Animals
Adult male and female mice of 10 weeks old were used for experiments. Eleven male and eight female wild-type C57BL/6J (Charles River Laboratory), four male Htr1a-IRES2-Cre-D knock-in mice (Htr1a-Cre; 030160, Jax Laboratory) as well as four male PVa-cre::Ai14 (PVa-cre; 008069, Jax Laboratory, Ai14; 007914, Jax Laboratory) were used in this study. Animals were kept group-housed (4-6 animals per cage) in a controlled environment (reverse 12 h light/dark cycle) with ad libitum food and water. All procedures of handling animals were approved by the French government (Ministère de lʼEnseignement Supérieur, de la Recherche et de lʼInnovation, Saisine #20414 and #19355,) and in accordance with the guidelines of the European Communities Council Directives.
Stereotaxic surgery
Mice were anesthetized with isoflurane (5% for induction, 1.5%-2.0% afterward) in the motorized stereotaxic frame (MTM-3, WPI) for the entire surgery and their body temperature was maintained with a heating pad. In order to label the insular neurons projecting to the downstream targets of our interest, 0.4 % of retrograde fluorescent tracer, Cholera toxin subunit B (CTB) coupled to Alexa Fluor 555 or 647 (CTB-555 or CTB-647, Thermo Fisher Scientific, C34776 or C34778), were injected into the basolateral amygdala (BLA, 200 nL), central nuclei of amygdala (CeA, 100 nL), rostral LH (rLH, 200 nL) and caudal LH (cLH, 200 nL) of the right hemisphere of the mouse brain (Figure 3b and Figure 4b). The following anteroposterior (AP) / mediolateral (ML) / dorsoventral (DV) stereotaxic coordinates were used (mm from Bregma): BLA: -1.45±0.15 / +3.30 / -4.90, CeA: -0.80 / +2.35 / ‑5.20, rLH: -0.70 / +1.00 / ‑5.25, cLH: -1.70 / +1.00 / -5.25.
To identify the synaptic targets of 5-HT1A-expressing (5-HT1A+) insula neurons, we injected 200 nL of a mixture of two adeno-associated viral vectors (AAVs, 1:1) into the anterior or posterior insula of Htr1a-Cre mice. The first construct was cre-dependently expressing mCherry (AAV9-Syn1-DIO-mCherry-WPRE, Vector Builder AAV9MP(VB181107-1041gwu)) while the second cre-dependently expressed synaptophysin fused to the enhanced yellow fluorescent protein eYFP (AAV9-EF1a-DIO-Syp-EYFP-WPRE, Vector Builder AAV9MP(VB181107-1041gwu)). In order to confirm this synaptophysin signal, we used an inverse viral strategy to express synaptophysin-mCherry in 5-HT1A-expressing insula neurons. We injected 250 nL of a mixture of two AAVs (1:2) into the anterior or posterior part of the insula of Htr1a-Cre mice. One construct cre-dependently expressed synaptophysin-mCherry (AAV8/2-hEF1a-DIO-Syp-mCherry, MGH, AAV-RN1) and the other construct cre‑dependently expressed eYFP (AAV9-EF1a-DIO-eYFP-WPRE-hGH, UPENN, AV‑9‑27056). The following AP / ML / DV coordinates were used (mm from Bregma): anterior insula: +1.70 / +3.10 / -3.50, posterior insula: -0.35 / +4.20 / -4.20.
Injections were performed using glass pipettes (3-000-203-G/X, Drummond) made by a puller (PC-100, Narishige) to deliver the retrograde tracer or viral vector at a rate of 5-8 nL/s using a Nanoject III (3-000-207, Drummond). After completion of the injection, the pipette was raised 100 µm, left for additional 10 min to allow diffusion of the retrograde tracer or the virus at the injection site, and then slowly withdrawn. After surgery, the mouse body temperature was maintained using a heat lamp until the animal fully recovered from anesthesia. Mice returned to their home cages for 1 week to allow CTB retrograde transport or 4 weeks to allow viral expression.
Immunohistochemistry
Brains were perfused and post-fixed with 4% paraformaldehyde (antigenfix, F/P0014, MM France) at 4°C for overnight, and cryo-protected in 30% sucrose in PBS. After embedded in optimum cutting temperature compound (OCT), horizontal sections (50 μm thick) or coronal sections (50 μm thick) were obtained using a frozen sliding microtome (12062999, Thermo Scientific) and stored in PBS at 4°C until processing for histology.
As these brain slices contain both anterior and posterior insula, the horizontal sections 3.60 mm ventral to Bregma were used for immunostaining (Figure 1d). Two successive brain sections with 50 μm dorsoventral difference were chosen and one section per brain was used for 5-HT1A staining and counting, and the next section was used for 5-HT2A staining and counting. After washed in PBS, the brain sections were blocked with 10% normal goat serum (NGS) and 0.5% Triton X-100 in PBS for 1 hour at room temperature. 5% NGS and 0.5% Triton X-100 in PBS was also used for the primary and secondary antibody dilution. Guinea pig polyclonal anti-NeuN (1:500; 266004, SYSY) [35], mouse monoclonal anti-GAD67 (1:500; MAB5406, Millipore) [36–38], rabbit polyclonal anti-5-HT1A receptor (1:500; ADI-905-741, Enzo) [39] and rabbit polyclonal anti-5-HT2A receptor (1:250; ab66049, Abcam) [40–44] were used as primary antibodies. The primary antibodies used in this study were validated and widely used in previous publications [35–44]. Alexa Fluor 488 goat anti-rabbit IgG (A11034, Fisher Scientific), Alexa Fluor 555 goat anti-guinea pig IgG (A21435, Fisher Scientific) and Alexa Fluor 647 goat anti-mouse IgG (A21236, Fisher Scientific) were used as secondary antibodies. Incubation of the primary antibodies was carried out at 4°C overnight, followed by incubation of secondary antibodies for 3 hours at room temperature with 1:1000. Washing using PBS for 5 min x 3 times was performed between every step. Lastly, Hoechst33342 (written DNA in the figures, 1:5000; 11544876, Fischer scientific) diluted in PBS was applied on the slice for 15 min before coverslips were applied.
Imaging and cell counting
Images were captured through a 10x dry objective (NA 0.70), a 40x oil-immersed objective (NA 1.30) and a 63x oil-immersed objective (NA 1.40) from a Leica SP8 confocal microscope (Leica Microscopy). First, all processed brain slices, as well as all slices containing an injection site were imaged using 10x or 20x objectives. For cell counting of NeuN, GAD-67, PV and insula projectors, z-stacks of ROIs (z step: 0.5 μm, 3-4 frame average, 1024 x 1024 pixels) were scanned using the 40x objective (Figure 1e-f and 3c and Supplementary Figure 2a). Retrograde tracers injections in two different targets is not an appropriate technique to quantify collateralization to two downstream regions due to three main limitations: (1) tracers have limited efficiency in retrograde transport (2) different CTB retrograde tracers have different efficiencies and (3) injections are performed to prioritize specificity over coverage of the target regions [45]. However, the overlap of CTB-555 and CTB-647 can instruct us whether collateralization exists. Interestingly, we detected few, but some cell bodies containing both CTB-555 and CTB-647. Specifically, we found that 3.84±1.35% of insula-amygdala labeled neurons were both IC-BLA and IC‑CeA projectors (n=5 mice, data not shown), and that 3.70±0.14% of insula‑LH labeled neurons were both IC‑rLH and IC-cLH projectors (n= 4 mice, data not shown). Therefore, neurons projecting to both BLA and CeA, or to both rLH and cLH exist, but their exact proportion should be studied using different methods, such as synaptophysin expression using viral vectors [45]. Example immunofluorescent pictures of 5-HT1A+ or 5-HT2A+ cells were captured with the 63x objective (Figure 1g-h, 3d-e, 5c-d and 5g-h and Supplementary Figure 2b-c). In order to detect synaptophysin-eYFP or synaptophysin-mCherry signal, z-stacks of BLA, CeA, rLH and cLH have been scanned with 63x objective in a picture format of 1024x1024 pixels (Number of z-stacks: 30, z-steps: ~1±0.10 μm, 2-3 frame average). All images have been processed using the open source Fiji software (ImageJ, NIH), and cell counting has been performed manually.
Brain Atlases
Three different atlases of the mouse brain were used in this study. Data of mRNA expression level in different brain regions were extracted from the Allen Mouse Brain Atlas (Supplementary Figure 1, Video 1 and 2, http://mouse.brain-map.org/). The mRNA expression level in regions of interests (ROIs) of in situ hybridization was calculated by multiplying the expression density and intensity [24].
[Please see the supplementary files section to view the equations.]
The Franklin & Paxino’s Brain Atlas was used to confirm the injection sites of CTB- or virus-injected brains (Figure 2a, 3b, and 4b) and to determine the scanning regions in the insular cortex (Figure 1d) and the dorsal raphe nucleus (Figure 1c) [46].
To compute the overall proportions of 5-HT1A and 2A expressing neurons among insular neurons (Figure 6b), we calculated the percentage of glutamatergic and GABAergic neurons using the Blue Brain Cell Atlas (https://bbp.epfl.ch/nexus/cell-atlas/) [47]. This recent study provides the first 3D cell atlas of the entire mouse brain, providing cell positions algorithmically constructed from Nissl staining and in situ hybridization of specific genetic markers within the whole brain. We included the total number of neurons, of excitatory neurons and of inhibitory neurons in the entire insular cortex, including: gustatory areas layer 1, 2/3, 4 5 6a and 6b, as well as dorsal, posterior and ventral agranular insular area. From their database, 73% of insular neurons were defined as excitatory (glutamatergic) and 27% were defined as inhibitory (GABAergic).
Statistical analysis
Histogram bars represent the mean ± SEM and were plotted in Prism 7 software (Graphpad). Two-tailed non-parametric t-test was used to compare percentages of glutamatergic and GABAergic neurons expressing 5-HT1A and 2A receptors in the insula. Statistical significance was defined at a p-value lower than 0.05. *p<0.05, **p<0.01, ***p<0.001.