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. 2020 Mar 18;40(12):2573–2588. doi: 10.1523/JNEUROSCI.2887-19.2020

Figure 4.

Figure 4.

Ablation of MnPOVgat neurons does not prevent fever induced by LPS. a, Schematic figure of AAV-DTA injection in the MnPO of Vgat-IRES-cre-GFP, Vgat-IRES-cre, or WT mice. b, A heatmap of the injection sites of all experimental mice. c, There was no difference in baseline Tb of Vgat-Cre mice injected with AAV-DTA (n = 7) or WT mice (n = 7) as controls (DTA_Vgat, 36.71 ± 0.03°C; DTA_WT, 36.52 ± 0.04°C; two-way ANOVA, p = 0.95) during 24 h and baseline locomotor activity of DTA_Vgat mice (DTA_Vgat, 2.62 ± 0.12 cpm; DTA_WT, 1.41 ± 0.08 cpm; p = 0.35, two-way ANOVA). d, The top panels show the mCherry (red) expression in the non-Vgat neurons in the MnPO of a Vgat-IRES-cre-GFP reporter mouse injected with the AAV-DTA, the central panels show the remained GABAergic (Vgat) neurons (green) in the POA region and the bottom panels show the efficiency of AAV-DTA to promote ablation of MnPOVgat in two coronal sections from the same Vgat-IRES-cre-GFP reporter mouse; no colocalization of Vgat neurons (green) and mCherry (red) was seen. e, In response to LPS injection (0.02 mg/kg), Vgat-Cre-DTA mice and WT mice showed fever responses when compared with Vgat-cre-DTA mice treated with saline, but the two groups injected with LPS (Vgat-cre or WT) were not different from each other. Vgat-cre-DTA mice treated with LPS show a reduction of LMA when compared with the same mice treated with saline, but no difference was found in LMA comparing the two groups treated with LPS. f, The Vgat-cre-DTA mice show an increase in Tb and LMA during cage exchange stress when compared with WT mice subjected to the same protocol, but no difference was found between the groups with handling. g, During the peak of the fever response, no difference was found between Vgat-cre-DTA or WT-DTA groups treated with LPS; however, Vgat-cre-DTA mice show a greater increase in Tb (fever response) after LPS when compared with same mice treated with saline (DTA_Vgat LPS, 37.15 ± 0.05°C; DTA_WT LPS, 37.11 ± 0.07°C; DTA_Vgat saline, 36.08 ± 0.04°C; ***p < 0.001, ****p < 0.0001, F(2,435) = 147.8, two-way ANOVA, followed by Bonferroni's post hoc test; n = 7 DTA_Vgat LPS; n = 7 DTA_Vgat saline; and n = 7 DTA_WT LPS). Vgat-cre-DTA mice and WT-DTA treated with LPS show similar patterns of LMA during the peak of fever response; however, Vgat-cre-DTA mice show a reduction of LMA when compared with the same mice treated with saline (DTA_Vgat LPS, 0.24 ± 0.03 cpm; DTA_WT LPS, 0.24 ± 0.04 cpm; DTA_Vgat saline, 0.88 ± 0.12 cpm; ***p < 0.001, ****p < 0.0001, F(2,400) = 17.07, two-way ANOVA; n = 7 DTA_Vgat LPS; n = 7 DTA_Vgat saline; and n = 7 DTA_WT LPS). h, During the first 2 h of stress, the ablation of MnPOVgat neurons also induced an increase in Tb and in LMA during cage exchange when compared with WT-DTA mice also subjected to the same stress protocol, but no difference was found between the groups with handling (DTA_Vgat STRESS, 37.56 ± 0.12°C; WT_STRESS, 37.0 ± 0.1°C; DTA_Vgat HANDLED, 36.19 ± 0.1°C; DTA_WT HANDLED, 36.31 ± 0.09°C; ***p < 0.001, ****p < 0.0001, F(3,442) = 102.4, two-way ANOVA, followed by Bonferroni's post hoc test; n = 5 Vgat-cre; n = 6 DTA_WT STRESS; and n = 4 DTA_WT HANDLED; DTA_Vgat STRESS, 5.8 ± 0.7 cpm; DTA_WT STRESS, 3.7 ± 0.5 cpm; DTA_Vgat HANDLED, 1.0 ± 0.3 cpm; DTA_WT HANDLED, 1.36 ± 0.3 cpm; ***p < 0.001, ****p < 0.0001, F(3,450) = 67.89, two-way ANOVA, followed by Bonferroni's post hoc test; n = 5 Vgat-cre; n = 6 DTA_WT STRESS; and n = 4 DTA_WT HANDLED). 3V, third ventricle.