Research Article

A Neural Circuit for Memory Specificity and Generalization

Science  15 Mar 2013:
Vol. 339, Issue 6125, pp. 1290-1295
DOI: 10.1126/science.1229534

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  1. Fig. 1

    Distinct mPFC neurons project to different synaptic targets. (A) Design of SynaptoTag AAV used for tracing synaptic connections. The synapsin promoter in the AAV drives bicistronic expression of soluble mCherry and a presynaptic EGFP–synaptobrevin-2 fusion protein (EGFP-Syb2). ITR, inverted terminal repeat; IRES, internal ribosome entry site; WPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) SynaptoTag AAV mapping of mPFC projections. Representative low-resolution (left and middle panels) and high-resolution images (right panels) illustrate synaptic targets for mPFC neurons. Red mCherry labeling marks axonal fibers, whereas green EGFP labeling marks synapses projecting from the mPFC (yellow, coincident red and green labeling). BLA, basolateral nucleus of the amygdala; IL, infralimbic cortex; PL, prelimbic cortex; N. acc, nucleus accumbens (for complete sections, see fig. S1). (C) Retrograde labeling of mPFC neurons after injection of Alexa Fluor-488 and -594 labeled cholera toxin-B (CTB-488 and CTB-594) into the NR (, green) and the mediodorsal thalamic nucleus (MD, red), respectively. Low-power micrographs (left panels) show injection areas, whereas high-power images (right panels) depict the three major mPFC regions. Most traced neurons were dominated by the presence of one fluorophore (for additional mPFC projections, see fig. S2).

  2. Fig. 2

    mPFC projection to the NR controls memory specificity. (A) Design of AAVs used for inactivating synaptic transmission in subsets of projection neurons with specific synaptic targets. Double-floxed inverted TetTox-AAV (2xFlx-TetTox AAV) encodes bicistronic expression of EGFP for visualizing infected neurons and of TetTox for blocking synaptic transmission. The coding region of the double-floxed inverted TetTox-AAV is not translated until cre-recombinase flips the inverted coding region into the correct orientation. WGA-cre AAV mediates bicistronic expression of mCherry and WGA-cre. When this AAV infects a neuron, WGA-cre is trans-neuronally transferred to connected neurons, whereas mCherry is only expressed in the infected neuron. (B) Coronal brain section of a mouse injected with 2xFlx-TetTox AAV in the mPFC and with WGA-cre AAV in the dorsomedial striatum. The green EGFP fluorescence in the mPFC indicates that trans-synaptically transported WGA-cre activated expression of TetTox and EGFP in the mPFC. For high-magnification images, additional examples, and quantification of the trans-synaptic transport efficiency, see figs. S3 to S5. (C) Experimental protocol for analyzing the behavioral effects of selective TetTox expression in mPFC neurons that project to specific targets. 2xFIx-TetTox AAV was stereotactically injected into the mouse mPFC, and WGA-cre AAV was injected into the striatum, mediodorsal thalamic nucleus, NR, or mPFC (control, no WGA-cre AAV injection). Mice were tested 4 weeks later for contextual fear conditioning (context test), fear conditioning in an altered context to measure memory precision, and cued fear conditioning (tone test). For additional information, see fig. S6. (D) Fear conditioning measured with the experimental strategy described in (C) in multiple independent experiments (in the left panel, numbers in bars denote the number of mice analyzed). The discrimination index was calculated as the difference between the percentage of freezing in the training context and the altered context, divided by the sum of the two percentages. Data are means ± SEM (error bars); statistical significance (*P < 0.05; **P < 0.01) was assessed by (i) two-way mixed-model analysis of variance (ANOVA) with Bonferroni's post-hoc test comparing the freezing levels or (ii) one-way ANOVA followed by Turkey's post-hoc test for the discrimination index. Horizontal dashed lines indicate the level of control groups.

  3. Fig. 3

    The NR bidirectionally controls fear memory generalization. (A) Representative coronal brain section showing local expression of EGFP (green) after stereotactic injection into the NR of lentiviruses encoding EGFP and TetTox or the neuroligin-2 knockdown (NL2 KD). (B) Schema of the effects of TetTox expression or of the neuroligin-2 knockdown on the activity of neurons in the NR. The neuroligin-2 knockdown decreases inhibition of NR neurons, thereby activating these neurons, whereas TetTox blocks synaptic outputs from NR neurons. (C) Effect of neuroligin-2 knockdown on the frequency of spontaneous inhibitory miniature synaptic events (mIPSCs), recorded in acute NR slices from mice that were injected with neuroligin-2 knockdown lentivirus (numbers in bars denote the number of neurons and mice analyzed, respectively). (D) Experimental protocol for testing fear memory after TetTox expression or neuroligin-2 knockdown in the NR. (E) Bidirectional changes in fear memory generalization by neuronal silencing with TetTox or neuronal activation with the neuroligin-2 knockdown. Mice injected with lentivirus expressing only EGFP were used as controls (numbers of mice are indicated in bars). (F and G) Same as (C) and (D), except that mice were injected with control or TetTox virus after fear conditioning training. (H and I) Effect of fear conditioning training and of TetTox expression or neuroligin-2 knockdown in the NR on the activity levels of neurons in different target brain regions. Control, TetTox, or the neuroligin-2 knockdown lentiviruses were injected into the NR of adult mice. Mice were subjected to fear conditioning training (+Training) or received no training (naïve) and were sacrificed 90 min after training. Brain sections were stained for c-Fos (red) to measure neuronal activation and NeuN to label all neuronal nuclei (blue). (H) Representative images of the hippocampal CA1 region. (I) Quantification of c-Fos expression in the indicated brain regions (n = 12 to 18 brain sections from four mice in each group; for additional data, see figs. S11 and S12). Data shown are means ± SEM (error bars). Statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001) was assessed by two-tailed Student's t test [(C) and (G)], two-way ANOVA followed by Bonferroni's post-hoc test [(E), comparing freezing levels, and (I)], or one-way ANOVA followed by Turkey's post-hoc test [discrimination index in (E)]. Horizontal dashed lines indicate the level of control groups.

  4. Fig. 4

    Firing pattern of NR neurons dictates memory generalization. (A) (Top) Coronal brain section illustrating expression of ChIEF-tdTomato (red fluorescent channelrhodopsin) in the NR. (Bottom) High-magnification micrograph showing ChIEF-tdTomato expressing NR neurons and their axonal fibers. (B) Experimental protocol for testing the effect of different optogenetic stimulation patterns of NR neurons on fear conditioning behavior, with the stimulation patterns illustrated below the time diagram. NR neurons were stimulated throughout the 6-min training period by either a 4-Hz tonic stimulation or a 30-Hz phasic stimulation administered for 0.5 s every 5 s. Stimulus light pulses lasted 15 ms. (C) Tonic and phasic optogenetic stimulation produced opposite effects on fear memory generalization. Control mice also expressed channelrhodopsin and contained an implanted optical fiber, but were not stimulated. Data shown are means ± SEM (error bars); numbers in bars indicate the number of mice analyzed. Statistical significance (*P < 0.05; **P < 0.01) was assessed by two-way mixed-model ANOVA followed by Bonferroni's post-hoc test comparing the freezing levels or by one-way ANOVA followed by Turkey's post-hoc test for the discrimination index. Horizontal dashed lines indicate the level of control groups.

  5. Fig. 5

    Model for the mechanism of the NR's control of memory generalization. (A) Schematic diagram of the synaptic interactions between the mPFC, NR, and hippocampus in controlling memory generalization. VTA, ventral tegmental area; EC, entorhinal cortex. (B) Illustration of the modular composition of memory features. We posit that memories differentially incorporate a composite of specific attributes. The more prominent a feature is, the more likely it is to be included in memory, as illustrated here with a baseball containing additional features besides "ballness." We propose that NR neurons control memory generalization by regulating the number of features that are incorporated into a memory. For a more detailed discussion, see fig. S13.

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