Additional file 3 of Expression of serotonin 1A and 2A receptors in molecular- and projection-defined neurons of the mouse insular cortex

Additional file 3: Fig. S1. mRNA expression level of Htr1a and Htr2a in different brain structures (data from Allen Brain Atlas, n = 1 mouse). a-b. mRNA expression of Htr1a in isocortical (a) and subcortical regions (b). c-d. mRNA expression of Htr2a in isocortical (c) and subcortical region (d). Fig. S2. Expression of 5-HT1A or 2A in parvalbumin-expressing neurons in the insular cortex. a. An image of PV labelled by tdTomato and nucleus staining in the anteriorinsula of PV-cre::Ai14 mouse brain. b-c. Representative images of parvalbumin-expressing (PV+) neurons containing 5-HT1A (b) or 2A (c) in the insula. Yellow arrowsindicate the expression of 5-HT1A or 2A. d. Percentage of 5-HT1A or 2A expression in PV+ neurons in the anterior (ICa) and posterior insula (ICp). Total PV+ neurons,1A+: ICa + ICp = 91 + 69 = 160; 2A+: ICa + ICp = 141 + 121 = 262; n = 4 mice e. 5-HT1A or 2A expressed in ~ 30% of PV+ neurons in the insula. Fig. S3. No sexdifference in 5-HT1A or 2A expression in six populations of the insular neurons. a and c. Percentage of glutamatergic (Glu) and GABAergic (GABA) neuronsexpressing 5-HT1A (a) or 5-HT2A (c) in the male and female insula (data of Figure 1j and 5f). Total cell numbers (n=2 mice/group, data of Figure 1j and 5f): (a) 5-HT1AR: Male: Glu=2051, GABA=458; Female: Glu=2274, GABA=428; (c) 5-HT2AR: Male: Glu=2794, GABA=641; Female: Glu=2433, GABA=478. b and d. Percentage of 5-HT1A (b) or 5-HT2A (d) -expressing insular neurons projecting to the basolateral amygdala (IC-BLA), central amygdala (IC-CeA), and rostral or caudalparts of lateral hypothalamus (IC-rLH and IC-cLH, data of Figure 3g, 4d, 5j and 5l) in male and female brains. Total cell numbers (mice number): (b) 5-HT1AR: Male:IC-BLA=852 (n=4), IC-CeA=711 (n=4), IC-rLH=402 (n=3), IC-cLH=289 (n=2); Female: IC-BLA=185 (n=3), IC-CeA=391 (n=3), IC rLH=209 (n=2), IC-cLH=377(n=3); (d) 5-HT2AR: Male: IC-BLA=370 (n=4), IC-CeA=802 (n=4), IC-rLH=368 (n=3), IC-cLH=313 (n=2), Female: IC-BLA=378 (n=3), IC-CeA=247 (n=3), ICrLH=492 (n=2), IC-cLH=509 (n=3). Fig. S4. Map of the injection sites of the retrograde tracers (CTB), represented on horizontal brain sections ofthe Paxinos atlas (3d edition). To identify the injection site, we searched the horizontal sections where the CTB signal had the largest spread in the target region, and theinjection point was defined at its center (in medio-lateral and antero-posterior axis). Fig. S5. Evidence of synaptic contacts of 5-HT1A+ insular neurons in the amygdalaand lateral hypothalamus. a. Experimental design to detect synaptic terminals of 5-HT1A-expressing (5-HT1A+) insula neurons through cre-dependent expression ofeYFP and synaptophysin-mCherry in the anterior or posterior insula of Htr1a-Cre mice. b. (Left) Confocal image of the cre-dependent viral vector injection site in acoronal section of the anterior (DI: dysgranular insula, AID: agranular insula dorsal part, AIV: agranular insula ventral part) and the posterior insula (GI: granular insula;AIP: agranular insula posterior part). Note eYFP expression in the soma of 5-HT1A+ neurons. (Right) Imaging locations of synaptophysin- mCherry in the basolateral and central amygdala (BLA and CeA) as well as the rostral and caudal part of the lateral hypothalamus (rLH and cLH). Distances are in the anteroposterioraxis from Bregma. c-d. Representative images of eYFP for labelling axonal projection and synaptophysin-mCherry for visualizing synaptic terminals in the BLA (c) andCeA (d), originating from 5-HT1A+ neurons of the posterior insula. Yellow arrows indicate axonal projection labelled by eYFP. e-f. Confocal images of synaptophysinmCherryin the BLA, CeA, rLH and cLH, expressed in the 5-HT1A+ neurons of the anterior (e) and posterior insula (f).