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.

Fig. 1|. Pairing either acetophenone or M72 photostimulation with footshock produces conditioned odor aversion.

a, Mice were presented with different combinations of an odor CS (acetophenone) and a US (footshock) during training and tested 24 h later in a neutral apparatus containing the CS (acetophenone (A)) at one end and a distinct odor (carvone (C)) at the other (CS + US group, n = 15 mice; home cage (HC), n = 8 mice; US only, n = 15 mice; CS only, n = 8 mice; US│CS group, n = 12 mice). b, In a 10-min test, only conditioned mice (CS + US) avoided acetophenone (two-tailed, one sample t-test versus zero preference: CS + US group, t₁₄ = 4.66, P = 0.004). The shaded region represents s.e.m. for the CS + US group (red) and control groups (gray). c, Summary data showing that conditioned mice differed from each control group (unequal variance, Kruskal–Wallis, χ²(4) = 15.73, P = 0.0034; *Mann–Whitney U planned comparisons: CS + US versus HC, U = 16, P = 0.0032; CS + US versus US only, U = 39.5, P = 0.0017; CS + US versus CS only, U = 23, P = 0.0159, CS + US versus US│CS, U = 36, P = 0.0074). Error bars represent s.e.m. d, M72 glomeruli expressing ChR2-YFP (green) in the olfactory bulb of M72 mice. Scale bar, 500 μm. e, Mice were presented with different combinations of M72 photostimulation (CS; blue denotes artificial) and footshock (US) during training and tested 24 h later in a neutral apparatus containing acetophenone and carvone (CS + US group, n = 18 mice; CS only, n = 8 mice; US│CS group, n = 8 mice). f, In this test, only conditioned mice (CS + US) avoided acetophenone (two-tailed, one sample t-test versus zero preference: t₁₇ = 5.31, P < 0.0001). The shaded region represents s.e.m. for the CS + US group (pink) and control groups (gray). g, Summary data showing that conditioned mice differed from each control group (ANOVA, F_2,31 = 8.91, P = 0.0009, post hoc *P< 0.05). Error bars represent s.e.m.

Fig. 2|. Generation of artificial memories by pairing M72 photostimulation with photostimulation of distinct LHb inputs into the VTA.

a,b, During training, M72 OSN afferent photostimulation was explicitly paired (CS + US, n = 12 mice) or not (US | CS, n = 8 mice) with photostimulation of the LHb terminal field in the medial VTA, and preference for M72-activating odorant (acetophenone) versus non-M72-activating odorant (carvone) was tested 24 h later. c, Low- and high-magnification (inset) images showing labeled LHb–VTA projections (pink) following infection of LHb with AAV-ChR2. Scale bars, 400 μm (low magnification), 100 μm (high magnification). d, In the test, only conditioned mice (CS + US) avoided acetophenone (two-tailed, one sample t-test versus zero preference: t₁₁ = 4.96, P = 0.0004). The shaded region represents s.e.m. for the CS + US group (pink) and US│CS group (gray). e, Summary data showing that preference in conditioned mice differed from that in control mice. CS and US shown in blue denote that these stimuli were artificial (two-tailed t-test: t₁₈ = 2.67, *P = 0.016). Error bars represent s.e.m. f,g, During training, M72 photostimulation was explicitly paired (CS + US, n = 10 mice) or not (US│CS, n = 10 mice) with photostimulation of the LDT terminal field in the lateral VTA, and preference for M72 odorant (acetophenone) versus non-M72 odorant (carvone) was tested 24 h later. h, Low- and high-magnification (inset) images showing labeled LDT–VTA projections (sky blue) following infection of LDT with AAV-ChR2. Scale bars, 400 μm (low magnification), 100 μm (high magnification). i, In the test, only conditioned mice (CS + US) were attracted to acetophenone (two-tailed t-test versus zero preference: t₉ = 2.92, P = 0.017). The shaded region represents s.e.m. for the CS + US group (blue) and US │CS group (gray). j, Summary data showing that preference in conditioned mice differed from that in control mice. CS and US shown in blue to denote that these stimuli were artificial (two-tailed t-test: t₁₈ = 2.45, *P = 0.025). Error bars represent s.e.m.

Fig. 3|. Real and artificial memories engage similar neural circuits.

a,b, In the real memory condition, during training mice were presented with either acetophenone alone (CS only, n = 6 mice) or acetophenone paired with footshock (CS + US, n = 6 mice). In the artificial memory condition, during training mice received either M72 photostimulation alone (CS only, n = 8 mice) or M72 photostimulation paired with photostimulation of LHb–VTA projections (CS + US, n = 7 mice). c, One day later, CS-induced Fos induction was analyzed in the central olfactory system and in regions involved in associative memory. These are sagittal sections, and numbers indicate distance from midline. Scale bar, 1 mm. d,e, Fold changes in c-Fos expression in CS + US versus CS-only groups in the real (left) and artificial (right) memory conditions compared to training-naive control mice (broken black line). The shaded area represents s.e.m. f,g, Sagittal sections showing Fos induction (white) in the HC, CS and CS + US conditions. Scale bar, 100 μm. Fos induction was elevated in the BLA in mice that formed a real memory (two-tailed t-test: CS + US > CS only, t₁₀ = 2.97, *P = 0.014). h,i, Sagittal sections showing Fos induction (white) in the HC, CS and CS + US conditions. Scale bar, 100 μm. Fos induction was elevated in the BLA in mice that formed an artificial memory (two-tailed t-test: CS + US > CS only, t₁₂ = 3.24, *P = 0.0071). Broken black lines represent c-Fos expression levels in training-naive control mice. Error bars represent s.e.m. AO, anterior olfactory nucleus; DTT, dorsal tenia tecta; Pir, piriform cortex; Tu,olfactory tubercle; LOT, nucleus of the lateral olfactory tract; PLCo, osterolateral cortical amygdaloid nucleus; PMCo, posteromedial cortical amygdaloid nucleus; APir, amygdalopiriform transition area; LEnt, lateral entorhinal cortex; BMA, basomedial amygdaloid nucleus; Ce, central amygdaloid nucleus; La, lateral amygdaloid nucleus; CA1, field CA1 of the hippocampus; RTn, rostromedial tegmental nucleus; VO, ventral orbital cortex; Cg, cingulate cortex; V1, primary visual cortex.

Fig. 4|. Silencing the BLA prevents expression of real and artificial odor memories.

a,c, AAV-hM4Di-mCherry was injected into the BLA of WT mice. Broken lines indicate anatomical boundaries. Scale bars, 100 μm (high magnification) and 400 μm (low magnification). b, During training, acetophenone was paired with footshock. Before testing, mice were treated with vehicle (VEH; n = 11) or C21 (n = 9). d, Only vehicle-treated mice avoided acetophenone (two-tailed, one sample t-test versus zero preference: t₁₀ = 3.70, P < 0.0041). The shaded regions represent s.e.m. e, Summary data showing that preference in vehicle-treated and C21-treated mice differed (two-tailed t-test: t₁₈ = 2.24, *P = 0.038). Error bars represent s.e.m. f,h, M72-ChR2 mice received infusions of AAV-hM4Di-mCherry into the BLA and AAV-ChR2 into the LHb. Optrodes were implanted above the M72 glomerulus and VTA. Broken lines indicate anatomical boundaries. Scale bars, 200 μm (top left, olfactory bulb), 400 μm (top right, LHb) and 400 μm (bottom, BLA). g, During training, M72 photostimulation was paired with photostimulation of LHb–VTA projections. Before testing, mice were treated with vehicle (n = 7) or C21 (n = 7). i, Only vehicle-treated mice avoided acetophenone (two-tailed, one sample t-test versus zero preference: t₆ = 2.76, P = 0.033). The shaded region represents s.e.m. j, Summary data showing that preference in vehicle-treated and C21-treated mice differed (two-tailed t-test: t₁₂ = 2.42, *P = 0.033). Error bars represent s.e.m.