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A temporal shift in the circuits mediating retrieval of fear memory

Abstract

Fear memories allow animals to avoid danger, thereby increasing their chances of survival. Fear memories can be retrieved long after learning1,2, but little is known about how retrieval circuits change with time3,4. Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning. Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval. Accordingly, the conditioned tone responses of PVT neurons increased with time after training. The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval5,6,7, sends dense projections to the PVT8. Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points. In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits. Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear. Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalar networks for retrieval and maintenance of long-term fear memories.

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Figure 1: The dMT is necessary for retrieval of fear at late, but not early, time points after conditioning.
Figure 2: c-Fos expression induced by fear retrieval at different time points after conditioning.
Figure 3: Time-dependent increases in tone responses of PVT neurons following fear conditioning.
Figure 4: Time-dependent shift of retrieval circuits after conditioning.

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Acknowledgements

We thank G. Manzano-Nieves for help with the optogenetic experiments, A. C. Felix-Ortiz for technical advice, and K. M. Tye for comments on the manuscript. We thank K. Deisseroth for viral constructs and the UNC Vector Core Facility for viral packaging. This study was supported by the NIH grants R01-MH058883, and P50-MH086400, and a grant from the University of Puerto Rico President’s Office to G.J.Q.; the MBRS-RISE Program (R25-GM061838) to K.Q.L.; and NSF grant DBI-0115825 and RCMI grant 8G12-MD007600 for the Confocal Microscope Facility.

Author information

Authors and Affiliations

Authors

Contributions

F.H.D.-M. performed behavioural, immunocytochemical and optogenetic experiments. F.H.D.-M. and K.Q.-L. performed single-unit recording in anaesthetized rats. K.Q.-L. performed single-unit recording experiments in behaving rats. F.H.D.-M., K.Q.-L. and G.J.Q. designed the study, interpreted results, and wrote the paper.

Corresponding author

Correspondence to Fabricio H. Do-Monte.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Conditioning levels in muscimol and optogenetic experiments.

ae, In the muscimol experiments, levels of freezing to tones (pre-treatment) for the habituation phase (first two blocks) and conditioning phase (last three blocks) were similar for saline (Sal., white circles) and muscimol (Mus., green circles) groups at 0.5 h (a), 6 h (b), 24 h (c), 7 d (d) and 28 d (e). fk, In the optogenetic experiments, freezing levels were similar for the eNpHR–eYFP groups (red circles) and the control groups (white circles) before manipulation of the following regions or pathways: PL somata (f); PL–PVT projections (g); PL–BLA projections (h); PVT–CeA projections (i); BLA somata (j); and PVT–CeA projections during inter-trial interval (ITI) (k). Data are shown as mean ± s.e.m. in blocks of two trials.

Extended Data Figure 2 Neural activity in dMT, but not the MAPK cascade or protein synthesis in dMT, is necessary for memory maintenance following reactivation.

a, Freezing to tones during habituation (Hab.; first two blocks of day 1), conditioning (Cond.; last three blocks of day 1), test 1 (day 7), and test 2 (drug-free test; day 14) performed 7 days after dMT infusion of saline (Sal., white circles, n = 10) or muscimol (Mus., green circles, n = 14), in rats never given bar-press training. Infusion of Mus. into the dMT impaired fear retrieval during test 1 (t = −4.35, P < 0.001), and also 1 week later during test 2 (t = −2.14, P = 0.04). b, Freezing to tones during habituation (Hab.; first two blocks of day 1), conditioning (Cond.; last three blocks of day 1) and drug-free test (day 8) performed 24 h after dMT infusion of saline (Sal., n = 5) or muscimol (Mus., n = 7). Rats were infused in their home cage without fear reactivation. Mus. infused this way had no effect on fear retrieval the following day (t = −0.88, P = 0.39). c, Intra-dMT infusion of MAPK inhibitor U0126 (1 µg per 0.5 µl per side, n = 11) immediately after a two-tone test on day 7 did not alter freezing levels during a drug-free test performed the following day, compared to a vehicle control (n = 9, t = 0.37, P = 0.71). d, Intra-dMT infusion of the protein synthesis inhibitor anisomycin (Aniso., 62.5 µg per 0.5 µl per side, n = 7) immediately after a two-tone test on day 7 did not alter freezing levels during a drug-free test performed the following day, compared to vehicle control (n = 5, t = 1.33, P = 0.21). One-way repeated-measures ANOVA was used on day 1. Unpaired t-test between Sal. and Mus. groups were used on days 7, 8 and 14. Data are shown as mean ± s.e.m. in blocks of two trials; *P < 0.05.

Extended Data Figure 3 Conditioning levels for c-Fos experiments, and the effects of PL inactivation at early versus late time points.

a, Freezing levels for conditioned (n = 4) and un-shocked control (n = 5) groups during fear conditioning and retrieval at the 6 h time point. The conditioned group showed a significant increase in freezing compared to controls (F(5,35) = 76.12, P < 0.001). Hab., habituation; Cond., conditioning. b, Freezing levels for conditioned (n = 3) and control (n = 4) groups during fear conditioning and retrieval at the 24 h time point. The conditioned group showed a significant increase in freezing compared to controls (F(5,25) = 40.07, P < 0.001). c, Freezing levels for conditioned (n = 5) and control (n = 6) groups during fear conditioning and retrieval at the 7 d time point. The conditioned group showed a significant increase in freezing compared to controls (F(5,45) = 49.88, P < 0.001). Rats were perfused for c-Fos immunocytochemistry 90 min after the fear retrieval test. Repeated-measures ANOVA followed by Tukey’s post-hoc test. d, Top, representative micrograph showing the site of fluorescent Mus. injection into the PL. Bottom, orange areas represent the minimum (darker colour) and the maximum (lighter colour) spread of muscimol into the PL. e, PL inactivation impaired fear retrieval at 6 h (F(1,11) = 7.92, P = 0.01; Sal., n = 6; Mus., n = 7). f, In separate animals, PL inactivation also impaired fear retrieval at 7 d after conditioning (F(1,14) = 13.8, P = 0.002; Sal., n = 8; Mus., n = 8). The retrieval test was performed 30 min after infusion of Sal. or Mus. (black arrows). One-way ANOVA followed by Tukey’s post-hoc test. Data are shown as mean ± s.e.m. in blocks of two trials; *P < 0.05.

Extended Data Figure 4 Conditioning levels for unit recording experiments, and waveform characteristics across recording sessions.

a, Freezing levels in response to tones during habituation (first two blocks), conditioning (last three blocks), test 1 (2 h) and test 2 (24 h) in rats never given bar-press training. Rats showed similar levels of freezing during retrieval at 2 h and 24 h after conditioning (n = 11). Data are shown as mean ± s.e.m. in blocks of two trials. b, Top, waveforms of three representative PVT neurons recorded during pre-conditioning (left), 2 h post-conditioning (middle), and 24 h post-conditioning (right). Bottom, principal component (PC) analysis of these cells’ waveforms at all three time points. c, Average valley-to-peak time (left), and average waveform amplitude (right) for all neurons (n = 54), shown as a percentage of pre-conditioning values. Of 54 neurons, 53 were unchanged (100% of pre-conditioning value) at both time points (2 h and 24 h) for one or both waveform parameters. One neuron showed 90% of pre-conditioning valley-to-peak time at both 2 h and 24 h, and ranged from 88 to 100% of pre-conditioning amplitude.

Extended Data Figure 5 Average firing rate and latency data for laser illumination of PL somata and PL terminals in the PVT expressing eNpHR-eYFP.

a, b, Average peri-stimulus time histogram of PL neurons that decreased (24 out of 50) (a) or increased (8 out of 50) (b) their firing rate during laser illumination of PL somata. c, Latency of PL neuronal responses to laser illumination of PL somata (paired Student’s t-test, P = 0.02). d, e, Average peri-stimulus time histogram of PVT neurons that decreased (13 out of 47) (d) or increased (9 out of 47) (e) their firing rate during laser illumination of PL terminals in the PVT. f, Latency of PVT neuronal responses to laser illumination of PL terminals in the PVT (paired Student’s t-test, P = 0.11). Peri-stimulus time histograms are shown in bins of 1 s. Response latency was measured in bins of 100 ms; *P < 0.05.

Extended Data Figure 6 Location of eNpHR–eYFP expression and optical fibres.

a, Left, representative micrograph showing eNpHR–eYFP expression in the PL. Right, placements of optical fibre tips in the PL. b, Left, representative micrograph showing the expression of eNpHR–eYFP in the PL and its terminals in dMT. Right, placement of optical fibre tips in the PVT. c, Left, representative micrograph showing the expression of eNpHR–eYFP in the PL and its terminals in the amygdala. Right, placement of optical fibre tips in the BLA. d, Left, representative micrograph showing the expression of eNpHR–eYFP in the PVT and its terminals in the amygdala. Right, placement of optical fibre tips in the CeA. Micrographs were obtained 8–10 weeks after virus infusion. cc, corpus callosum; IL, infralimbic cortex; MD, mediodorsal thalamus; Op., optical tract; sm, stria medullaris.

Extended Data Figure 7 PL neurons projecting to the PVT versus the BLA are located in distinct layers.

a, Left, schematic of retrobead injections. Middle, micrograph showing the site of retrobead infusion into the PVT (green), and right, micrograph showing the site of retrobead infusion into the BLA (red) in the same rat. b, Left, PL neurons retrogradely labelled from PVT infusion (green). Middle, PL neurons retrogradely labelled from BLA infusion (red). Right, overlay image showing the absence of co-labelling between PL neurons projecting to the PVT (green, deep layers) and PL neurons projecting to the BLA (red, superficial layers). Scale bar, 100 µm.

Extended Data Figure 8 Silencing BLA somata impaired fear retrieval at early, but not late, time points after conditioning.

a, Representative micrograph showing eNpHR–eYFP expression in the BLA. b, Green areas represent the minimum (darker colour) and the maximum (lighter colour) expression of eNpHR–eYFP in the BLA. c, Dots represent the location of optical fibre tips within the BLA. d, Illumination of BLA soma (yellow bar) reduced freezing at 6 h (F(1,9) = 54.6, P < 0.001), but not at 7 d (F(1,9) = 10.1, P = 0.91) or 8 d (P = 0.33), in the eNpHR–eYFP group (n = 5) compared to eYFP control group (n = 6). Repeated-measures ANOVA followed by Tukey’s post-hoc test. Data are shown as mean ± s.e.m. in blocks of 2 trials; *P < 0.05. ‘×’ symbols indicate baseline (pre-tone) freezing levels.

Extended Data Figure 9 Silencing PVT projections to the CeA during the inter-trial interval did not impair fear retrieval.

a, Representative micrograph showing the expression of eNpHR–eYFP in the PVT and its terminals in the amygdala. b, Top, green areas represent the minimum (darker colour) and the maximum (lighter colour) expression of eNpHR–eYFP in the PVT. Bottom, dots represent the location of optical fibre tips within the CeA. c, Illumination (40 s) of PVT inputs to the CeA during the interval between tones (3 min) did not affect freezing at 6 h (F(1,8) = 0.75, P = 0.40), 7 d (F(1,8) = 0.04, P = 0.84), or 8 d (P = 0.93), compared to the eYFP control group (n = 5 per group). Repeated-measures ANOVA followed by Tukey’s post-hoc test. Data are shown as mean ± s.e.m. in blocks of 2 trials; P < 0.05. ‘×’ symbols indicate baseline (pre-tone) freezing levels.

Extended Data Figure 10 Effects of laser illumination on locomotion, anxiety and food-seeking behaviour in rats expressing eNpHR–eYFP.

ae, Rats were tested in an open field during a 9 min session (3 min acclimation, 3 min laser off, 3 min laser on). We measured the total distance travelled (left) and the percentage of time spent in the centre of the apparatus (middle) to assess locomotor activity and anxiety, respectively. We also compared the rate of pressing for food (right) in a 15 min session (5 min acclimation, 5 min laser off, 5 min laser on). Silencing of PL somata (eNpHR–eYFP, n = 7; control, n = 5) (a), PL inputs to the PVT (eNpHR–eYFP, n = 5; control, n = 5) (b), PVT inputs to the CeA (eNpHR–eYFP, n = 7; control, n = 5) (d), or BLA somata (eNpHR–eYFP, n = 5; control, n = 6) (e) did not affect locomotion, anxiety or food-seeking. However, silencing PL–BLA projections (c) increased locomotion (F(1,12) = 12.4, P = 0.004; eNpHR–eYFP, n = 6; control, n = 8) and decreased food-seeking (F(1,15) = 6.0, P = 0.02; eNpHR–eYFP, n = 7; control, n = 10). Repeated-measures ANOVA followed by Tukey’s post-hoc test. Data are shown as mean ± s.e.m.; *P < 0.05.

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Do-Monte, F., Quiñones-Laracuente, K. & Quirk, G. A temporal shift in the circuits mediating retrieval of fear memory. Nature 519, 460–463 (2015). https://doi.org/10.1038/nature14030

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