HFS of the MGB induces thalamocortical LTP in the ACx.

(A) Schematic of the experimental setup. Stimulation electrodes were inserted in the MGB, and the recording electrodes were placed into the ACx.

(B) Representative raw extracellular multiunit recordings form the MGB and ACx. Noise bursts of 100 ms duration and 70dB intensity were presented during electrode insertion. Robust auditory responses were recorded when the electrodes reached layer IV of the ACx and the ventral division of MGB.

(C) HFS protocol. Each burst consisted of five 0.5-ms pulses at 100 Hz. Each block included 10 bursts at 5 Hz, with an inter-block interval of 10 s. Four blocks were delivered in total.

(D) HFS-induced LTP in the auditory thalamocortical pathway. Upper: Representative waveforms of fEPSPs evoked by ES in MGB, with pre-HFS responses shown in gray (labeled 1) and post-HFS responses shown in black (labeled 2). Scale bar: 10 ms, 0.1 mV. Lower: Population data of normalized fEPSP slopes recorded in the ACx before and 1 hour after HFS (n = 13 recording sites from 10 young adult mice, One-way RM ANOVA, ***, p < 0.001).

(E) The preparation (upper) and the paradigm for induction of LTP in natural auditory responses (lower).

(F) Raster plots showing auditory responses in the ACx before and after thalamocortical LTP induction.

(G) HFS-induced LTP of auditory responses. Upper: Representative waveforms of fEPSPs evoked by noise bursts before (Gray, 1) and after (Black, 2) HFS in the MGB. Scale bar: 0.1 s, 0.2 mV. Lower: Population data of normalized fEPSP slopes recorded in the ACx before and after HFS (n=17 from 6 mice, One-way RM ANOVA, ***, p < 0.001).

Thalamocortical LTP is CCK-dependent.

(A) Upper panel: Cre-dependent expression of EYFP in CCK-positive thalamocortical projection neurons in CCK-Cre mice. Histological validation shows viral expression in the ACx and MGB (Green: EYFP). Lower panel: The post-synaptic marker PSD95 was labeled in red. CCK-B receptors are labeled by far-red-fluorescent dye and represented in magenta. Thalamocortical terminals (Yellow dots) and their co-localization with CCKBR are indicated by white arrowheads. Scale bars: Upper, 300 µm; Lower, 20 µm.

(B) Induction of LTP in the thalamocortical pathway by HFLS of CCK-positive thalamocortical fibers in the ACx. Upper left: Experiment design schematic. Upper right: Representative waveforms of laser-evoked fEPSPs before (Gray, 1) and after (Black, 2) HFLS. Scale bar: 40 ms, 20 µV. Lower panel: Normalized slopes of laser-evoked fEPSPs for 16 mins before and 1h after HFLS in the ACx (n=25 from 5 mice, one-way ANOVA, ***, p < 0.001).

(C) Left panel: Schematic diagram of viral injections. Mice were injected with AAV expressing shRNA targeting Cck (anti-Cck: rAAV-hSyn-EGFP-5’miR-30a-shRNA(Cck)-3’-miR30a-WPREs) or a scrambled control sequence (anti-Scramble: rAAV-hSyn-EGFP-5’miR-30a-shRNA(Scramble)-3’-miR30a-WPREs). Right panel: Post-hoc verification of viral expression in the MGB and thalamocortical projections distributed in the ACx. Scale bars: 200 µm.

(D) Upper left: Schematic diagram of the experimental design. Upper right: Representative fEPSP traces before (1,3) and after (2,4) HFS in the anti-Scramble (gray) and anti-Cck (blue) groups. Scale bar: 10 ms, 0.1 mV. Lower panel: Normalized fEPSP slopes before and after HFS in the anti-Cck group (blue, ns, p = 0.859, n = 16 from 10 mice) and anti-Scramble group (Gray, ***, p < 0.001, n = 17 from 8 mice; two-way ANOVA comparison between groups after HFS:, ***, p < 0.001).

(E) Upper panel: AAV9-syn-CCKsensor was injected into the ACx, and AAV9-Syn-FLEX-ChrimsonR-tdTomato/AAV9-Syn-ChrimsonR-tdTomato was injected into the MGB of CCK-ires-Cre/CCK-KO mice. Lower panel: Enlarged images showing the expression of AAV9-hSyn-Chrimson-tdTomato in the MGB, its thalamocortical projections to the ACx, along with the expression of CCK-sensor in the ACx. Scale bars: 200 μm.

(F) Upper: Schematic diagram of the experimental design. An optical fiber was attached to the surface of ACx to activate thalamocortical terminals (left) or inserted into the MGB to activate the cell body (right). Another optical fiber was placed in the ACx to monitor fluorescence intensity. Lower: Traces of fluorescence signals of the CCK-sensor before and after HFLS (red light) in CCK-Cre or CCK-KO mice. The averaged ΔF/F0% is presented as solid lines, with the SEM indicated by the shadow area. Fluorescence increased after HFLS in CCK-Cre mice (Orange, terminal activation; red, soma activation), whereas HFLS failed to induce an increase in CCK-KO mice (green, terminal activation; blue, soma activation).

(G) Bar charts showing averaged ΔF/F0% from different groups before and after HFLS (Bonferroni multiple comparisons adjustment: Upper, before vs. after HFLS at terminal in CCK-Cre: ***, p < 0.001, n = 16, N = 8; before vs. after HFLS at terminal in CCK-KO: ns, p = 0.616, n = 14, N = 7; After HFLS at terminal in CCK-Cre vs. CCK-KO: **, p = 0.004; Lower, before vs. after HFLS at soma in CCK-Cre: ***, p < 0.001, n = 21, N = 11; before vs. after HFLS at soma in CCK-KO: ns, p = 0.723, n = 20, N = 10; After HFLS at soma in CCK-Cre vs. CCK-KO: **, p = 0.003).

(H) Upper: Schematic diagram of viral injections. AAV9-syn-CCKsensor was injected into ACx, and anti-CCK (rAAV-hSyn-mCherry-5’miR-30a-shRNA(Cck)-3’-miR30a-WPREs) /anti-Scramble (rAAV-hSyn-mCherry-5’miR-30a-shRNA(Scarmble)-3’-miR30a-WPREs) shRNAs was injected into the MGB of C57 mice. Lower: Enlarged images show the expression of shRNAs in the MGB, its thalamocortical projections to the ACx, along with the expression of CCK sensor in the ACx. Scale bars: 200 μm.

(I) Upper: Experiment design schematic. A stimulation electrode was inserted into MGB for HFS. Another optical fiber was placed in ACx to monitor the fluorescence intensity. Lower: Traces of fluorescence signals of the CCK-sensor before and after HFS in the anti-Cck group (blue) or anti-Scramble group (gray). The Averaged ΔF/F0% is presented as solid lines, with the SEM indicated by the shadow area. Fluorescence increased after HFS of MGB in the anti-Scramble group (gray), whereas the HFS failed to induce an increase in the anti-Cck group (blue).

(J) Bar charts showing the averaged ΔF/F0% from different groups before and after HFS (Bonferroni multiple comparisons adjustment: before vs. after HFS in anti-Scramble group: ***, p < 0.001, n = 21 from 11 mice; before vs. after HFS in anti-Cck group: ns, p = 0.999, n = 22 from 11 mice; After HFLS in anti-Scramble group vs. anti-Cck group: ***, p < 0.001).

Cholecystokinin correlates with developmental thalamocortical LTP in the MGB

(A) Expression of Cck mRNA in the MGB of different age groups and in CCK-KO mice. Scale bars: Upper, 200 µm; Lower, 20 µm.

(B) Quantification of Cck expression in the MGB. Upper panel: The total number of Cck-expressing cells in the MGv (see Methods) for each group (One-way ANOVA, Bonferroni multiple comparisons adjustment: P14 vs. P20, ***, p < 0.001; P20 vs. 8W, ns, p = 1.0; 8W vs. 18M, *, p = 0.018; 18M vs. CCK-KO, *, p = 0.05; 8W vs. CCK-KO, ***, p < 0.001. n of P14 = 4 from 2 mice; n of P20 = 4 from 2 mice; n of 8W = 4 from 2 mice; n of 18M = 4 from 2 mice; n of CCK-KO = 8 from 4 mice). Lower panel: Normalized Cck mRNA intensity per neuron (see Methods) across developmental stages (One-way ANOVA, Bonferroni multiple comparisons adjustment: P14 vs. 8W, ***, P < 0.001; P20 vs. 8W, ***, P < 0.001; 8W vs. 18M, ***, P < 0.001; n of P14 = 228 neurons; n of P20 = 1104 neurons; n of 8W = 929 neurons; n of 18M = 400 neurons).

(C) HFS-induced thalamocortical LTP in different age groups. Normalized fEPSP slopes before and after HFS in the MGB at P20 (upper, ns, p = 0.542, n=15 from 10 mice), 8W (middle, ***, p < 0.001, n = 13 from 10 mice), and 18M(lower, ns, p = 0.380, n = 18 from 8 mice).

(D) Immunostaining of CCKBR in the ACx neurons at different ages (P20, 8W and 18M). CCK: red; DAPI: blue. Scale bars: 20 µm.

(E) Upper: Injection of AAV9-syn-CCKsensor into ACx of aged mice (18M). Lower: Enlarged images displaying the CCK-sensor expression in the ACx. Scale bar: 500 μm.

(F) Upper: Experiment design for HFS application in the MGB, and fluorescence monitoring in the ACx. Lower: Traces of fluorescence signals of the CCK-sensor before and after HFS in aged mice (blue). Fluorescence signals from the anti-Scramble group (gray, adult control, 3 ∼ 4 months) are included for comparison. The averaged ΔF/F0% is shown as solid lines, with the shaded areas representing SEM.

(G) Bar charts showing the averaged ΔF/F0% before and after HFS for aged mice and adult control mice (Statistical analysis with Bonferroni multiple comparisons adjustment: before vs. after HFS in aged mice: ns, p = 0.699, n = 12 from 6 mice; after HFS in aged mice vs. adult control group: **, p = 0.01).

Exogenous application of CCK rescues thalamocortical connectivity.

(A) Schematic representation of the experimental setup, showing electrode placement and drug injection sites.

(B) Experimental protocol. CCK or ACSF was injected into the ACx, followed by HFS in the MGB. fEPSPs in response to MGB ES were recorded before and after the intervention.

(C) CCK-4 injection restored HFS-induced thalamocortical LTP in aged mice. Upper: Representative traces of fEPSPs recorded before (1,3) and after (2,4) drug injection followed by HFS in the CCK-4 (red) and ACSF (gray) groups. Scale bar: 10 ms, 0.1 mV. Lower panel: Normalized fEPSP slopes before and after CCK-4 (red, ***, p < 0.001, n = 13 from 6 mice) or ACSF (gray, ns, p = 0,404, n = 12 from 7 mice) injection and subsequent HFS in aged mice (two-way ANOVA, CCK vs. ACSF after the intervention: **, p = 0.006).

(D) Upper: AAV9-hSyn-ChrimsonR-tdTomato was injected into the MGB of C57 mice. Lower panel: Histological confirmation of viral expression in the ACx and MGB. Scale bars: 100 μm.

(E) Upper panel: Schematic Diagram of the Experimental Setup. A 620-nm laser was used to activate thalamocortical terminals expressing opsins in the ACx. Glass pipette electrodes in the ACx recorded field EPSPs evoked by laser stimulation. Lower panel: Experimental protocol. CCK or ACSF was injected into the ACx, followed by 200 auditory stimulations. fEPSPs in response to laser stimulation were recorded before and after the intervention.

(F) Upper: Representative fEPSP traces evoked by laser before (1,3) and after (2,4) the intervention (drug injection followed by auditory stimulation). Scale bars: 40 ms, 0.1 mV. Lower: Normalized slopes of laser-evoked fEPSPs for 16 mins before and 1h after the intervention in the CCK-4 and ACSF groups (two-way RM ANOVA, before vs. after: CCK group, ***, p < 0.001, n = 10 from 10 mice; ACSF group, ns, p = 0.899, n =19 from 10 mice; CCK vs. ACSF after the intervention, p < 0.001).

Exogenous application of CCK enhances frequency discrimination.

(A) Upper: Schematic diagram of the experimental setup for the PPI test. lower: Experimental timeline showing tone exposure and CCK/ACSF injection into the ACx of young adult mice.

(B) Left: Schematic representation of the PPI acoustic startle test protocol. A continuous background pure tone (70 dB, 9.8 kHz or 16.4 kHz) was presented during the test, except when prepulse tones and startle noise bursts were delivered. PPI trials were presented in a pseudorandom order. Each prepulse test consisted of an 80 ms prepulse at 70dB (Δf, pure-tone frequency was 0%, 2%, 4%, 8%, 16%, or 32% lower than the background tone), followed by a 20 ms white noise startle pulse at 120 dB, after which the background tone resumed. Each trial was repeated 15 times. Middle: Averaged startle waveforms from a representative mouse. Right: Calculated PPI (as defined below) for the same example.

(C) Mean PPI (%) of the startle responses in young adult mice exposed to a 9.8 kHz tone after CCK-8S (red) or ACSF (gray) injection into the ACx (CCK, n=7, ACSF n=7, two-way ANOVA, p < 0.05, post-hoc, Tukey test, CCK vs. ACSF at Δf = -2%, **, p < 0.001). Prepulse frequencies were 0%, 2%, 4%, 8%, 16%, or 32% lower than the background tone, 9.8 kHz. The difference in the mean PPI between the CCK infusion and ACSF infusion groups is listed as a function of the Δf in the lower panel.

(D) Mean PPI (%) of startle responses in young adult mice exposed to a 16.4 kHz tone after CCK-8S (red) or ACSF (gray) injection into the ACx (CCK n=8, ACSF n=7, two-way ANOVA, p < 0.05, post-hoc, Tukey test, CCK vs. ACSF at Δf = -2%, **, p < 0.001). Prepulse frequencies were 0%, 2%, 4%, 8%, 16%, or 32% lower than the background tone, 16.4 kHz. The difference in the mean PPI between the CCK infusion and ACSF infusion groups is listed as a function of the Δf in the lower panel.

(E) Experimental timeline for tone exposure and CCK/saline i.p. injection in aged mice.

(F) Mean PPI (%) of startle responses in aged mice exposed to a 9.8 kHz tone after i.p. injection of CCK-4 (red) or saline (gray). Prepulse frequencies were 0%, 2%, 4%, 8%, 16%, or 32% lower than the background tone, 9.8 kHz. The difference in mean PPI between the CCK-4 injection and saline injection groups is listed as a function of the Δf in the lower panel (Bonferroni-adjusted multiple comparisons with CCK vs. saline at Δf =: -2%, **, p = 0.010; -4%, **, p = 0.006; -8%, **, p = 0.006; -16%, *, p = 0.022; -32%, *, p = 0.027; Frequency-discrimination task in CCK-4 group vs. saline group, two-way ANOVA, **, p = 0.004; CCK-4, N = 15; saline, N = 15).

Thalamocortical LTP is CCK-dependent, related to Figure 2.

(A) Neuronal responses recorded as field potentials evoked by laser stimulation of CCK-positive thalamocortical fibers in the ACx. Left, Schematic of the optical fiber position in the ACx. Right, Representative traces showing that fEPSPs amplitudes increased with higher laser power. Scale bar: 20 ms, 20 µV.

(B) HFLS of thalamocortical fibers induces synchronized fEPSPs. fEPSPs in the ACx reliably followed laser pulses at 80Hz. Scale bar: 20 ms, 20 µV.

(C) HFLS of CCK-positive neurons in the MGB induced LTP in the thalamocortical pathway. Upper left: Schematic diagram of the experimental design. Upper right: Representative laser-evoked fEPSP waveforms recorded before (Gray, 1) and after (Black, 2) HFLS in the MGB. Scale bar: 50 ms, 20 µV. Lower panel: Normalized fEPSP slopes recorded for 16 mins before and 1h after HFLS in the MGB (one-way ANOVA, ***, p < 0.001, n=18 from 6 mice).

(D) CCKBR antagonist blocked HFS-induced thalamocortical LTP. Upper left: Schematic diagram of the experimental design. Upper right: Representative fEPSP waveforms recorded before (1,3) and after (2,4) HFS in the MGB. The ACSF group is indicated in gray, and the L365 group is indicated in blue. Scale bar: 10 ms, 0.1 mV. Lower panel: Normalized fEPSP slopes before and after HFS in the L365 group (blue, ns, p = 0.779, n = 22 from 5 mice) and the ACSF group (gray, ***, p < 0.001, n = 12 from 5 mice).

Exogenous CCK restores thalamocortical LTP in aged mice and improves frequency discrimination ability, related to Figure 4 and Figure 5.

(A) CCK injection into the ACx alone did not induce thalamocortical LTP. Upper left: Schematic diagram of the experimental design showing electrodes and drug-injection pipette placement. Upper right: Experimental protocol. After baseline fEPSPs recording, CCK was injected into the ACx without stimulation, followed by a resumption of ES for subsequent fEPSPs recordings. Lower: Normalized fEPSP slopes in response to ES recorded before and after CCK-4 injection(one-way RM ANOVA, ns, p = 0.956, n = 20 from 5 mice).

(B) CCK injection into the ACx without auditory stimulation failed to potentiate laser-evoked fEPSPs. Upper left: Schematic diagram of the experimental design showing the placement of electrodes, optical fiber, and drug-injection pipette. Upper right: the Experimental protocol. After baseline fEPSPs recording, CCK was injected into the ACx without stimulation, followed by a resumption of laser stimulation (LS) for subsequent fEPSPs recordings. Lower: Normalized fEPSP slopes in response to laser recorded before and after CCK-4 injection (one-way RM ANOVA, ns, p = 0.468, n = 22 from 4 mice).

(C) Left: Schematic drawing of the experiment setup. Right: Experimental paradigm.

(D) Left: Representative tuning curves recorded before (gray line) and after (red line) the sequential process of CCK-8S infusion and a non-CF tone exposure. The arrow indicates the exposed frequency (EF). Right: Representative tuning curves recorded before (gray line) and after (blue line) the sequential process of ACSF infusion and a non-CF tone exposure. The arrow indicates the exposed frequency (EF).

(E) Population data showing reductions in response thresholds after CCK-8S (red line) infusion followed by tone exposure, but not after ACSF infusion (blue line) (CCK-8S, n=15; ACSF, n=12; two-way ANOVA, p < 0.001, post-hoc, Tukey test, CCK-8S vs. ACSF, * p < 0.05, ** p < 0.001).

(F) Schematic diagram of the experimental design showing the placement of an optical fiber in the ACx for fluorescence monitoring using CCK sensors.

(G) Traces of fluorescence signals of the CCK-sensor before and after i.p. injection of CCK-4 (1 mg/kg) or saline in CCK-Cre mice or CCK-KO mice. Averaged ΔF/F0% is shown as lines, with the SEM indicated by the shadow area. The fluorescence increased after CCK-4 application (solid) in both CCK-Cre mice (red) and CCK-KO mice (blue), whereas saline application (dashed) did not induce an increase.

(H): Bar charts showing the averaged ΔF/F0% across groups. Left panel: CCK-Cre mice, two-way ANOVA with Bonferroni multiple comparisons adjustment, before vs. after CCK-4 treatment: red, **, p = 0.002, N = 7; before vs. after saline treatment: blue, ns, p = 0.951, N = 9; After CCK-4 vs. after saline: *, p = 0.025. Right panel: CCK-KO mice, two-way ANOVA with Bonferroni multiple comparisons adjustment, before vs. after CCK-4 treatment: red, ***, p < 0.001, N = 7; before vs. after saline treatment: blue, ns, p = 0.877, N = 8; After CCK-4 vs. after saline: ***, p = 0.001).