding author upon request.

Code availability

Custom code for behavioral analysis is available from the corresponding author upon request.

References

  1. Eichenbaum, H. Still searching for the engram. Learn. Behav. 44, 209–222 (2016).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Martin, S. J. & Morris, R. G. New life in an old idea: the synaptic plasticity and memory hypothesis revisited. Hippocampus 12, 609–636 (2002).

    Article  CAS  Google Scholar 

  5. Buck, L. & Axel, R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175–187 (1991).

    Article  CAS  Google Scholar 

  6. Buck, L. B. The molecular architecture of odor and pheromone sensing in mammals. Cell 100, 611–618 (2000).

    Article  CAS  Google Scholar 

  7. Firestein, S. How the olfactory system makes sense of scents. Nature 413, 211–218 (2001).

    Article  CAS  Google Scholar 

  8. Mombaerts, P. et al. Visualizing an olfactory sensory map. Cell 87, 675–686 (1996).

    Article  CAS  Google Scholar 

  9. Ressler, K. J., Sullivan, S. L. & Buck, L. B. Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245–1255 (1994).

    Article  CAS  Google Scholar 

  10. Wang, F., Nemes, A., Mendelsohn, M. & Axel, R. Odorant receptors govern the formation of a precise topographic map. Cell 93, 47–60 (1998).

    Article  CAS  Google Scholar 

  11. Jones, S. V., Choi, D. C., Davis, M. & Ressler, K. J. Learning-dependent structural plasticity in the adult olfactory pathway. J. Neurosci. 28, 13106–13111 (2008).

    Article  CAS  Google Scholar 

  12. Morrison, F. G., Dias, B. G. & Ressler, K. J. Extinction reverses olfactory fear-conditioned increases in neuron number and glomerular size. Proc. Natl Acad. Sci. USA 112, 12846–12851 (2015).

    Article  CAS  Google Scholar 

  13. Jiang, Y. et al. Molecular profiling of activated olfactory neurons identifies odorant receptors for odors in vivo. Nat. Neurosci. 18, 1446–1454 (2015).

    Article  CAS  Google Scholar 

  14. Smear, M., Resulaj, A., Zhang, J., Bozza, T. & Rinberg, D. Multiple perceptible signals from a single olfactory glomerulus. Nat. Neurosci. 16, 1687–1691 (2013).

    Article  CAS  Google Scholar 

  15. Lammel, S. et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature 491, 212–217 (2012).

    Article  CAS  Google Scholar 

  16. Cousens, G. & Otto, T. Both pre- and posttraining excitotoxic lesions of the basolateral amygdala abolish the expression of olfactory and contextual fear conditioning. Behav. Neurosci. 112, 1092–1103 (1998).

    Article  CAS  Google Scholar 

  17. Thompson, K. J. et al. DREADD agonist 21 (C21) is an effective agonist for muscarinic based DREADDs in vitro and in vivo. ACS Pharmacol. Transl Sci. 1, 61–72 (2018).

    Article  CAS  Google Scholar 

  18. Walker, D. L., Paschall, G. Y. & Davis, M. Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning and expression. Learn. Mem. 12, 120–129 (2005).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Gore, F. et al. Neural representations of unconditioned stimuli in basolateral amygdala mediate innate and learned responses. Cell 162, 134–145 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Yiu, A. P. et al. Neurons are recruited to a memory trace based on relative neuronal excitability immediately before training. Neuron 83, 722–735 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Rashid, A. J. et al. Competition between engrams influences fear memory formation and recall. Science 353, 383–387 (2016).

    Article  CAS  Google Scholar 

  26. Ramirez, S. et al. Creating a false memory in the hippocampus. Science 341, 387–391 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Ohkawa, N. et al. Artificial association of pre-stored information to generate a qualitatively new memory. Cell Rep. 11, 261–269 (2015).

    Article  CAS  Google Scholar 

  29. Dudai, Y. in Science of Memory: Concepts (eds Roediger, H. L. III, Dudai, Y. & Fitzpatrick, S. M.) 13–16 (Oxford Univ. Press, 2007).

  30. Shinkman, P. G., Swain, R. A. & Thompson, R. F. Classical conditioning with electrical stimulation of cerebellum as both conditioned and unconditioned stimulus. Behav. Neurosci. 110, 914–921 (1996).

    Article  CAS  Google Scholar 

  31. Steinmetz, J. E., Lavond, D. G. & Thompson, R. F. Classical conditioning in rabbits using pontine nucleus stimulation as a conditioned stimulus and inferior olive stimulation as an unconditioned stimulus. Synapse 3, 225–233 (1989).

    Article  CAS  Google Scholar 

  32. Hegoburu, C., Parrot, S., Ferreira, G. & Mouly, A. M. Differential involvement of amygdala and cortical NMDA receptors activation upon encoding in odor fear memory. Learn. Mem. 21, 651–655 (2014).

    Article  CAS  Google Scholar 

  33. Sevelinges, Y., Gervais, R., Messaoudi, B., Granjon, L. & Mouly, A. M. Olfactory fear conditioning induces field potential potentiation in rat olfactory cortex and amygdala. Learn. Mem. 11, 761–769 (2004).

    Article  Google Scholar 

  34. McGann, J. P. Associative learning and sensory neuroplasticity: how does it happen and what is it good for? Learn. Mem. 22, 567–576 (2015).

    Article  CAS  Google Scholar 

  35. Herry, C. & Johansen, J. P. Encoding of fear learning and memory in distributed neuronal circuits. Nat. Neurosci. 17, 1644–1654 (2014).

    Article  CAS  Google Scholar 

  36. Ozawa, T. et al. A feedback neural circuit for calibrating aversive memory strength. Nat. Neurosci. 20, 90–97 (2017).

    Article  CAS  Google Scholar 

  37. Di Scala, G., Mana, M. J., Jacobs, W. J. & Phillips, A. G. Evidence of Pavlovian conditioned fear following electrical stimulation of the periaqueductal grey in the rat. Physiol. Behav. 40, 55–63 (1987).

    Article  Google Scholar 

  38. Kim, E. J. et al. Dorsal periaqueductal gray–amygdala pathway conveys both innate and learned fear responses in rats. Proc. Natl Acad. Sci USA 110, 14795–14800 (2013).

    Article  CAS  Google Scholar 

  39. Han, S., Soleiman, M. T., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Elucidating an affective pain circuit that creates a threat memory. Cell 162, 363–374 (2015).

    Article  CAS  Google Scholar 

  40. Sato, M. et al. The lateral parabrachial nucleus is actively involved in the acquisition of fear memory in mice. Mol. Brain 8, 22 (2015).

    Article  Google Scholar 

  41. Tang, J. et al. Pavlovian fear memory induced by activation in the anterior cingulate cortex. Mol. Pain 1, 6 (2005).

    Article  Google Scholar 

  42. Johansen, J. P. & Fields, H. L. Glutamatergic activation of anterior cingulate cortex produces an aversive teaching signal. Nat. Neurosci. 7, 398–403 (2004).

    Article  CAS  Google Scholar 

  43. Tye, K. M. & Deisseroth, K. Optogenetic investigation of neural circuits underlying brain disease in animal models. Nat. Rev. Neurosci. 13, 251–266 (2012).

    Article  CAS  Google Scholar 

  44. Carey, R. M. & Wachowiak, M. Effect of sniffing on the temporal structure of mitral/tufted cell output from the olfactory bulb. J. Neurosci. 31, 10615–10626 (2011).

    Article  CAS  Google Scholar 

  45. Doucette, W. et al. Associative cortex features in the first olfactory brain relay station. Neuron 69, 1176–1187 (2011).

    Article  CAS  Google Scholar 

  46. Rojas-Libano, D. & Kay, L. M. Interplay between sniffing and odorant sorptive properties in the rat. J. Neurosci. 32, 15577–15589 (2012).

    Article  CAS  Google Scholar 

  47. Shusterman, R., Smear, M. C., Koulakov, A. A. & Rinberg, D. Precise olfactory responses tile the sniff cycle. Nat. Neurosci. 14, 1039–1044 (2011).

    Article  CAS  Google Scholar 

  48. Wesson, D. W., Donahou, T. N., Johnson, M. O. & Wachowiak, M. Sniffing behavior of mice during performance in odor-guided tasks. Chem. Senses 33, 581–596 (2008).

    Article  Google Scholar 

  49. Kepecs, A., Uchida, N. & Mainen, Z. F. The sniff as a unit of olfactory processing. Chem. Senses 31, 167–179 (2006).

    Article  Google Scholar 

  50. Vetere, G. et al. Chemogenetic interrogation of a brain-wide fear memory network in mice. Neuron 94, 363–374 (2017).

    Article  CAS  Google Scholar 

  51. Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotaxic Coordinates (Academic Press, 2012).

  52. Renier, N. et al. Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165, 1789–1802 (2016).

    Article  CAS  Google Scholar 

  53. Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).

    Article  CAS  Google Scholar 

  54. Preibisch, S., Saalfeld, S. & Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25, 1463–1465 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Canadian Institute of Health Research (CIHR) grants to P.W.F. (FDN143227) and S.A.J. (MOP74650) and by National Institute of Mental Health grants to K.J.R. (R01-MH108665) and F.G.M. (F31-MH105237). S.A.J. is a CIHR Canada Research Chair in Memory Function and Dysfunction. P.W.F. is a CIHR Canada Research Chair in Cognitive Neurobiology. The authors thank A. Ramsaran and J. Johansen for comments on an earlier draft of this manuscript.

Author information

Authors and Affiliations

Authors

Contributions

G.V. and P.W.F. conceived the study and designed the experiments. G.V., L.M.T., and S.M. conducted the behavioral and immunohistochemical experiments. G.V. and L.R. conducted data and statistical analyses. P.E.S. conducted the iDISCO tissue clearing. P.W.F., G.V., K.J.R., F.G.M., and S.A.J. wrote the paper.

Corresponding authors

Correspondence to Sheena A. Josselyn or Paul W. Frankland.

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The authors declare no competing interests.

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Journal peer review information: Nature Neuroscience thanks Mark Baxter and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 Testing apparatus.

a, Odor preference was assessed in a rectangular box. Filter paper containing acetophenone or carvone was placed in the lid of a Petri dish. This was covered by a Petri dish (with 9 drilled holes), and then bedding. b, Photograph and schematic of test apparatus.

Supplementary Figure 2 Distance traveled in test sessions. These graphs depict the distance traveled during the test session in each of the experiments.

a, Acetophenone paired with shock (ANOVA, F4,46 = 13.10, P < 0.0001). (The data shown here corresponds to groups shown in Fig. 1b, c: CS + US group, n = 15 mice; naïve/home cage, n = 8 mice; US only, n = 15 mice; CS only, n = 8 mice; US│CS group, n = 12 mice). b, M72 photostimulation paired shock (ANOVA, F2,26 = 10.86, P = 0.0004). (The data here corresponds to groups shown in Fig. 1f, g: CS + US group, n = 18 mice; CS only, n = 8 mice; US│CS group, n = 8 mice). c, Acetophenone paired with food (corresponds to data shown in Supplementary Fig. 6, n = 12). d, M72 photostimulation paired with LHb stimulation (2 tailed t-test: t14 = 0.59, P = 0.56). (The data shown here corresponds to groups shown in Fig. 2d, e: CS + US, n = 12 mice; US│CS, n = 8 mice). e, M72 photostimulation paired with LDT stimulation (2 tailed t-test: t18 = 1.29, P = 0.21). (The data shown here corresponds to groups shown in Fig. 2i, j: CS + US, n = 10 mice; US│CS, n = 10 mice). f, Acetophenone paired with shock (2 tailed t-test: t14 = 0.24, P = 0.81) (real memory condition in BLA silencing experiment, corresponding to groups shown in Fig. 4d, e). g. M72 photostimulation paired with LHb photostimulation (2 tailed t-test: t14 = 1.26, P = 0.23) (real memory condition in BLA silencing experiment, corresponding to groups shown in Fig. 4i, j). *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001 by Newman-Keuls. Error bars = s.e.m.

Supplementary Figure 3 Schematic showing responses of the representative glomerular array (circles) to acetophenone and carvone.

Acetophenone activates the M72 glomerulus (and other glomeruli). Carvone activates other glomeruli, but not the M72 glomerulus.

Supplementary Figure 4 Training protocol pairing M72 photostimulation with shock.

Each train included 40 pulses (pulse width 100 ms) delivered at 4 Hz. During the last 1 s of the train, a 0.7 mA shock was delivered. During training, mice received 10 photostimulation-shock pairs.

Supplementary Figure 5 Training protocol pairing M72 photostimulation (artificial CS) with photostimulation of either LHb-VTA or LDT–VTA projections (artificial US).

(Top) M72 stimulation occurred continuously through training, and consisted of 100 ms pulses, delivered at 4 Hz. (Bottom) Phasic VTA stimulation consisted of trains (0.5 s duration) of 5 ms pulses delivered at 30 Hz. Inter-train interval was 2 s.

Supplementary Figure 6 Formation of an odor-reward memory.

a, Schematic showing apparatus and training protocol. Mice were food-deprived one day prior to training. During training, acetophenone was paired with food, and preference (acetophenone vs. carvone) was evaluated one day later. b, In the test, mice (n = 12) spent more time on the acetophenone side of the apparatus (2 tailed, one sample t test vs. zero preference: t11 = 2.89, P = 0.015). Shading represents s.e.m. c, Summary data showing preference scores for individual mice. Error bar = s.e.m.

Supplementary Figure 7 VTA photostimulation alone does not act as a US during conditioning.

During training, acetophenone was paired with VTA photostimulation in mice (n = 12) not expressing ChR2. a, During testing mice did not exhibit conditioned aversion to acetophenone (2 tailed, one sample t test vs. zero preference: t11 = 0.47, P = 0.65). Shading represents s.e.m. b, Summary data showing preference scores for individual mice. Error bar = s.e.m.

Supplementary Figure 8 Recall induced cFos expression.

During the training phase mice received either CS alone (that is, acetophenone [n = 6] or M72 stimulation [n = 8]) or CS + US (that is, acetophenone paired with shock [n = 6] or M72 stimulation paired with LHb-mVTA stimulation [n = 7]). During testing, the CS only was presented, and c-Fos induction assessed in 18 brain regions (see Fig. 3a, b). The numbers of c-Fos+ nuclei are shown for mice in the ‘real’ (left) and ‘artificial’ (right) memory conditions. Error bars = s.e.m. Groups (CS + US vs. CS only) were compared by unpaired t-tests (2 tailed) in the real and artificial memory conditions. a, APir (real): t10 = 0.52, P = 0.62; APir (artificial): t13 = 0.56, P = 0.59. b, Pir (real): t10 = 0.26, P = 0.80; Pir (artificial): t13 = 0.20, P = 0.84. c, PLCo (real): t10 = 0.17, P = 0.86; PLCo (artificial): t13 = 0.20, P = 0.84. d, LEnt (real): t10 = 0.29, P = 0.77; LEnt (artificial): t13 = 1.17, P = 0.26. e, PMCo (real): t10 = 0.00, P = 0.99; PMCo (artificial): t13 = 0.71, P = 0.49. f, Tu (real): t9 = 1.34, P = 0.21; Tu (artificial): t11 = 1.70, P = 0.12. g, DTT (real): t9 = 0.68, P = 0.52; DTT (artificial): t10 = 0.25, P = 0.80. h, LOT (real): t10 = 0.99, P = 0.35; LOT (artificial): t11 = 3.25, P = 0.0077. i, AO (real): t10 = 0.99, P = 0.34; AO (artificial): t9 = 0.53, P = 0.61. j, V1 (real): t10 = 1.41, P = 0.19; V1 (artificial): t13 = 0.06, P = 0.95. k, VO (real): t9 = 0.86, P = 0.41; VO (artificial): t10 = 0.81, P = 0.43. l, BMA (real): t10 = 0.02, P = 0.99; BMA (artificial): t10 = 0.22, P = 0.83. m, CA1 (real): t10 = 0.36, P = 0.73; CA1 (artificial): t10 = 1.13, P = 0.29. n, RTn (real): t9 = 0.95, P = 0.37; RTn (artificial): t8 = 2.90, P = 0.020. o, Cg (real): t9 = 1.43, P = 0.19; Cg (artificial): t9 = 0.58, P = 0.58. p, Ce (real): t10 = 2.05, P = 0.068; Ce (artificial): t13 = 1.91, P = 0.079. q, La (real): t10 = 1.13, P = 0.29; La (artificial): t12 = 0.92, P = 0.38. Abbreviations: APir = amygdalopiriform transition area; Pir = piriform cortex; PLCo = posterolateral cortical amygdaloid nucleus; LEnt = lateral entorhinal cortex; PMCo = posteromedial cortical amygdaloid nucleus; Tu = olfactory tubercle; DTT = dorsal tenia tecta; LOT = nucleus of the lateral olfactory tract; AO = anterior olfactory nucleus; V1 = primary visual cortex; VO = ventral orbital cortex; BMA = basomedial amygdaloid nucleus; CA1 = field CA1 of the hippocampus; RTn = rsotromedial tegmental nucleus; Cg = cingulate cortex; Ce = central amygdaloid nucleus; La = lateral amygdaloid nucleus.

Supplementary Figure 9 Regional cFos expression is highly correlated in real vs artificial conditions.

a, CS only condition. Regional (n = 18 regions) c-Fos expression is highly correlated in mice in the real vs artificial conditioning groups (Pearson’s, r = 0.80, P < 0.0001). b, CS + US condition. Regional (n = 18 regions) c-Fos expression is highly correlated in mice in the real vs artificial conditioning groups (Pearson’s, r = 0.77, P = 0.0002).

Supplementary Figure 10 C21 treatment alone does not affect conditioned odor aversion.

During training, acetophenone was paired with shock in mice not expressing the DREADD, hM4Di. Before testing, mice were treated with VEH (n = 12) or C21 (n = 12). a. Both VEH-treated (one sample, 2 tailed t test vs. zero preference: t11 = 2.92, P = 0.014) and C21-treated (one sample, 2 tailed t test vs. zero preference: t11 = 2.40, P = 0.033) mice avoided acetophenone. Shaded area depicts s.e.m. b. Summary data showing preference in VEH- and C21-treated mice did not differ (2 tailed t test, t22 = 0.34, P = 0.73). Error bars = s.e.m.

Supplementary Figure 11 C21 treatment reduces recall-induced activation of hM4Di-infected neurons in the BLA.

a, b. During training, acetophenone was paired with shock. During testing, VEH-treated mice (n = 4) or C21-treated mice (n = 3) were presented with the CS (acetophenone) and c-Fos was quantified in hM4Di+ neurons in the BLA. C21 treatment reduced c-Fos expression (2 tailed t test, t5 = 3.03, P = 0.029). c, d. During training, M72 stimulation was paired with LHb-VTA stimulation. During testing, VEH-treated mice (n = 4) or C21-treated mice (n = 3) were presented with the CS (M72 stimulation) and c-Fos was measured in hM4Di+ neurons in the BLA. C21 treatment reduced c-Fos expression (2 tailed t test, t5 = 3.15, P < 0.05). In all graphs, error bars = s.e.m. Scale bars (in b and d) = 50 µm.

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Vetere, G., Tran, L.M., Moberg, S. et al. Memory formation in the absence of experience. Nat Neurosci 22, 933–940 (2019). https://doi.org/10.1038/s41593-019-0389-0

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