Article Figures & Data
Figures
- Figure 1.
Adolescent thalamocortical inhibition decreases excitatory transmission to pyramidal cells in layer II/III of the mPFC. a, Adolescent experimental timeline and schematic. Whole-cell patch-clamp recordings were made from pyramidal cells in layer II/III of the mPFC from hM4D and control mice. These pyramidal cells receive excitatory inputs from the thalamus as well as inhibitory inputs from local interneurons; created with https://biorender.com. b, Example image illustrating hM4D-mCherry expression in the mediodorsal thalamus (orange outline) in adolescent animals. Histology images were collected from each animal for each cohort, with at least six slices taken for each animal. Scale bar, 200 µm. c, Example image illustrating the recording location in the prelimbic (PrL) subregion of the medial prefrontal cortex. All recordings for this experiment were conducted in this region ∼200–350 µm from the pial surface (L II/III). Each circle represents the approximate location of a recorded cell. Scale bar, 500 µm. d, Top, Baseline EPSC activity at P35, all events cease after application of AMPA and NMDA receptor antagonists, CNQX and APV. Bottom, Representative traces showing sEPSCs of hM4D animals and mCherry controls. e, Top, Baseline IPSC activity at P35, all events cease after application of GABA receptor antagonist bicuculline. Bottom, Representative traces showing sIPSCs of HM4D animals and mCherry controls. f, sEPSC frequency (right) and median instantaneous frequency (middle) are significantly reduced following adolescent thalamic inhibition relative to control mice, but sEPSC amplitude (left) is unchanged; mCherry control, n = 42 cells and 8 animals; hM4D, n = 28 cells and 7 animals; HM4D + Saline n = 24 cells and 5 animals; frequency, mCherry control 5.750(4.183) Hz, hM4D 2.867(2.900) Hz, and hM4D + Saline 5.875(4.862) Hz, contrasts of estimated marginal means revealed a significant difference between hM4D animals and both mCherry, z = 4.188, df = ∞ *pbonferroni < 0.001, and HM4D + Saline, z = 4.515 df = ∞ *pbonferroni < 0.001 but no significant difference between mCherry and HM4D + Saline, z = −1.005, df = ∞ *pbonferroni = 0.945; median instantaneous frequency, mCherry control 9.292(6.699) Hz, hM4D 4.472(4.044) Hz, hM4D + Saline 11.349(9.957) Hz; contrasts of estimated marginal means revealed a significant difference between hM4D animals and both mCherry, z = 4.142, df = ∞ *pbonferroni < 0.001, and HM4D + Saline, z = 5.392, df = ∞ *pbonferroni < 0.001 but no significant difference between mCherry and HM4D + Saline, z = −1.953, df = ∞ *pbonferroni = 0.153; amplitude, mCherry control 21.691(10.399) pA, hM4D 22.066(14.875) pA, and HM4D + Saline 29.665(10.517) pA. All reported values are median(interquartile range). g, Graph of the cumulative firing distribution for sEPSCs in L II/III pyramidal cells in the mPFC; IEI represents the interevent interval between spontaneous events. h, sIPSC amplitude (left), median instantaneous frequency (middle), and frequency (right) are unchanged following adolescent thalamic inhibition relative to control mice; mCherry control, n = 42 cells and 8 animals; hM4D, n = 28 cells and 7 animals; HM4D + Saline n = 24 cells and 5 animals; amplitude, mCherry control 18.503(9.833) pA, hM4D 18.526(9.687) pA, and hM4D + Saline 16.586(6.272); median instantaneous frequency, mCherry control 4.994(8.177) Hz, hM4D 2.829(4.170) Hz, and hM4D + Saline 4.107(3.993); frequency, mCherry control 3.158(3.867) Hz, hM4D 1.433(2.017) Hz and HM4D + Saline 2.425(1.825). All reported values are median(interquartile range). i, Graph of the cumulative firing distribution for sIPSCs in L II/III pyramidal cells in the mPFC; IEI represents the interevent interval between spontaneous events. *p < 0.05.
- Figure 2.
Adolescent thalamocortical inhibition decreases excitatory and inhibitory transmission to layer II/III NAc-projecting cells in the mPFC. a, Adolescent experimental timeline and schematic. Whole-cell patch-clamp recordings were made from accumbens-projecting pyramidal neurons in layer II/III of the mPFC from hM4D and control mice; created with https://biorender.com. b, Example image illustrating rGFP-positive, accumbens-projecting cells in the prelimbic subregion of the medial prefrontal cortex. c, Each circle represents the approximate location of a recorded cell. Scale bar, 500 µm. d, Representative traces showing sEPSCs of hM4D animals and mCherry controls. e, Representative traces showing sIPSCs of hM4D animals and mCherry controls. f, sEPSC frequency (right) is significantly reduced in accumbens-projecting cortical neurons following adolescent thalamic inhibition relative to control mice, but sEPSC amplitude (left) and median instantaneous frequency (middle) are unchanged; mCherry control, n = 21 cells and 5 animals; hM4D, n = 28 cells and 6 animals; frequency: mCherry control 5.367(2.717) Hz, hM4D 2.975(1.871) Hz; median instantaneous frequency, mCherry control 8.514(4.842) Hz and hM4D 5.579(4.803) Hz; amplitude: mCherry control 21.051(7.992) pA and hM4D 23.648(12.495) pA. All reported values are median(interquartile range). g, Graph of the cumulative firing distribution for sEPSCs in L II/III accumbens-projecting pyramidal cells in the mPFC; IEI represents the interevent interval between spontaneous events. h, sIPSC frequency (right) is significantly reduced in accumbens-projecting cortical cells following adolescent thalamic inhibition relative to control mice, but sIPSC amplitude (left) and median instantaneous frequency (middle) are unchanged; mCherry control, n = 21 cells and 5 animals; hM4D, n = 28 cells and 6 animals; frequency, mCherry control 3.350(2.350) Hz, hM4D 1.417(1.762) Hz; median instantaneous frequency, mCherry control 5.195(4.817) Hz and hM4D 2.776(2.753) Hz; amplitude, mCherry control 14.618(3.768) pA and hM4D 17.597(7.919) pA. All reported values are median(interquartile range). i, Graph of the cumulative firing distribution for sIPSCs in L II/III accumbens-projecting pyramidal cells in the mPFC. *p < 0.05.
- Figure 3.
Adolescent thalamocortical inhibition decreases excitatory and inhibitory transmission to MD-projecting cells in layer V/VI of the mPFC. a, Adolescent experimental timeline and schematic. Whole-cell patch-clamp recordings were made from cortical-projecting pyramidal cells in layer V/VI of the mPFC from hM4D and control mice. These pyramidal cells receive excitatory inputs from the thalamus and superficial layers of the cortex as well as inhibitory inputs from local interneurons; created with https://biorender.com. b, Example image illustrating rGFP-positive cells in the prelimbic subregion of the medial prefrontal cortex. All recordings for this experiment were conducted in this region ∼500–600 µm from the pial surface (L V/VI). cR Each circle represents the approximate location of a recorded cell. Scale bar, 300 µm. d, Representative traces showing sEPSCs of hM4D animals and mCherry controls. e, Representative traces showing sIPSCs of hM4D animals and mCherry controls. f, sEPSC frequency (right) and median instantaneous frequency (middle) are significantly reduced in thalamic-projecting cortical cells following adolescent thalamic inhibition relative to control mice, but sEPSC amplitude (left) is unchanged; mCherry control, n = 19 cells and 4 animals; hM4D, n = 26 cells and 5 animals; frequency, mCherry control 6.717(4.392) Hz and hM4D 2.833(1.408) Hz; median instantaneous frequency, mCherry control 11.173(7.187) Hz and hM4D 4.204(1.997) Hz; amplitude, mCherry control 21.764(9.897) pA and hM4D 24.043(13.508) pA. All reported values are median(interquartile range). g, Graph of the cumulative firing distribution for sEPSCs in L V/VI thalamic-projecting pyramidal cells in the mPFC; IEI represents the interevent interval between spontaneous events. h, sIPSC frequency (right) and median instantaneous frequency (middle) is significantly reduced in thalamic-projecting cortical neurons following adolescent thalamic inhibition relative to control mice, but sIPSC amplitude (left) is unchanged; mCherry control, n = 19 neurons and 4 animals; hM4D, n = 26 neurons and 5 animals; frequency: mCherry control 4.917(1.825) Hz and hM4D 2.100(1.350) Hz; median instantaneous frequency: mCherry control 7.896(4.100) Hz and hM4D 2.755(2.081) Hz; amplitude: mCherry control 19.267(7.264) pA and hM4D 15.720(7.127) pA. All reported values are median(interquartile range). i, Graph of the cumulative firing distribution for sIPSCs in L V/VI thalamic-projecting pyramidal neurons in the mPFC. *p < 0.05.
- Figure 4.
Adolescent thalamocortical inhibition increases the amplitude of excitatory and inhibitory currents to layer V/VI NAc-projecting cells in the mPFC. a, Adolescent experimental timeline and schematic. Whole-cell patch-clamp recordings were made from accumbens-projecting pyramidal neurons in layer V/VI of the mPFC from hM4D and control mice; created with https://biorender.com. b, Example image illustrating rGFP-positive, accumbens-projecting cells in the prelimbic subregion of the medial prefrontal cortex. c, Each circle represents the approximate location of a recorded cell. Scale bar, 500 µm. d, Representative traces showing sEPSCs of hM4D animals and mCherry controls. e, Representative traces showing sIPSCs of hM4D animals and mCherry controls. f, sEPSC amplitude (left) is significantly increased in accumbens-projecting cortical neurons following adolescent thalamic inhibition relative to control mice, but sEPSC frequency (right) and median instantaneous frequency (middle) are unchanged; mCherry control, n = 22 neurons and 5 animals; hM4D, n = 9 neurons and 4 animals; frequency, mCherry control 4.450(2.012) Hz, hM4D 3.167(4.217) Hz; median instantaneous frequency, mCherry control 7.371(4.043) Hz and hM4D 5.290(12.190) Hz; amplitude, mCherry control 20.460(11.638) pA and hM4D 29.858(5.869). All reported values are median(interquartile range). g, Graph of the cumulative firing distribution for sEPSCs in L II/III accumbens-projecting pyramidal neurons in the mPFC; IEI represents the interevent interval between spontaneous events. h, sIPSC frequency (right) is significantly reduced in accumbens-projecting cortical neurons following adolescent thalamic inhibition relative to control mice, but sIPSC amplitude (left) and median instantaneous frequency (middle) are unchanged; mCherry control, n = 21 neurons and 5 animals; hM4D, n = 28 neurons and 6 animals; frequency, mCherry control 1.883(1.817) Hz, hM4D 2.50(2.350) Hz; median instantaneous frequency, mCherry control 2.997(2.405) Hz and hM4D 4.005(4.349) Hz; amplitude, mCherry control 13.362(2.306) pA and hM4D 17.495(3.331). All reported values are median(interquartile range). i, Graph of the cumulative firing distribution for sIPSCs in L V/VI accumbens-projecting pyramidal neurons in the mPFC. *p < 0.05.
- Figure 5.
Adolescent thalamocortical inhibition increases inhibitory transmission in layer II/III callosal-projecting cells in the mPFC. a, Adolescent experimental timeline and schematic. Whole-cell patch-clamp recordings were made from cortical-projecting pyramidal neurons in layer II/III of the mPFC from hM4D and control mice; created with https://biorender.com. b, Example image illustrating rGFP-positive, cortical-projecting cells in the prelimbic subregion of the medial prefrontal cortex. Virus was injected into the dextral (right) hemisphere and cells were recorded only in the sinistral (left) hemisphere. c, Each circle represents the approximate location of a recorded cell. Scale bar, 500 µm. d, Representative traces showing sEPSCs of hM4D animals and mCherry controls. e, Representative traces showing sIPSCs of hM4D animals and mCherry controls. f, sEPSC frequency (right), sEPSC amplitude (left), and sEPSC median instantaneous frequency are unchanged in cortical-projecting cortical neurons following adolescent thalamic inhibition relative to control mice; mCherry control, n = 38 neurons and 5 animals; hM4D, n = 26 neurons and 5 animals; frequency, mCherry control 3.042(1.713) Hz and hM4D 2.95(2.933) Hz; median instantaneous frequency, mCherry control 4.807(3.944) Hz and hM4D 5.042(5.890) Hz; amplitude, mCherry control 17.876(7.549) pA and hM4D 23.951(8.397) pA. All reported values are Median(Interquartile Range). g, Graph of the cumulative firing distribution for sEPSCs in cortical-projecting pyramidal neurons in the mPFC; IEI represents the interevent interval between spontaneous events. h, sIPSC median instantaneous frequency (middle) is significantly increased and frequency (right) is trending toward an increase in cortical-projecting cortical neurons following adolescent thalamic inhibition relative to control mice, while amplitude (left) is unchanged; mCherry control, n = 38 neurons and 5 animals; hM4D, n = 26 neurons and 5 animals; frequency, mCherry control 1.233(0.954) Hz and hM4D 2.208(1.887) Hz; median instantaneous frequency, mCherry control 1.937(1.676) Hz and hM4D 5.551(2.498); amplitude, mCherry control 15.735(5.268) pA and hM4D 15.099(5.667) pA. All reported values are median(interquartile range). i, Graph of the cumulative firing distribution for sIPSCs in cortical-projecting pyramidal neurons in the mPFC. *p < 0.05, #p < 0.10.
- Figure 6.
Adolescent thalamic inhibition only alters Excitation-Inhibition balance in layer II/III callosally projecting cells in the mPFC. a, Left panel, Schematic of viral injections. Center panel, Excitatory/inhibitory ratio in nonspecific layer II/III cells for amplitude (left), median instantaneous frequency (middle), and frequency (right). There was no significant difference in E/I ratio between mCherry 1.251(0.443) pA/pA, hM4D 1.432(0.854) pA/pA, and hM4D + Saline 1.633(0.738) pA/pA for amplitude (f = 1.966; df = 2, 5.47; p = 0.173). There was no significant difference in E/I ratio between mCherry 1.693(1.858) Hz/Hz, hM4D 1.427(1.310) Hz/Hz, and hM4D + Saline 2.185(1.269) Hz/Hz for median instantaneous frequency (f = 0.770; df = 2, 17.63; p = 0.478). There was no significant difference in E/I ratio between mCherry 1.723(1.571) Hz/Hz, hM4D 1.270(1.784) Hz/Hz, and hM4D + Saline 2.123(1.217) Hz/Hz for frequency (f = 1.196; df = 2, 16.55; p = 0.327). Right panel, Diagram of significant changes in excitation and inhibition after adolescent thalamocortical inhibition. b, Left panel, Schematic of viral injections. Center panel, Excitatory/inhibitory ratio in layer II/III accumbens projecting cortical cells for amplitude (left), median instantaneous frequency (middle), and frequency (right). There was no significant difference in E/I ratio between mCherry 1.316(0.587) pA/pA and hM4D 1.524(0.505) pA/pA for amplitude (f = 0.543; df = 1, 6.59; p = 0.487). There was no significant difference in E/I ratio between mCherry 1.686(1.368) Hz/Hz and hM4D 1.999(1.715) Hz/Hz for median instantaneous frequency (f = 0.836; df = 1, 7.91; p = 0.388). There was no significant difference in E/I ratio between mCherry 1.663(0.963) Hz/Hz and hM4D 1.865(1.406) Hz/Hz for frequency (f = 0.750; df = 1, 8.10; p = 0.411). Right panel, Diagram of significant changes in excitation and inhibition after adolescent thalamocortical inhibition. c, Left panel, Schematic of viral injections. Center panel, Excitatory/inhibitory ratio in layer V/VI thalamic projecting cortical cells for amplitude (left), median instantaneous frequency (middle), and frequency (right). There was no significant difference in E/I ratio between mCherry 1.195(0.500) pA/pA and hM4D 1.376(0.858) pA/pA for amplitude (f = 3.625; df = 1; 42; p = 0.064). There was no significant difference in E/I ratio between mCherry 1.414(0.485) Hz/Hz and hM4D 1.660(1.143) Hz/Hz for median instantaneous frequency (f = 0.975; df = 1, 5.21; p = 0.367). There was no significant difference in E/I ratio between mCherry 1.414(0.485) Hz/Hz and hM4D 1.503(1.136) Hz/Hz for frequency (f = 1.115; df = 1, 5.31; p = 0.337). Right panel, Diagram of significant changes in excitation and inhibition after adolescent thalamocortical inhibition. d, Left panel, Schematic of viral injections. Center panel, Excitatory/inhibitory ratio in layer V/VI accumbens projecting cortical cells for amplitude (left), median instantaneous frequency (middle), and frequency (right). There was no significant difference in E/I ratio between mCherry 1.467(0.624) pA/pA and hM4D 1.730(0.618) pA/pA for amplitude (f = 2.220; df = 1, 28; p = 0.147). There was no significant difference in E/I ratio between mCherry 2.060(1.840) Hz/Hz and hM4D 2.432(1.435) Hz/Hz for median instantaneous frequency (f = 0.412; df = 1, 12.33; p = 0.533). There was no significant difference in E/I ratio between mCherry 1.829(1.623) Hz/Hz and hM4D 2.658(1.765) Hz/Hz for frequency (f = 0.014; df = 1, 12.10; p = 0.908). Right panel, Diagram of significant changes in excitation and inhibition after adolescent thalamocortical inhibition. e, Left panel, Schematic of viral injections. Center panel, Excitatory/inhibitory ratio in cortical projecting cortical cells for amplitude (left), median instantaneous frequency (middle), and frequency (right). There was no significant difference in E/I ratio between mCherry 1.209(0.359) pA/pA and hM4D 1.473(0.403) pA/pA for amplitude (f = 1.099; df = 1, 59; p = 0.299). There was a significant decrease in E/I ratio between mCherry 2.513(1.915) Hz/Hz and hM4D 1.517(1.670) Hz/Hz for median instantaneous frequency. There was also a significant decrease in E/I ratio between mCherry 2.594(1.607) Hz/Hz and hM4D 1.564(1.544) Hz/Hz for frequency. All reported values are median(interquartile range). Right panel, Diagram of significant changes in excitation and inhibition after adolescent thalamocortical inhibition. *p < 0.05.
- Figure 7.
P20–P35 thalamocortical inhibition produces cognitive deficits in adult mice. a, Adolescent experimental timeline and schematic. J60 was administered from P20–P35 to mice expressing hM4D or a control fluorophore (GFP) in the thalamus. Behavioral testing was conducted 55 d later, at P90. Created with https://biorender.com. b, Representative image of hM4D expression in the MD of adult mice. Scale bar, 300 µm. c, Representative viral spread of hM4D receptor in mice that underwent the ASST task. d, Schematic of the ASST. Mice were trained to associate a cinnamon scent with a reward irrespective of bedding medium (IA). After meeting criterion, an EDSS occurred and mice had to associate the paper bedding with a reward irrespective of scent. Red is representative of the rewarded stimulus. e, Adolescent-inhibited hM4D animals are no different than controls in the initial acquisition (IA) of the ASST (left, control: n = 11 animals, 14.27 ± 2.276 trials; hM4D, n = 13 animals; hM4D, 12.08 ± 2.276 trials; two-sided unpaired t test; t = 0.9647; df = 22; p = 0.3452) but take significantly more trials in the EDSS than controls (right, control: n = 11 animals, 10.73 ± 1.871 trials; hM4D, n = 13 animals; hM4D, 14.92 ± 1.871 trials; unpaired t test; t = 2.442; df = 22; *p = 0.0354). f, Adolescent-inhibited hM4D animals committed more perseverative errors (P. Err.) than control animals (left, control: n = 11 animals, 1.091 ± 0.7759 trials; hM4D, n = 13 animals; hM4D, 3.000 ± 0.7759 trials; unpaired t test; t = 2.461; df = 22; *p = 0.0222). Perseverative errors are when the mouse continues with the same response strategy following a rule switch. Adolescent-inhibited hM4D animals committed more random errors (R. Err.) than control animals (right, control: n = 11 animals, 1.182 ± 0.9010 trials; hM4D, n = 13 animals; hM4D, 3.692 ± 0.9010 trials; unpaired t test; t = 2.786; df = 22; *p = 0.0108) nonperseverative errors are generally considered to be random.
Tables
mCherry hM4D hM4D + NaCl All statistical tests are linear mixed effects models A. C. A. C. A. C. Amplitude Median Inst. Freq. Frequency L II/III N 8 42 7 28 5 24 E (F(2,18.71) = 2.414; p = 0.117) (F(2,90) = 15.553, p < 0.001)↓ (F(1,92) = 12.271; p < 0.001)↓ ♂ 2 5 3 I (F(2,16.30) = 0.269; p = 0.768) (F(2,17.03) = 0.785; p = 0.472) (F(2,16.39) = 1.141; p = 0.344) ♀ 6 2 2 E/I (F(2,5.47) = 1.966; p = 0.173) (F(2,17.63) = 0.770; p = 0.478) (F(2,16.55) = 1.196; p = 0.327) II/III→NAc. N 5 21 6 28 – E (F(1,6.19) = 3.313; p = 0.117) (F(1,6.77) = 1.926; p = 0.208) (F(1,47) = 9.354; p = 0.004)↓ ♂ 4 4 I (F(1,7.03) = 3.402; p = 0.107) (F(1,7.79) = 0.955; p = 0.358) (F(1,4.89) = 9.451; p = 0.028)↓ ♀ 1 2 E/I (F(1,6.59) = 0.543; p = 0.487) (F(1,7.91) = 0.836; p = 0.388) (F(1,8.10) = 0.750; p = 0.411) V/VI→MD N 4 19 5 26 – E (F(1,5.36) = 0.689; p = 0.442) (F(1,6.10) = 13.045; p = 0.011)↓ (F(1,6.73) = 12.960; p = 0.009)↓ ♂ 1 3 I (F(1,4.77) = 0.991; p = 0.367) (F(1,5.96) = 6.501; p = 0.044)↓ (F(1,6.39) = 7.829; p = 0.029)↓ ♀ 3 2 E/I (F(1,42) = 3.625; p = 0.064) (F(1,5.21) = 0.975; p = 0.367) (F(1,5.31) = 1.115; p = 0.337) V/VI→NAc. N 5 22 4 9 – E (F(1,29) = 8.976; p = 0.006)↑ (F(1,6.24) = 0.196; p = 0.673) (F(1,5.49) ≤ 0.001; p = 0.993) ♂ 4 2 I (F(1,4.25) = 14.809; p = 0.013)↑ (F(1,6.53) = 0.910; p = 0.374) (F(1,6.82) = 0.417; p = 0.539) ♀ 1 2 E/I (F(1,28) = 2.220; p = 0.147) (F(1,12.33) = 0.412; p = 0.533) (F(1,12.10) = 0.014; p = 0.908) II/III→II/III N 5 38 5 26 – E (F(1,6.37) = 2.865; p = 0.134) (F(1,7.30) = 0.399; p = 0.547) (F(1,7.41) = 0.012; p = 0.917) ♂ 2 2 I (F(1,62) = 0.112; p = 0.912) (F(1,5.95) = 8.023; p = 0.030)↑ (F(1,7.32) = 4.185; p = 0.078)↑ ♀ 3 3 E/I (F(1,59) = 1.099; p = 0.299) (F(1,9.34) = 5.744; p = 0.039)↓ (F(1,11.37) = 5.697; p = 0.035)↓ A, animal; C, cell; all significant results are bolded and italicized with directionality indicated by an arrow.
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