HFS of the MGB induces thalamocortical LTP in the ACx.

(A) Experimental setup. The stimulation electrode array (ES) was inserted into the MGB, while the recording electrode array was placed into the ACx.

(B) Raw extracellular multiunit recording signals form the MGB and the ACx are shown. 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, respectively.

(C) HFS protocol. Each burst includes five 0.5-ms pulses at 100 Hz, and each block consists of 10 bursts at 5 Hz. Four blocks were delivered with an inter-block interval of 10 s.

(D) HFS-induced LTP in the auditory thalamocortical pathway. Upper: Sample fEPSP waveforms evoked by ES in MGB before (Gray, 1) and after (Black, 2) HFS in MGB. Scale bar: 10 ms, 0.1 mV. Lower: Population data of normalized fEPSP slopes recorded in ACx before and after HFS (n = 13 from 10 young adult mice, One-way RM ANOVA, ***, p < 0.001).

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

(F) Raster displays of auditory responses in ACx were compared before and after the thalamocortical LTP induction.

(G) HFS-induced thalamocortical LTP of auditory responses. Upper: Sample fEPSP waveforms evoked by noise bursts before (Gray, 1) and after (Black, 2) HFS in MGB. Scale bar: 0.1 s, 0.2 mV. Lower: Population data of normalized fEPSP amplitude 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) The Cre-dependent virus-infected CCK-positive thalamocortical projection neurons in CCK-Cre mice. Histological confirmation of the virus expression in the ACx and MGB (Green: EYFP). The post-synaptic marker PSD95 was labeled in red. Lower right: 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 bar: Upper, 300 µm; Lower, 20 µm.

(B) Induction of LTP in the thalamocortical pathway by high-frequency laser stimulation (HFLS) of CCK-positive thalamocortical fibers in the ACx. Upper left: Experiment design diagram. Upper right: Representative waveforms of laser-evoked fEPSPs before (Gray, 1) and after (Black, 2) the 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 illustrating the injection of mice with AAV expressing short hairpin RNA (shRNA) targeting Cck (anti-Cck: rAAV-hSyn-EGFP-5’miR-30a-shRNA(Cck)-3’-miR30a-WPREs) or a scrambled 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 area and thalamocortical projections distributed in the ACx area (scale bars: 200 µm).

(D) Upper left: Experiment design diagram. Upper right: Representative traces of the evoked fEPSP before (1,3) and after (2,4) HFS in the two groups. Anti-Scramble is indicated in gray, and anti-Cck is indicated in blue. Scale bar: 10 ms, 0.1 mV. Lower panel: Normalized fEPSP slope before and after HFS in mice expressing anti-Cck (Blue, ns, p = 0.859, n = 16 from 10 mice) or anti-Scramble (Gray, ***, p < 0.001, n = 17 from 8 mice) shRNA (Anti-Cck vs. Anti-Scramble after HFS, two-way ANOVA, ***, p < 0.001).

(E) AAV9-syn-CCKsensor was injected into ACx, and AAV9-Syn-FLEX-ChrimsonR-tdTomato/AAV9-Syn-ChrimsonR-tdTomato was injected into the MGB of CCK-ires-Cre/CCK-KO mice. Enlarged images show the expression of AAV9-hSyn-Chrimson-tdTomato in the MGB (upper right) and thalamocortical projections with CCK-sensor expression (scale bars: 200 μm).

(F) Upper: Experiment design diagram. 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 shown as the mean value in solid line with the SEM represented by the shadow area. Fluorescence increased after HFLS in CCK-Cre mice (Orange, terminal activation; red, soma activation), but HFLS failed to induce an increase in CCK-KO mice (green, terminal activation; blue, soma activation).

(G) Bar charts present the 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) AAV9-syn-CCK sensor 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 were injected into the MGB of C57 mice. Enlarged images show the expression of shRNAs in the MGB (lower right) and thalamocortical projections with CCK sensor (lower left) expression (scale bars: 200 μm).

(I) Upper: Experiment design diagram. 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 signal of the CCK-sensor before and after the HFS in the anti-Cck group or anti-Scramble group. The Averaged ΔF/F0% is shown as the mean value in solid line with the SEM represented by the shadow area. Fluorescence increased after HFS of MGB in the anti-Scramble group (gray), but the HFS failed to induce an increase in the anti-Cck group (blue).

(J) Bar charts present 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 MGB of different age groups and CCK-KO mice. Scale bar: Upper, 200 µm; Lower, 20 µm.

(B) Comparison of Cck expression level in different age groups and CCK-KO mice. Upper panel: The number of Cck-expression cells in 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 of single cells (see Methods) in different age groups (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 at different developmental stages. Normalized fEPSP slopes before and after HFS in the MGB at P20, 8W, and 18M (P20, upper, ns, p = 0.542, n=15 from 10 mice; 8W, middle, ***, p < 0.001, n = 13 from 10 mice; 18M, lower, ns, p = 0.380, n = 18 from 8 mice).

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

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

(F) Upper: Experiment design diagram. A stimulation electrode was inserted into MGB to apply HFS, and an optical fiber was placed in the ACx to monitor fluorescence intensity. Lower: Traces of fluorescence signal of the CCK-sensor before and after HFS in aged mice (blue). The fluorescence signals from anti-Scramble group (gray) in Figure 2I were utilized as adult control (3 ∼ 4 M). The averaged ΔF/F0% is shown as the mean value in solid line with the shadow area indicating the SEM. HFS failed to induce an increase in fluorescence in aged mice.

(G) Bar charts showing the averaged ΔF/F0% from the different groups before and after HFS (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) Experiment design diagram illustrating the electrode and drug-injection pipette placement.

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

(C) CCK-4 injection rescued the HFS-induction of thalamocortical LTP in aged mice. Upper: Representative traces of the evoked fEPSP before (1,3) and after (2,4) injection followed by HFS in the CCK-4 (red) and ACSF group (gray). Scale bar: 10 ms, 0.1 mV. Lower panel: Normalized fEPSP slope before and after CCK-4 (red) or ACSF (gray) injection in the ACx followed by HFS in the MGB of the old mice (CCK-4: n = 13 from 6 mice, ***, p < 0.001; ACSF: n = 12 from 7 mice, ns, p = 0,404; 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 virus expression in both the ACx and the MGB (Scale bars: 100 μm).

(E) Upper panel: Experiment design diagram. A laser with a wavelength of 620-nm was used to activate thalamocortical terminals expressing opsins in the ACx. Glass pipette electrodes in the ACx recorded the 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 in different groups. Scale bars: left, 40 ms, 0.1 mV; right, 40 ms, 20 µV. Lower: Normalized slopes of laser-evoked fEPSPs for 16 mins before and 1h after injection followed by auditory stimulation (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 behavior setup. lower: Experimental procedures of passive tone exposure with CCK/ACSF injection into the ACx of young adult mice.

(B) Left: Prepulse inhibition acoustic startle test protocol: a continuous background pure tone at 70 dB (9.8 kHz or 16.4 kHz), was presented during the test, except when the prepulse tones and startle noises were delivered. Prepulse inhibition 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, and then the background tone resumed after the startle. Each test was repeated 15 times. Middle: examples of averaged startle waveforms from a sampled mouse. Right: Prepulse inhibition (PPI as defined below) for the examples.

(C) Mean PPI (%) of the startle responses in C57 mice exposed to 9.8 kHz tone after CCK-8S (red line) or ACSF (gray line) injection into the ACx (CCK, n=7, ACSF n=7, two-way ANOVA, p<0.05, post-hoc, Tukey test, CCK vs. ACSF in 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 below.

(D) Mean PPI of startle responses in C57 mice exposed to a 16.4 kHz tone after CCK-8S (red line) or ACSF (gray line) injection into the ACx (CCK n=8, ACSF n=7, two-way ANOVA, p<0.05, post-hoc, Tukey test, CCK vs. ACSF in 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 below.

(E) Experimental procedures of passive tone exposure with 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 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 below (Bonferroni-adjusted multiple comparisons with CCK vs. Saline in 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 in field potentials evoked by laser stimulation of CCK-positive thalamocortical fibers in the ACx. Left, Schematic showing the position of laser fiber in the ACx. Right, evoked fEPSPs increased with higher laser power. Scale bar: 20 ms, 20 µV.

(B) fEPSPs elicited by the high-frequency (HF) laser pulse train demonstrated that fEPSPs in the ACx could follow the laser stimulation with 80Hz. Scale bar: 20 ms, 20 µV.

(C) HF laser stimulation (HFLS) of CCK-positive neurons in the MGB induced LTP in the thalamocortical pathway. Upper left: Experiment design diagram. Upper right: Sample laser-evoked fEPSP waveforms before (Gray, 1) and after (Black, 2) HFLS. Scale bar: 50 ms, 20 µV. Lower panel: Normalized slopes of laser-evoked fEPSPs 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: Experiment design diagram. Upper right: Representative traces of the evoked fEPSP before (1,3) and after (2,4) induction in the two groups. ACSF group is indicated in gray, and L365 group is indicated in blue. Scale bar: 10 ms, 0.1 mV. Lower panel: Normalized fEPSP slope before and after drug application followed by HFS in the MGB (L365 group, blue, ns, p = 0.779, n = 22 from 5 mice; ACSF group, gray, ***, p < 0.001, n = 12 from 5 mice).

Exogenous application of CCK rescued thalamocortical LTP in aged mice and enhanced frequency discrimination ability, related to Figure 4 and Figure 5.

(A) Upper left: Experiment design diagram showing electrodes and drug-injection pipette placement. Upper right: Experimental protocol. CCK was injected into the ACx without stimulation. Lower: Normalized fEPSP slope in response to ES before and after CCK-4 injection in the ACx (one-way RM ANOVA, ns, p = 0.956, n = 20 from 5 mice).

(B) Upper left: Experiment design diagram showing electrodes, optical fiber, and drug-injection pipette placement. Upper right: the Experimental protocol. CCK was injected into the ACx without stimulation. Lower: Normalized fEPSP slope in response to laser before and after CCK-4 injection in the ACx (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: Sample tuning curves before (gray line) and after (red line) CCK-8S infusion and a non-CF tone exposure. The arrow above the horizontal axis indicates the exposed frequency (EF). Right: Sample tuning curves before (gray line) and after (blue line) ACSF infusion and a non-CF tone exposure. The arrow above the horizontal axis indicates the exposed frequency (EF).

(E) Population data showing the changes in responding thresholds after CCK-8S (red line) or ACSF (blue line) infusion followed by tone exposure (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) Left: Experiment design diagram showing the placement of an optical fiber in the ACx for monitoring the fluorescence intensity.

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

(H) : Bar charts showing the averaged ΔF/F0% from the different groups before and after drug application. (Left panel: Averaged ΔF/F0% of CCK-Cre mice, two-way ANOVA followed by 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: Averaged ΔF/F0% of CCK-KO mice, two-way ANOVA followed by 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).