Fig 1: Distribution and localization of Kir6.2 immunoreactivity (ir) in the SCN. (A) Distribution of the Kir6.2 (A1–3) and AVP (A4-6) immunoreactivity in the rostral (A1, A4), middle (A2, A5), and caudal (A3, A6) sections of the SCN. Scale bar: 200 µm. (B) Selective colocalization of Kir6.2-ir with AVP-ir (B1–3) but not with VIP-ir (B4–6). Note the colocalization of Kir6.2-ir and AVP-ir in and around the soma (B2) and in varicosities along the process (marked by arrowheads, B3). Note the reciprocal apposition of VIP-ir bouton-like swellings against a Kir6.2-ir soma (B5) and Kir6.2-ir bouton-like swellings against VIP-ir somata (B6). Note also bouton-like swellings (yellow) double-stained with Kir6.2 (green) and AVP (red) apposing Hoechst-stained cells (blue) in the ventrolateral region of the SCN (B7). Scale bar: 100 µm (B1, B4); 10 µm (B2, B3, B5–7). (C) Lack of colocalization of Kir6.2-ir with markers for three afferent inputs SERT-ir (C1, C2), vGluT2-ir (C3, C4), NPY-ir (C5, C6). Insets: co-distribution of SERT-ir, vGluT2-ir, and NPY-ir with Kir6.2-stained somata in the ventromedial region of the mid-SCN section. Scale bar: 100 µm (C1, C3, C5); 10 µm (C2, C4, C6, insets). OC: optic chiasm. 3 V: third ventricle. Asterisks mark Hoechst-stained nuclei.
Fig 2: Double labeling of PCDH11X/Y. (A–D) Merged images showing co-localization of all PCDH11X/Y antibodies in the fetal cerebral cortex, 13 PCW female (A, Procad1a: green, anti-PCDH11X/Y: red; B, Procad1a: green, X11: red; C, Ex6: green, X11: red; D, Ex6: green, anti-PCDH11X/Y: red). (E and F) Merged images showing co-localization of PCDH11X/Y with DCX and NPY in the fetal cerebral cortex, 13 PCW female (E, Procad1a: green, DCX: red; F, Procad1a: green, NPY: red). Arrowheads in (E and F) highlight the co-expression of PCDH11X/Y in the SP and IZ with DCX and NPY, respectively. (G–K) Adult cerebral cortex, female (G, Procad1a: green; H, anti-PCDH11X/Y: red; I, merged image; J, calretinin: green, anti-PCDH11X/Y: red; K, calbindin: green, anti-PCDH11X/Y: red). Arrowheads in (J and K) highlight the expression of PCDH11X/Y in the absence of calretinin and calbindin, respectively. Scale bars: (A–J) 100 µm; (K): 25 µm. CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone.
Fig 3: Effect of anti-NGF treatment on expression of CGRP-ir and NPY-ir neurons in dorsal root ganglia. The percentage of CGRP-ir (A) or NPY-ir (B) neurons in the upper lumbar (L1–L3) and lower lumbar (L4–L6) DRG was evaluated in sham + saline mice (black bars, left columns, OVX + saline mice red bars, middle columns) and OVX + anti-NGF (blue bars, right columns). Calcitonin gene-related peptide–ir neurons in the lower lumbar DRG in OVX + saline with significantly increased compared with sham + saline mice, and anti-NGF treatment significantly decreased the expression of CGRP-ir neurons in OVX mice (A). In the upper lumbar DRG, CGRP-ir neurons just tended to be higher than sham-operated mice, but there was no significant difference between the OVX group with vehicle and with anti-NGF (A). In the expression of NPY-ir neurons in both upper and lower lumbar DRG, no effects of OVX-surgery or anti-NGF treatment were observed (B). Data are expressed as mean ± SEM. **P < 0.01; OVX + saline vs sham + saline; †P < 0.05; OVX + saline vs OVX + anti-NGF, 1-way ANOVA, followed by the Tukey multiple comparisons. ANOVA, analysis of variance; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; NGF, nerve growth factor; NPY, neuropeptide-Y; OVX, ovariectomized.
Fig 4: Enhanced survival of interneurons in the juvenile hippocampus.a In situ hybridization on coronal sections of adult brains using a cRNA probe for GAD67 (GAD1) (top panels). Quantification of GAD67+ cells in the different hippocampal regions of control and mutant animals (bottom panel) showing no differences in the number of interneurons in distinct hippocampal regions (p = 0.6947 for CA1/CA2; p = 0.8548 for DG; p = 0.9578 for CA3, two-way ANOVA, Sidak’s multiple comparison test, n = 3 controls, n = 4 mutants). Dotted lines in a–d represent the subdivisions in different regions: CA1/CA2, CA3 and DG. b In situ hybridization on coronal sections of juvenile brains using a cRNA probe for GAD67 (top panels). Quantification of GAD67+ cells in the different hippocampal regions of control and mutant animals (bottom panel) showing increased number of interneurons in CA1/CA2 (p = 0.0118), but not CA3, DG or total number of interneurons in mutant animals (p > 0.9999; 0.8129 and 0.3429, respectively; two-way ANOVA, Sidak’s multiple comparison test; Mann–Whitney for total, n = 4 controls and n = 4 mutants). c In situ hybridization on coronal sections of juvenile brains using a cRNA probe for somatostatin (SST) (top panels). Quantification of SST+ cells of control and mutant animals (bottom panel) showing no differences in the number of interneurons in mutant animals (n = 3 controls and n = 3 mutants) in CA1/CA2, CA3 and DG or in their total numbers (p = 0.9655 for CA1/CA2; p = 0.9998 for DG; p > 0.9999 for CA3, two-way ANOVA, Sidak’s multiple comparison test; p > 0.9999 for total, Mann–Whitney). d Immunofluorescence for NPY on coronal sections of juvenile brains (top panels). Quantification of NPY+ cells of control and mutant animals showing increased number of interneurons in CA1/CA2 (p = 0.003), but not CA3 or DG (p = 0.9621 and p = 0.5889 respectively, two-way ANOVA, Sidak’s multiple comparison test), as well as an increase in the total number of NPY+ interneurons in mutant animals (p = 0.0017; Mann–Whitney; n = 4 controls and n = 4 mutants). High magnification of a CA1 portion (white square) (right panels) highlighting NPY+ neurons (yellow arrows). Scale bar: 100 µm. Scale bars in a–d low magnifications: 500 µm. Asterisks indicate statistical significance (*p < 0.05; **p < 0.01). All data are presented as mean ± SEM.
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