Homosynaptic LTP stimulus minutes before, after, or 24 hrs after a weak associative conditioning produces lasting memory.

a) Diagram showing the experimental timeline. b) Left: High frequency stimulation (HFS) of the thalamic inputs (Th) to the lateral amygdala (LA) applied either 24 hours (WTh+24hHFSTh, corresponding to panel a, top branch), or immediately after a weak thalamic associative conditioning (WTh+HFSTh, corresponding to panel a, middle branch), significantly increased the CS-evoked freezing (n=9; One-way ANOVA, F (2, 23) = 8.202, p-value=0.0020). Right: HFS of the thalamic input immediately before (HFSTh+WTh, corresponding to panel a, bottom branch) (n=6) or after a weak associative conditioning (n=8) (WTh+HFSTh, corresponding to panel a, middle branch) is equally effective in increasing the CS-evoked freezing. Colors of the bar graphs represent the experimental protocols for each group of mice (colored boxes in panel a). Subscripts with blue font indicate stimulation of the blue-shifted channelrhodopsin oChIEF using the selective procedure. c) Diagram showing the experimental setup of the in vivo electrophysiology recording (Rec) in anesthetized mice. Evoked field EPSP was produced by blue light stimulation (450nm) of thalamic inputs expressing oChIEF. d) Plot of average in vivo field EPSP slope (normalized to baseline period) in the LA before and after HFS (n=5). Right inset: Superimposed traces of in vivo field responses to single optical stimulus before (dashed line) and after (solid line) HFS. Scale bar, 0.1 mV, 5ms. Results are reported as mean ±S.E.M. **, p<0.01. Ctx: Cortical input; Th: Thalamic input; LA: lateral amygdala; HFS: High Frequency Stimulation; EPSP: excitatory postsynaptic potential; WTh: Recall session after a weak thalamic associative conditioning.

Submilliwatt yellow light renders a red-shifted channelrhodopsin insensitive to blue light.

a) Diagram showing ex vivo electrophysiology recordings in slices where ChrimsonR-expressing thalamic inputs to the lateral amygdala (red lines) were optically activated. Synaptic responses were evoked by pulses of 450nm blue light (450nm), or pulses of blue light co-illuminated with a 561nm yellow light pulse (co-illumination). b, c) Bar graph (normalized to blue light (450nm) stimuli) (b), and example recording (scale bar, 50 pA, 20ms) (c), of optically driven synaptic responses to pulses of blue light (450nm) or pulses of blue light co-illuminated with yellow light (450nm with yellow underline). d) Left: Diagram showing the experimental set up of electrophysiology recordings in freely moving mice where ChrimsonR-expressing thalamic inputs (Th) to the lateral amygdala (LA) were optically activated. Right: Comparison of a representative waveform average of the response to pulses of red light (638nm), pulses of red light co-illuminated with a 500ms yellow light pulse (638nm with yellow underline), pulses of blue light (450nm), and pulses of blue light co-illuminated with a 500ms yellow light pulse (450nm with yellow underline) (n=3). e) Left: Diagram showing the experimental set up of electrophysiology recordings in freely moving mice where oChIEF-expressing thalamic inputs (Th) to the lateral amygdala (LA) were optically activated. Right: Comparison of a representative waveform average of the response to pulses of red light (638nm), pulses of blue light (450nm), and pulses of blue light co-illuminated with a 500ms yellow light pulse (450nm with yellow underline) (n=3). f) Left: Diagram showing the experimental setup of electrophysiology recordings in anesthetized mice where ChrimsonR-expressing thalamic inputs (Th) to the lateral amygdala (LA) were optically activated. Middle: Comparison of a representative waveform average of the response to pulses of blue light (450nm), pulses of blue light co-illuminated with a 500ms yellow light pulse (A), pulses of blue light following the yellow light pulse by 50 ms (B), or 500 ms (C) (n=4). Right: Comparison of the waveform average responses to pulses of blue light (450nm), pulses of blue light co-illuminated with a 500ms yellow light pulse (450nm with yellow underline), and pulses of blue light after sequentially applying NBQX and TTX, and later in a euthanized mouse (Dead), (n=4). Scale bar, 0.2 mV, 10ms.

Heterosynaptic LTP stimulus produces lasting memory if delivered within minutes after a weak associative conditioning.

a,b) Diagram showing the experimental timeline of the heterosynaptic LTP protocol manipulation following a weak thalamic associative conditioning. HFS with yellow upperline indicates that the delivery of high frequency stimulation with blue light overlapped with long pulses of yellow light. This co-illumination prevents the activation of ChrimsonR-expressing thalamic inputs (Th) by blue light, while the oChIEF-expressing cortical inputs remain unaffected. Note that yellow light specifically renders ChrimsonR, and not oChIEF, insensitive to blue light. c) Left: High frequency stimulation (HFS) of the thalamic input expressing ChrimsonR immediately following a weak associative conditioning on the same input (WTh+HFSTh, corresponding to panel b) (n=9) was ineffective in producing the CS-evoked freezing. HFS with yellow upperline indicates that HFS with blue light overlapped with long pulses of yellow light. This was to prevent the activation of ChrimsonR-expressing thalamic inputs by blue light, as described above and detailed in figure 2. The same HFS protocol in mice that additionally, expressed oChIEF in the cortical inputs (WTh+HFSCtx, corresponding to panel a, bottom branch) (n=10), significantly increased the CS-evoked freezing (heterosynaptic LTP) (Unpaired t-test, p-value=0.0100). Middle: HFS on the cortical input, induced 24 hours after a weak associative conditioning (WTh+24hHFSCtx, corresponding to panel a, top branch) was ineffective in producing the CS-evoked freezing. (n=9; Paired t-test, p-value=0.2193). Right: Comparison of the effect of homosynaptic LTP protocol (WTh+HFSTh) (same dataset from figure 1b) and heterosynaptic LTP protocol (WTh+HFSCtx) (same dataset from panel c, left) (Unpaired t-test, p-value=0.9740). Results are reported as mean ±S.E.M. **, p<0.01. Subscripts with red font and blue font indicate stimulation of the red-shifted channelrhodopsin ChrimsonR and the blue-shifted channelrhodopsin oChIEF, respectively.

Homosynaptic and heterosynaptic LTP protocols produce lasting memory when applied within minutes after a non-associative conditioning.

a) Diagram showing the experimental timelines for fiber photometry from thalamic inputs (Th) expressing GCaMP7s. b) Averaged trace of the thalamic input activity in response to footshock (onset indicated by the dotted line), n=5. c) Diagram showing the experimental timelines for fiber photometry from the lateral amygdala (LA) neurons expressing GCaMP8m with intact or lesioned thalamic inputs. d) Averaged trace of the LA neurons activity in response to footshock (onset indicated by the dotted line) in mice with lesion (dash line) or no lesion (solid line) in the lateral thalamus (Th), n=6 per group. e) Diagram showing the experimental timelines of the homosynaptic LTP protocol manipulation following an unpaired thalamic conditioning. f) Unpaired conditioning on the thalamic inputs (UTh, corresponding to panel e, top branch) produced no CS-evoked freezing, while if unpaired conditioning was immediately followed by high frequency stimulation (HFS) on the same inputs (UTh+HFSTh, corresponding to panel e, bottom branch) it significantly increased the CS-evoked freezing (homosynaptic LTP), (n=11 per group; Mann-Whitney test, p-value=0.0002). Subscripts with blue font indicate stimulation of the blue-shifted channelrhodopsin oChIEF using the selective procedure. g) Diagram showing the experimental timelines of the heterosynaptic LTP protocol manipulation following an unpaired thalamic conditioning. h) High frequency stimulation (HFS) of the thalamic input expressing red-shifted channelrhodopsin ChrimsonR immediately following an unpaired conditioning on the same input (UTh+HFSTh, corresponding to panel g, top branch) was ineffective in producing the CS-evoked freezing, while the same protocol in mice that, in addition, expressed oChIEF in the cortical inputs (UTh+HFSCtx, corresponding to panel g, bottom branch), significantly increased the CS-evoked freezing (heterosynaptic LTP) n=11 per group; Mann-Whitney test, p-value=0.0002). During HFS, blue light pulses overlapped with long pulses of yellow light. This co-illumination prevents the activation of ChrimsonR-expressing thalamic inputs (Th) by blue light, while the oChIEF-expressing cortical inputs remain unaffected. Note that yellow light specifically renders ChrimsonR, and not oChIEF, insensitive to blue light. Subscripts with red font and blue font indicate stimulation of the red-shifted channelrhodopsin ChrimsonR and the blue-shifted channelrhodopsin oChIEF, respectively. i) Diagram showing the experimental setup of the in vivo electrophysiology recordings (Rec) in anesthetized mice where the thalamic input expressing ChrimsonR and/or cortical input expressing oChIEF were optically activated independently. j) Left: Plot of average in vivo field EPSP slope (normalized to baseline period) in LA evoked by optical activation of thalamic inputs, before and after footshock delivery (n=5; Paired t-test, p-value=0.2916). Middle: Plot of average in vivo field EPSP slope (normalized to baseline period) in LA evoked by optical activation of cortical inputs (Ctx), before and after high frequency stimulation (HFS) of these inputs (n=6; Paired t-test, p-value=0.0031). Right: Plot of average in vivo field EPSP slope (normalized to baseline period) in LA evoked by optical activation of thalamic inputs (Th), before and after HFS delivery on the cortical inputs (heterosynaptic LTP) (n=5; Paired t-test, p-value=0.0074). HFS with yellow upperline indicates that the delivery of high frequency stimulation with blue light overlapped with long pulses of yellow light. This co-illumination prevents the activation of ChrimsonR-expressing thalamic inputs (Th) by blue light,, while the oChIEF-expressing cortical inputs remain unaffected. Superimposed traces of in vivo field response to single optical stimulus before (dash line) and after (solid line) the induced protocols. Results are reported as mean ±S.E.M. **, p<0.01; ***, p<0.001.

Heterosynaptic LTP protocol when applied within minutes after a weak associative conditioning produces a long-lasting memory accompanied by the synaptic potentiation of the conditioned inputs.

a) Diagram showing the experimental setup of the in vivo electrophysiology recordings (Rec) in freely moving mice where the thalamic input expressing ChrimsonR and cortical input expressing oChIEF were optically activated independently. b) Left: Plot of average in vivo field EPSP slope (normalized to baseline period) in LA evoked by optical activation of cortical inputs, before and 24 hours after a weak thalamic conditioning followed immediately by HFS delivery on the same cortical inputs. Right: as left, except field EPSP was evoked by activation of thalamic inputs. The potentiation of the field EPSP of cortical (homosynaptic LTP) (n=4; Paired t-test, p=0.0082) as well as thalamic inputs (heterosynaptic LTP) (n=4; Paired t-test, p=0.0336) is evident 24 hours after the delivery of HFS. Superimposed traces of in vivo field response to single optical stimulus before (dash line) and after (solid line) HFS. c) The behavioral responses of the mice tested for their homo- and hetero-synaptic plasticity in panel b. Note a significant CS-evoked freezing 24 hours after a weak thalamic conditioning followed immediately by HFS delivery on the cortical inputs (heterosynaptic LTP). These mice did not show a CS-evoked freezing prior to the protocol (BL) (n=4; Paired t-test, p=0.0478). d) Positioning of the stimulating electrodes (Th. Stim and Ctx. Stim.) and the recording electrode. e) (left) Weak stimulation of the thalamic input (purple circle) results in a transient LTP. No change was observed in the basal synaptic transmission of the cortical input (control pathway, yellow circle). Strong stimulation of the cortical input following the weak stimulation of the thalamic input stabilized synaptic potentiation of the thalamic input (right). Dash line indicates the onset of HFS induction. f) A paired-comparison of the decay of synaptic potentiation of the thalamic input with (WTh+SCtx) or without (WTh) the strong stimulation of the cortical input. (n=4; Welch’s t test, p=0.0062). Results are reported as mean ±S.E.M. *, p<0.05; **, p<0.01.

a) Representative image of a coronal section of mice expressing AAV-oChIEF-tdTomato in the lateral thalamus (Left). Scale bars, 1 mm. Axonal expression of AAV-oChIEF-tdTomato in lateral amygdala (Right). Scale bars, 500 μm. b) The CR is significantly higher after strong conditioning (n=8) compared to weak conditioning (n=9), and unpaired conditioning (n=7). Optical CS alone did not elicit any CR (n=4; Two-way ANOVA, F (3, 16) = 34,68, p-value<0.0001). c) Optic fiber placement of individual mice from figure 1. Results are reported as mean ±S.E.M. ****, p<0.0001.

a) Representative image of a coronal section of mice expressing AAV-oChIEF-tdTomato in the LA-projecting cortical region and AAVChrimsonR-tdTomato in the LA-projecting thalamic region. Scale bars, 1 mm. Axonal expression of AAV-oChIEF-tdTomato and AAVChrimsonR-tdTomato in lateral amygdala. Scale bars, 500 μm. b) Optic fiber placement of individual mice from figure 2.

a) Representative image of a coronal section of mice expressing AAV-GCaMP7s in the lateral thalamus (Left). Scale bars, 1 mm. Axonal expression of AAV-GCaMP7s in lateral amygdala. Scale bars, 500 μm. b) Optic fiber placement of indi-vidual mice from figure 3. c) Overlay of the maximum extent of the lesion in the thalamic-lesioned group (n=6). d) Optic fiber placement of individual mice from figure 3 f,h.

a) Comparison of the freezing levels evoked by high frequency stimulation of the thalamic input in naive mice (HFSTh, n=4) compared to mice subjected to unpaired conditioning (U; n=11) and to unpaired conditioning filled by HFS (U + HFSTh, n=11; Two-way ANOVA, F (2,12)= 10.04, p-value=0.0027). b)A paired comparison of the freezing levels evoked by thalamic axons activation when the thalamic LTP is applied 24 hours after the unpaired conditioning. the (n=7, Wilcox test, p-value=0.0625). c) Optic fiber placement of individual mice from figure 3. Results are reported as mean ±S.E.M.

a,b) Diagram and histology of the brain sections showing the AAVs injection sites, and the optetrode implantation sites. c) Representative example of the EPSP amplitude recorded in the LA by stimulation of cortical axons(left), and thalamic axons (right) before and after the weak conditioning and cortical LTP.