Extended Data Fig. 3: Leukocyte homing during stress.
From: Brain motor and fear circuits regulate leukocytes during acute stress
a, Schematic of Cre-mediated flip-excision activation of the activating chemogenetic virus pAAV-hSyn-DIO-hM3D(Gq)-mCherry. Representative injection site of pAAV-hSyn-DIO-hM3D(Gq)-mCherry in the PVH of a CRHCre mouse stained with anti-mCherry primary antibody and AF594-labelled secondary antibody (PVH, paraventricular hypothalamus; 3V, third ventricle). Scale bar indicates 400 µm. b, Determination of plasma corticosterone levels and quantification of circulating blood leukocytes 1 h after high-dose intraperitoneal injection of clozapine-N-oxide (CNO) versus vehicle in WT mice (n = 5 mice per group). Two-tailed unpaired t-test. c, Determination of plasma corticosterone levels and quantification of circulating blood leukocytes 4 h after high-dose intraperitoneal injection of clozapine-N-oxide (CNO) versus vehicle in WT mice (n = 4 mice per group). Two-tailed unpaired t-test. d, Determination of plasma corticosterone levels and quantification of circulating blood leukocytes 4 h after intraperitoneal injection of clozapine-N-oxide (CNO) versus vehicle in CRHCre mice that had previously received an mCherry control virus injection into the PVH (two experiments with changed groups combined, total of n = 6 mice per group). Two-tailed unpaired t-test. e, Determination of plasma corticosterone levels and quantification of circulating blood leukocytes 5 h after injection of vehicle or CNO into CRHCre mice that had received an hM3D(Gq) injection into the PVH 3 weeks prior, pre- and post adrenalectomy (ADX) (leukocyte analyses: n = 7 pre-ADX vehicle, n = 6 pre-ADX CNO, n = 7 post-ADX vehicle, n = 6 post-ADX CNO; corticosterone analyses: n = 7 pre-ADX vehicle, n = 7 pre-ADX CNO, n = 3 post-ADX vehicle, n = 4 post-ADX CNO)Two-way ANOVA. f, Schematic of Cre-mediated flip-excision activation of the apoptosis-inducing virus AAV-flex-taCasp3-TEVp. Representative images of the PVH of CRHCre:R26-stop-EYFP mice injected with either pAAV-hSyn-DIO-hM3D(Gq)-mCherry control virus (left image) or AAV-flex-taCasp3-TEVp ablating virus (right image) stained with anti-GFP primary antibody and AF488-labelled secondary antibody (PVH, paraventricular hypothalamus; 3V, third ventricle). Scale bars indicate 250 µm. g, Quantification of circulating blood neutrophils in the experiments presented in Fig. 2a-2g and 2i-2k (n numbers indicated in Fig. 2a-2g and 2i-2k). Two-way ANOVA or unpaired t-test as appropriate. Data are mean ±s.e.m.; ★P < 0.05, ★★P < 0.01, ★★★P < 0.001, statistical tests used as indicated.
