f 7000 fluorescence spectrophotometer  (Hitachi Ltd)

Journal: Pain

Article Title: A glutamatergic S1-VPL-S1 corticothalamocortical loop amplifies mechanical hypersensitivity in neuropathic pain

doi: 10.1097/j.pain.0000000000004068

Figure Lengend Snippet: Activation and projection of S1HL Glu neurons to VPL Glu in SNI mice. (A) Schematic diagram of the SNI model. (B) Representative images of the Fos expression and (C) quantitative analysis of the Fos-positive neuron density in the contralateral S1HL on D7 and D30 postsurgery, n = 5 mice per group, ***P < 0.001. (D and E) Representative images of Fos (green) positive neurons coexpressed with DAPI (blue), CamKIIa (cyan), and Gad2 (red) (D), and the percentage analysis in the S1HL (E). n = 4 mice per group. (F) Schematic of virus carrying Synaptophysin-mCherry injection in S1HL to track downstream projections in CamKIIa-Cre mice. (G) Representative images of the virus injection site in S1HL (left) and the main thalamus regions with axon terminals labelled by mCherry (right). (H) The statistical analysis of synaptophysin fluorescence intensity ratio in VPL (82.22%) and PO (66.08%) compared with the local injection site. (I) Schematic of CTB-555 injection in VPL to track upstream neurons. (J) Image of the virus injection site in VPL (left) and the traced upstream neurons in S1HL L6 (right). (K and L) Representative images of CTB (red) positive neuron coexpressed with DAPI (blue), CamKIIa (cyan), or Gad2 (green) (K), and the percentage analysis in the S1HL (L). n = 3 mice. (M) Representative images of VGluT2 (red)-positive neuron and VGAT (cyan)-positive neuron in the thalamus. Data are presented as mean ± SEM. PO, posterior thalamic nucleus; S1HL, hindlimb area of S1; SNI, spared nerve injury; VPL, ventral posterolateral thalamic nucleus.

Article Snippet: The probes targeting Fos (Cat# 316921-C1), CaMKIIa (Cat# 445231-C2), Slc17a6 (Cat# 1170921-C2), Slc32a1 (Cat# 319191-C3), and Gad2 (Cat# 439371-C3) mRNA were provided by ACD Bio (Newark, CA, USA) (for detailed procedures, refer to the ACD Bio RNAscope Multiplex Fluorescent Reagent Kit V2 User Manual).

Techniques: Activation Assay, Expressing, Virus, Injection, Fluorescence

Journal: Pain

Article Title: A glutamatergic S1-VPL-S1 corticothalamocortical loop amplifies mechanical hypersensitivity in neuropathic pain

doi: 10.1097/j.pain.0000000000004068

Figure Lengend Snippet: The S1HL Glu -VPL Glu circuit mainly projects back to glutamatergic neurons in S1HL. (A) Schematic of the strategy to perform anterograde tracing of the S1HL Glu -VPL Glu circuit. (B) Representative image of the whole-brain tracing results of projecting terminals expressing synaptophysin from S1HL Glu -VPL Glu circuit. (C) Analysis of the relative fluorescence density of whole-brain axonal projections from S1HL Glu -VPL Glu circuit. Fluorescence density was calculated as the percentage of fluorescence intensity in each downstream nucleus relative to the total fluorescence intensity of all nuclei. (D) Representative image of projecting terminals expressing synaptophysin from S1HL Glu -VPL Glu in S1HL sublayers. n = 5 mice. (E) Schematic of the strategy of anterograde transsynaptic tracing of the S1HL Glu -VPL Glu -S1HL circuit. (F) The mCherry-labeled neurons in S1HL. (G and H) Schematic of retrograde monosynaptic rabies virus (RV) tracing of S1HL in CaMKIIa-Cre (G) and Gad2-Cre (H) mice. (I and K) Representative image of the injection site in S1HL in CaMKIIa-Cre (I left) and Gad2-Cre (K left) mice and the close-up of the boxed region (I right and K right). Arrows denote the starter neurons. (J and L) The RV-labeled upstream neuron in VPL in CaMKIIa-Cre (J) and Gad2-Cre (L) mice. (M and N) Representative images of mCherry (red) positive neuron coexpressed with DAPI (blue), CamKIIa (cyan), and Gad2 (green) (M), and the percentage analysis in the S1HL (N). n = 3 mice. Data are presented as mean ± SEM. AC, auditory cortex; EC, entorhinal cortex; IC, insular cortex; M1, primary motor cortex; M2, secondary motor cortex; mPFC, medial prefrontal cortex; PAG, periaqueductal gray; RVM, rostral ventromedial medulla; S1BF, primary somatosensory cortex barrel field; S1DZ, primary somatosensory cortex dysgranular zone; S1HL, hindlimb area of S1; VC, visual cortex; VPL, ventral posterolateral thalamic nucleus.

Article Snippet: The probes targeting Fos (Cat# 316921-C1), CaMKIIa (Cat# 445231-C2), Slc17a6 (Cat# 1170921-C2), Slc32a1 (Cat# 319191-C3), and Gad2 (Cat# 439371-C3) mRNA were provided by ACD Bio (Newark, CA, USA) (for detailed procedures, refer to the ACD Bio RNAscope Multiplex Fluorescent Reagent Kit V2 User Manual).

Techniques: Anterograde Tracing, Expressing, Fluorescence, Labeling, Virus, Injection

Journal: Nature

Article Title: Lifespan single-cell transcriptomic atlas of the human prefrontal cortex

doi: 10.1038/s41586-026-10271-7

Figure Lengend Snippet: a , Study design overview. snRNA-seq profiling of 284 post-mortem donors across the lifespan yielded 1,307,674 nuclei. UMAP visualization shows nuclei coloured by 8 classes and 27 subclasses. b , Analytical framework, including nucleus composition, transcriptomic changes, lineage trajectories and circadian rhythmicity. c , The mean proportion of age-specific gene expression variance explained by age across 26 subclasses. d , The fraction of nuclei across six age groups, coloured by 27 subclasses. e , The variance explained by age in subclass-specific nucleus abundance in development (dev) ( n = 53), young adulthood (YA; n = 54), middle adulthood (MA; n = 95) and late adulthood (LA; n = 82). The box plots show the median (centre lines) and 25th–75th percentiles (box limits), and the whiskers extend to ±1.5 × interquartile range. f , Estimated effect sizes of age-associated changes in nucleus abundance across age groups. P values were computed using two-sided t -tests ( n = 284) with Benjamini–Hochberg correction across 26 subclasses. g , RNAscope in situ hybridization showing labelling of IN_SST neurons (green), oligodendrocytes (red) and nuclei (DAPI, blue) in DLPFC tissue sections from donors aged 9 months, 38 years, 53 years and 86 years ( n = 4). Multiple fields of view per tissue section showed similar results. Scale bars, 20 μm. h , Counts of aDEGs stratified by development and late adulthood. The red and blue bars indicate genes increasing (up-aDEGs) and decreasing (down-aDEGs) with age. i , Gene Ontology (GO) biological process enrichment of upregulated and downregulated aDEGs using enrichR (two-sided Fisher’s exact test with Benjamini–Hochberg correction across all pathways). exc., excitatory; neg., negative; pos., positive; reg., regulation. j , MAGMA enrichment P values for upregulated and downregulated aDEGs across brain-related and non-brain-related traits. ADHD, attention deficit hyperactivity disorder; AUT, autism spectrum disorder; BD, bipolar disorder; EA, educational attainment; MDD, major depressive disorder; OCD, obsessive compulsive disorder; SCZ, schizophrenia. For f and j , P values were obtained using two-sided t -tests with Benjamini–Hochberg correction across traits and subclasses; * P < 0.05, # FDR-corrected P < 0.05 (Benjamini–Hochberg method). EN_L5_ET was excluded in c , e , f and h due to low nucleus counts. The diagram in a was partially created using BioRender; Girdhar, K. https://biorender.com/tsljhi0 (2026).

Article Snippet: RNAscope Multiplex Fluorescent v2 assay (ACD, UM 323100/Rev B) was performed on FFPE DLPFC sections (5 μm).

Techniques: Profiling, Gene Expression, RNAscope, In Situ Hybridization, Tissue

Journal: Nature

Article Title: Lifespan single-cell transcriptomic atlas of the human prefrontal cortex

doi: 10.1038/s41586-026-10271-7

Figure Lengend Snippet: The top row displays the colour scheme for each subclass. a , Stratification of lifespan trajectories of nuclei counts from all subclasses into log-increasing and log-decreasing groups. b , Examples of log-decreasing and log-increasing trends in IN PVALB (left) and EN_L2_3_IT (right) subclasses, respectively. c , crumblr results of univariate hypothesis testing on the leaves and multivariate hypothesis testing on the internal nodes shown on the hierarchical clustering, based on nucleus composition (left panel). Colour and size of each node shows the FDR value from multivariate hypothesis testing. The right panel shows estimated effect size of age-associated changes in nuclei counts across the lifespan for all subclasses (n = 284 donors). Data are presented as effect sizes from a model with age as a predictor; size of each dot depicts -log 10 (adjusted p-value); error bars represent 95% confidence intervals around effect size estimates from univariate testing. All tests were two-sided; P values from multivariate testing were adjusted using the Benjamini–Hochberg procedure. d , Representative RNAscope image from 9-month-old tissue showing two regions of interest (green and red boxes). Bottom row shows segmented nuclei: SST+ (green, left) and GPR37+ oligodendrocytes (red, right). Scale bar, 20 μm. e , Fraction of SST+ and GPR37+ nuclei across age groups (n = 8 biologically independent donors: 3 developmental [0.33, 0.75, 9 years], 1 young adult [38 years], 2 middle adult [51, 53 years], 2 late adult [65, 86 years]) from 3 regions of interest per sample, measured by RNAscope. Linear regression performed with log 2 (age + 1) as predictor; β represents the regression slope, p-value indicates statistical significance of the regression (two-sided test), and grey bands represent 95% confidence intervals around fitted regression line. f , Heatmap of z-scaled pathway enrichment scores by subclass and developmental stage. Subclasses annotated by cluster trend (log-increasing vs log-decreasing with age).

Article Snippet: RNAscope Multiplex Fluorescent v2 assay (ACD, UM 323100/Rev B) was performed on FFPE DLPFC sections (5 μm).

Techniques: RNAscope, Tissue