Journal: Advanced Science

Article Title: A Critical Time Window of Neocortex Modulation for Remote Fear Memory in Rats and Humans

doi: 10.1002/advs.202520707

Figure Lengend Snippet: Dopamine D1/D5 receptors modulate delayed mPFC actions on remote fear memory. (A) Rats were infused with vehicle or SCH23390/ SKF38393 /propranolol into the PrL 18 h after contextual fear memory training. (B) Remote but not recent memory decreased after SCH23390 was injected into the PrL 18 h after contextual fear memory training (drug × test day interaction: F (1, 18) = 5.03 , p = 0.038). (C) The microinjection of SKF38393 into the PrL 18 h after contextual fear memory training promoted the maintenance of remote fear memory (unpaired t‐test, p = 0.041). (D) The microinjection of propranolol into the PrL 18 h after contextual fear memory training had no effect on the expression of recent or remote fear memory ( p > 0.05). The data in B‐D are expressed as the mean ± SEM. * p ≤ 0.05. n = 8–12 rats/group. We used weak training (0.3 mA) for the agonist SKF38393 experiment to provide headroom for enhancement effects, and normal training (0.9 mA) for antagonist experiments. (E) Timeline of sEPSC recordings of PrL pyramidal neurons before and after administration of the D 1 /D 5 receptor antagonist SCH23390 or the D 1 /D 5 receptor agonist SKF38393 in the naive group and 18 h group after contextual fear memory training. (F) Example sEPSC traces from PrL pyramidal neurons in the naive group (pink: naive baseline; red: naive SCH23390) and 18 h group (light blue: 18 h baseline; dark blue: 18 h SCH23390). Scale bar: 20 pA (vertical), 1 s(horizontal). (G) Cumulative distribution of sEPSC inter‐event intervals and dot plots (inset) showed that PrL pyramidal neurons in the 18 h group had significantly lower sEPSC frequencies after SCH23390 administration ( F (1, 19) = 12.48, p = 0.0022). (H) Cumulative distribution of sEPSC amplitudes and dot plots (inset) showed that PrL pyramidal neurons in the 18 h group had lower sEPSC amplitudes after SCH23390 administration (18 h + SCH vs. 18 h baseline: paired t‐test, p = 0.042). (I) Example sEPSC traces from PrL pyramidal neurons in the naive group (pink: naive baseline; red: naive SKF38393 ) and 18 h group (light blue: 18 h baseline; dark blue: 18 h SKF38393 ). Scale bar: 20 pA (vertical), 1 s(horizontal). (J) Cumulative distribution of sEPSC inter‐event intervals and dot plots (inset) showed that the PrL pyramidal neurons both in the naive group and the 18 h group had significantly higher sEPSC frequencies after SKF38393 administration (Naive + SKF vs. Naive baseline: paired t‐test, p < 0.001; 18 h + SKF vs. 18 h baseline: paired t‐test, p = 0.036). (K) Cumulative distribution of sEPSC amplitudes and dot plots (inset) showed that PrL pyramidal neurons in the naive group or 18 h group had significantly higher sEPSC amplitude after SKF38393 administration (Naive + SKF vs. Naive baseline: paired t‐test, p = 0.039; 18 h + SKF vs. 18 h baseline: paired t‐test, p = 0.0021). The data in G, H, J, and K are expressed as the mean ± SEM, * p ≤ 0.05, n = 8–10 neurons and 8–10 sections from 3 rats per group. Frequencies in the inset are reported in Hz for clarity, derived from inter‐event intervals.

Article Snippet: For ex vivo pharmacology, drugs were bath applied: β‐adrenoceptor antagonist propranolol ((S)‐(−)‐propranolol hydrochloride, 295.80 g/mol; Sigma–Aldrich) at a concentration of 100 μ m , D 1 /D 5 receptor antagonist SCH23390 (SCH23390 hydrochloride, 324.24 g/mol; Tocris) at a concentration of 10 μ m , D 1 /D 5 receptor agonist SKF38393 ((R)‐(+)‐ SKF38393 hydrochloride, 291.77 g/mol; Sigma) at a concentration of 20 μ m , these drugs were freshly dissolved in ACSF.

Techniques: Injection, Microinjection, Expressing, Derivative Assay

Journal: Advanced Science

Article Title: A Critical Time Window of Neocortex Modulation for Remote Fear Memory in Rats and Humans

doi: 10.1002/advs.202520707

Figure Lengend Snippet: The delayed engagement of VTA‐mPFC projection modulates remote fear memory via dopamine D 1 /D 5 receptors in the PrL. (A) Retro‐AAV‐hSyn‐EGFP‐WPRE‐hGH (Retro‐EGFP) in the PrL. (B) (Left) The image shows Retro‐EGFP and DAPI staining in the PrL (scale bar = 1 mm). (Middle) The image shows Retro‐EGFP and DAPI staining in the VTA (scale bar = 200 µ m ). (Right) Retro‐EGFP‐labeled neuron from the PrL co‐labeled with DAPI in the VTA (scale bar = 50 µ m ). (C) Schematic of the experiment. Retro‐AAV‐hSyn‐Cre‐EGFP was injected into the PrL, and rAAV‐EF1α‐DIO‐hM4Di‐mCherry/rAAV‐EF1α‐DIO‐hM3Dq‐mCherry/rAAV‐EF1α‐DIO‐mCherry was injected into the VTA. Three weeks later, the rats underwent contextual fear memory training, and CNO was injected 17 h after CFC. Brains were removed 60 min after the remote fear memory test. (D) (Left) Retro‐Cre‐EGFP expressed at the PrL injection site (scale bar = 1 mm). (Right) Local magnification of Retro‐Cre‐EGFP expressed in the PrL (green: Retro‐Cre‐EGFP; blue: DAPI; scale bar = 50 µ m ). (E) (Left) Retro‐Cre‐EGFP and DIO‐ hM4Di‐mCherry (DIO‐mCherry for control) expressed in the VTA. (Right) Local magnification of virus expression in the VTA (green: Retro‐Cre‐EGFP; red: DIO‐ hM4Di ‐mCherry or DIO‐mCherry; blue: DAPI; scale bar = 50 µ m ). (F) After contextual fear memory training, CNO application at 17 h significantly decreased remote fear memory but had no effect on recent fear memory (two‐way ANOVA followed by Bonferroni‐corrected post hoc tests, F (1, 16) = 31.27, p < 0.001). n = 9 rats/group. (G) (Left) Retro‐EGFP‐Cre expressed at the PrL injection site (scale bar = 1 mm). (Right) Local magnification of Retro‐Cre‐EGFP expressed in the PrL (green: Retro‐Cre‐EGFP; blue: DAPI; scale bar = 50 µ m ). (H) (Left) Retro‐Cre‐EGFP and DIO‐ hM3Dq‐mCherry (DIO‐mCherry for control) expressed in the VTA. (Right) Local magnification of virus expression in the VTA (green: Retro‐Cre‐EGFP; red: DIO‐ hM3Dq‐mCherry or DIO‐mCherry; blue: DAPI; scale bar = 50 µ m ). (I) After contextual fear memory training, CNO application at 17 h promoted the maintenance of remote fear memory (unpaired t‐test, p < 0.05). n = 8 rats/group. (J) Schematic of the experiment. Retro‐Cre‐EGFP was injected into the PrL, and DIO‐ hM4Di‐mCherry/DIO‐hM3Dq‐mCherry was injected into the VTA. Three weeks later, the rats received a CNO injection 17 h after contextual fear memory training, and vehicle or SKF38393 /SCH23390 was injected into the PrL 1 h later. (K) (Left) Retro‐Cre‐EGFP expressed at the PrL injection site (scale bar = 1 mm). (Right) Local magnification of Retro‐Cre‐EGFP expressed in the PrL (green: Retro‐Cre‐EGFP; blue: DAPI; scale bar = 50 µ m ). (L) (Left) Retro‐Cre‐EGFP and DIO‐ hM4Di‐mCherry expressed in the VTA. (Right) Local magnification of virus expression in the VTA (green: Retro‐Cre‐EGFP; red: DIO‐ hM4Di‐mCherry; blue: DAPI; scale bar = 50 µ m ). (M) Chemogenetic inhibition of the VTA‐PrL projection impaired the expression of remote fear memory but not in rats that received SKF38393 in the PrL (two‐way ANOVA followed by Bonferroni‐corrected post hoc tests, F (1, 19) = 9.71, p = 0.006). n = 10–11 rats/group. (N) (Left) Retro‐Cre‐EGFP expressed at the PrL injection site (scale bar = 1 mm). (Right) Local magnification of Retro‐Cre‐EGFP expressed in the PrL (green: Retro‐Cre‐EGFP; blue: DAPI; scale bar = 50 µ m ). (O) (Left) Retro‐Cre‐EGFP and DIO‐hM3Dq‐mCherry expressed in the VTA. (Right) Local magnification of virus expression in the VTA (green: Retro‐Cre‐EGFP; red: DIO‐hM3Dq‐mCherry; blue: DAPI; scale bar = 50 µ m ). (P) Chemogenetic activation of the VTA‐PrL projection promoted the expression of remote fear memory but not in rats that received SCH23390 in the PrL (unpaired t‐test, p = 0.003). n = 8–10 rats/group. The data in F, I, M, and P are expressed as the mean ± SEM. * p ≤ 0.05.

Article Snippet: For ex vivo pharmacology, drugs were bath applied: β‐adrenoceptor antagonist propranolol ((S)‐(−)‐propranolol hydrochloride, 295.80 g/mol; Sigma–Aldrich) at a concentration of 100 μ m , D 1 /D 5 receptor antagonist SCH23390 (SCH23390 hydrochloride, 324.24 g/mol; Tocris) at a concentration of 10 μ m , D 1 /D 5 receptor agonist SKF38393 ((R)‐(+)‐ SKF38393 hydrochloride, 291.77 g/mol; Sigma) at a concentration of 20 μ m , these drugs were freshly dissolved in ACSF.

Techniques: Staining, Labeling, Injection, Control, Virus, Expressing, Inhibition, Activation Assay

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: SLC25A33 deficiency induces pericyte differentiation via mitochondrial ROS accumulation, which were regulated by the TGF‐β/Smad2 signaling pathway. (A) Flow cytometry analysis using MitoSOX Red shows increased mtROS production in SLC25A33‐suppressed HBVPs compared to control and SLC25A33‐overexpressing groups. (B) Quantification of mtROS fluorescence intensity confirms this increase. (C, D) qPCR analysis reveals upregulated α‐Sma and downregulated E‐cadherin mRNA levels in Slc25a33 ‐knockdown HBVPs. (E) Representative Western blot images of α‐SMA and E‐cadherin protein expression. (F, G) Densitometric quantification shows increased α‐SMA and decreased E‐cadherin protein levels. (H) qPCR analysis shows upregulated Tgfb1 mRNA in SLC25A33‐deficient HBVPs. (I–K) No significant changes are observed in Smad2 , Smad3 , or Smad4 mRNA levels among groups. (L) Representative western blots of TGF‐β, p‐Smad2, Smad2, p‐Smad3, Smad3, Smad4, and GAPDH. (M) TGF‐β protein levels are significantly elevated in the knockdown group. (N) Phosphorylated Smad2 (p‐Smad2) is markedly increased, indicating pathway activation. (O–R) No significant differences are detected in p‐Smad3, total Smad2, Smad3, or Smad4 protein expression. Control: HBVPs without treatment; SLC25A33 (−): HBVPs treated with LV‐ Slc25a33 ‐shRNA; SLC25A33 (+): HBVPs treated with LV‐ Slc25a33 ‐OE. Data are mean ± SEM; n = 3.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: Flow Cytometry, Analysis, Control, Fluorescence, Knockdown, Western Blot, Expressing, Activation Assay, shRNA

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: SLC25A33 modulates α‐SMA and E‐cadherin expression through the TGF‐β/Smad2 pathway in vivo. (A) Immunofluorescence staining shows α‐SMA (red) and E‐cadherin (green) expression in the stria vascularis. Nuclei are counterstained with DAPI (blue). (B, C) Quantification reveals increased α‐SMA and decreased E‐cadherin in the Slc25a33 ‐knockdown group ( n = 5, five typical sections from five individual mice were analyzed per group). (D, E) qPCR confirms upregulation of α‐Sma and downregulation of E‐cadherin mRNA levels in the Slc25a33 ‐knockdown group ( n = 3 mice). (F, G) TGF‐β and (H, I) p‐Smad2 immunoreactivity are enhanced in the knockdown group and reduced in the overexpression group ( n = 5, five typical sections from five individual mice were analyzed per group). (J) qPCR confirms upregulation of Tgf‐β mRNA levels in the knockdown group and downregulation levels in the overexpression group ( n = 3 mice). Control: wild‐type C57BL/6J mice without treatment; SLC25A33 (−): mice treated with AAV‐ie‐sh Slc25a33 ; SLC25A33 (+): mice treated with AAV‐ie‐ Slc25a33 ‐OE. Data are mean ± SEM.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: Expressing, In Vivo, Immunofluorescence, Staining, Knockdown, Over Expression, Control

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: Exogenous IGF‐1 rescues blood‐labyrinth barrier integrity and improves hearing function in aged mice. (A) TEM images reveal ultrastructural changes in the stria vascularis. In the IGF‐1‐inhibited group, pericytes exhibit abnormal morphology, loose association with endothelial cells, and widened tight junctions (yellow arrows). These defects are ameliorated by exogenous IGF‐1 administration. (B) Click‐evoked ABR thresholds are significantly elevated in IGF‐1‐suppressed mice but partially rescued by IGF‐1 supplementation. (C–E) At 12 kHz, ABR thresholds are significantly lower in the IGF‐1‐treated group compared to control and suppressed groups, indicating improved auditory sensitivity. (F) Proposed model of IGF‐1/SLC25A33‐mediated regulation of pericyte phenotype and BLB integrity in age‐related hearing loss. Under physiological conditions, pericytes maintain a quiescent state characterized by low α‐SMA and high E‐cadherin expression, contributing to BLB stability. With aging, declining IGF‐1 levels lead to reduced SLC25A33 expression, resulting in mtROS accumulation and activation of the TGF‐β/Smad2 signaling pathway. This drives pericyte transition toward a contractile, pro‐fibrotic phenotype (high α‐SMA, low E‐cadherin), disrupting BLB integrity and promoting cochlear dysfunction. Targeting this axis may offer therapeutic potential for ARHL. Control: wild‐type C57BL/6J mice without treatment; IGF‐1 (−): mice treated with NVP‐AEW541; IGF‐1 (+): mice treated with recombinant IGF‐1. Data are mean ± SEM; n = 6 mice.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: Morphology, Control, Sensitivity, Expressing, Stability, Activation Assay, Recombinant