Connectivity and molecular profiles of Foxp2- and Dbx1-lineage neurons in the accessory olfactory bulb and medial amygdala

Abstract

In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity.

Keywords: Dbx1; Foxp2; accessory olfactory system; medial amygdala; olfactory neuronal diversity; sex differences; vomeronasal organ.

Figure 1:. Dbx1-lineage chemosensory neurons in the VNO

(a) Triple immunofluorescence for EYFP (green), PDE4A (red) and DAPI (blue) in a unilateral coronal section of the VNO from a Dbx1^cre;RYFP mouse. b-d and f-h correspond to the image shown in a. (b-d) Recombined EYFP+ neurons (green) are shown within the total DAPI+ (blue) population across the sensory epithelium (SE). Solid white arrowheads in d highlight EYFP+ neurons. (e) Quantification of EYFP+ neuron number as a percentage of the total DAPI+ population. (f-h) EYFP+ neurons are observed in both the PDE4A+ apical layer (A) and the PDE4A− basal layer (B), demarcated by the white lines in g & h. Solid white arrowheads in h highlight EYFP+ PDE4A+ neurons (yellow) in the apical VNO. Empty white triangles highlight EYFP+ PDE4A− neurons (green) in the basal VNO. (i) Quantification of the proportions of PDE4A+ (apical) and PDE4A− (basal) neurons within the EYFP+ (Dbx1-lineage) population. (j-l) Double immunofluorescence for EYFP (green, j) and OMP (red, k), a marker of mature olfactory neurons. Solid white arrowheads in l highlight EYFP+ OMP+ (yellow) mature Dbx1-lineage VNO neurons and empty white triangles highlight EYFP+ OMP− (green) immature Dbx1-lineage neurons in the neurogenic niche. (m) Quantification of mature EYFP+ neuron number as a percentage of the total EYFP+ population. n = 18 mice (9 males and 9 females, 4–6 unilateral sections/mouse) in e, i, m. Abbreviations: BV – Blood Vessel, L – Lumen, NN – Neurogenic Niche, NSE – Non-Sensory Epithelium, SE – Sensory Epithelium. Scale bars equal 100 μm.

Figure 2:. Dbx1-lineage and Foxp2+ neurons in the AOB

(a, h, p) Low magnification images of unilateral, coronal sections from the anterior (a), middle (h) and posterior (p) olfactory bulb (OB) from a Dbx1^cre;RYFP mouse. Foxp2+ (red) and EYFP+ (green, Dbx1-lineage) neurons identified by immunofluorescence and co-labeled with DAPI (blue). White boxes in a, h, p highlight the accessory olfactory bulb (AOB), shown at higher magnification in b-e, i-l and q-t. (b-e) Higher magnification images of boxed region in a, show Foxp2+ neurons (red, c) and EYFP+ Dbx1-lineage neurons (green, d), in both the external plexiform (EplA) and mitral cell (MiA) layers of the anterior AOB. DAPI (blue) shown in e. Many Foxp2+ neurons (red) are present throughout the AOB, with fewer Dbx1-lineage neurons (green, EYFP+). Merged image (b) shows minimal overlap of the two populations. (f, g) Box-and-whisker plots quantifying number, per unilateral section of Foxp2+ (red circles, left Y-axis), EYFP+ (green squares, right Y-axis) and co-expressing (double positive, orange diamonds, right Y-axis) neurons in the EplA (f) and MiA (g). n = 19 unilateral sections from 5 adult mice (4 males, 1 female). (i-l) Higher magnification images of the middle AOB (boxed region in h). (i) Glomerular layer (GlA), EplA and MiA of the AOB are shown. (m-o) Quantification of neuron number in each layer of the middle AOB. n = 41 unilateral sections from 5 adult mice (4 males, 1 female). (q-t) Higher magnification images of the posterior AOB (boxed region in p). GlA and MiA are much smaller and the EplA is absent at the posterior level of the AOB. Quantification of each layer in the posterior AOB layer is shown in u, v. n = 10 unilateral sections from 5 adult mice (4 males, 1 female). Solid white arrowheads in c, d, j, k, r, highlight examples of quantified neurons. In a, h and p, crosses indicate section orientation (D – dorsal, M – medial, L – lateral). Abbreviations: EplA – External plexiform layer of the AOB, GlA – Glomerular layer of the AOB, MiA – Mitral cell layer of the AOB. Scale bars in a, h, p equal 200 μm, in b-t: 100 μm. *** p < 0.001, **** p < 0.0001 (Mann-Whitney U tests).

Figure 3:. AOB Foxp2-lineage neurons project to the MeA

(a) Schematic of anterograde viral transduction into the AOB of Foxp2^cre mice. Boxed regions within images (b, e) from The Mouse Brain in Stereotaxic Coordinates (Franklin & Paxinos, 2008) denote the AOB and MeA shown in (c, d) and (f, g), respectively. (c, d) Representative images from unilateral coronal sections of virally transduced AOBs show recombined EYFP+ neurons (green) from two different mice (c, d) in the AOB (middle level, Bregma +3.08 mm). (f, g) EYFP+ green fibers (white arrows) emanating from transduced AOB mitral cells (MiA) are observed along the input pathway to the posterior MeA (Bregma −1.46 mm) – ventral tissue boundary of the posterior MeA. Crosses in c, d indicate section orientation (D – dorsal, M – medial, L – lateral). Abbreviations: AOB – Accessory Olfactory Bulb, Hyp – hypothalamus, ic – internal capsule, MeA – Medial Amygdala, MiA – Mitral cell layer of the AOB, opt – optic tract. In all panels, scale bar equals 100 μm and DAPI-labelled nuclei are shown in blue. n = 6 mice (3 females and 3 males).

Figure 4:. Retrograde tracing of AOB inputs to MeA neurons

(a) Schematic illustrating the injection strategy of dual switch retrograde AAV transduction in the medial, cortical and posterior nuclei of the amygdala (MeA-CoA-PA) in Foxp2^cre mice. (b) Representative immunofluorescence image of a coronal section at the injection site shows strong viral expression in the posterior MeA (Bregma −1.94 mm). (c-e) Immunofluorescence images of the ipsilateral anterior AOB (coronal section, Bregma +3.56 mm) show Foxp2+ (recombined EGFP+, green, c) and Foxp2− (non-recombined tdTomato+, red, d) neurons. Merged image (e) reveals very minimal overlap of EGFP and tdTomato. (f-h) Immunofluorescence images of the ipsilateral middle AOB (coronal section, Bregma +3.20 mm), show similar recombination in the middle AOB. Crosses indicate section orientation (D – dorsal, M – medial, L – lateral). (i) Pie chart showing quantification of the percentage of Foxp2+ (green) and Foxp2- (red) ipsilateral AOB output neurons projecting to the MeA-CoA-PA. Abbreviations: CoA – Cortical Amygdala cp – cerebral peduncle, EplA – External plexiform layer of the AOB, MiA – Mitral cell layer of the AOB, MeA – Medial Amygdala and opt – optic tract. Scale bar equals 200 μm in b and 100 μm in all other panels. n = 7 mice (4 females and 3 males, 10–17 sections/mouse).

Figure 5:. Distribution of Dbx1-lineage and Foxp2+ neurons in the MeA in relation to CARTPT expression

(a) Image from The Mouse Brain in Stereotaxic Coordinates (Franklin & Paxinos, 2008) showing the posterior MeA (red box) in coronal view at the same antero-posterior level as the sections shown in panels (b-h). (b) Brightfield immunohistochemistry image showing CARTPT expression localized in the posterodorsal MeA. (c-e) Immunofluorescence image showing Dbx1-lineage neurons (EYFP+, green, c) and CARTPT+ cell bodies and fibers (red, d) in the MeA of a Dbx1^cre;RYFP mouse. CARTPT expression matches the distribution of Dbx1-lineage neurons which are observed embedded within a zone of CARTPT immunoreactivity (e). (f-h) Complementary distribution of Foxp2+ neurons (green, f), to the expression pattern of CARTPT (red, g), with Foxp2+ neurons predominantly observed outside the zone of CARTPT expression (h). Cross in b indicates section orientation (D – dorsal, M – medial, L – lateral), same followed in c-h. Abbreviations: cp – cerebral peduncle, Hyp – hypothalamus, MeA – Medial Amygdala and opt – optic tract. All scale bars equal 200 μm.

Figure 6:. Fluorescent in situ hybridization for neuropeptides and receptors in Foxp2+ and Dbx1-lineage neurons in the MeA

(a-a”) Expression and quantification of Tac2 mRNA (cyan)-expressing neurons within Foxp2 mRNA+ (yellow, a) and Dbx1-lineage (EYFP mRNA+, magenta, a’) neurons, as shown in coronal sections from the posterodorsal MeA. (a”) Box-and-whisker plots show percentage of co-expression of Tac2 mRNA within each population. (b-h”) Expression and quantification of the following other mRNAs: Ucn3 (b-b”), Npy (c-c”), Tacr1 (d-d”), Avp (e-e”), Ecel1 (f-f”), Htr2c (g-g”) and Trh (h-h”). Solid white arrowheads indicate neurons double positive for Foxp2 or EYFP mRNA, and the candidate gene mRNA; empty white triangles indicate neurons positive for Foxp2 or EYFP mRNA but not the candidate gene mRNA. Scale bars equal 25 μm for all images. * FDR-adjusted p value < 0.05 after multiple Mann-Whitney U-tests. All other comparisons not significant (FDR-adjusted p > 0.05). n = 4 male mice (2–4 sections/mouse, bilateral counts).

Figure 7:. Sex and lineage differences in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs)

(a) Representative epifluorescence and (b) DIC images show a representative recombined EYFP+ neuron (white arrows) in the posterior MeA targeted for ex vivo patch electrophysiology. (c) Representative current traces in whole-cell voltage clamp mode (−60 mV holding potential) from Dbx1-lineage (top) or Foxp2-lineage (bottom) neurons with traces from females and males shown separately. Orange circles indicate counted sEPSC events. (d) Quantification of frequency (events per second plotted as mean ± s.e.m.) of sEPSCs in Dbx1-lineage (left) or Foxp2-lineage (right) neurons in ACSF, from female (circles, white bar) or male (triangles, black bar) mice. * p < 0.05, ** p < 0.01, **** p < 0.0001, other pairwise comparisons not significant, α = 0.05 (2-way ANOVA & Sidak’s posthoc test). n = 10–13 neurons from 5–7 mice/group. Scale bars equal 50 μm in a, b.

Figure 8:. MeA Foxp2-lineage neurons receive excitatory and inhibitory inputs, with no sex differences

(a) Posterior MeA Foxp2-lineage neurons (EYFP+, yellow), identified by immunofluorescence in coronal sections from Foxp2^cre;RYFP mice and co-immunostained for the excitatory pre- and postsynaptic markers, VGLUT2 (magenta) and PSD95 (cyan), respectively. (b-e) Higher magnification images of boxed region in a, with EYFP (b, yellow), VGLUT2 (c, magenta) and PSD95 (d, cyan) channels shown separately. Merged image (e) shows Foxp2-lineage neurons expressing PSD95 and surrounded by VGLUT2 puncta (solid white arrowheads in b-e), indicating putative excitatory synapses. (f) Box-and-whisker plots quantifying Foxp2-lineage neurons positive for both VGLUT2 and PSD95; plotted as a percentage of total Foxp2-lineage (EYFP+) neurons in the posterior MeA, comparing females versus males. (g) Posterior MeA Foxp2-lineage neurons (EYFP+, yellow), co-immunostained for the inhibitory pre- and postsynaptic markers, VGAT (magenta) and Gephyrin (cyan), respectively. (h-k) Higher magnification images of boxed region in g, with EYFP (h, yellow), VGAT (i, magenta) and Gephyrin (j, cyan) channels shown separately. Merged image (k) shows Foxp2-lineage neurons expressing Gephyrin and surrounded by VGAT puncta (solid white arrowheads in h-k), indicating putative inhibitory synapses. (l) Box- and-whisker plots quantifying Foxp2-lineage neurons positive for both VGAT and Gephyrin; plotted as a percentage of total Foxp2-lineage (EYFP+) neurons in the posterior MeA, comparing females versus males. Crosses in a, g indicate section orientation (D – dorsal, M – medial, L – lateral). Abbreviations: cp – cerebral peduncle, itc – intercalated nucleus of the amygdala, MeA – Medial Amygdala and opt – optic tract. Scale bars in a, g equal 250 μm. In all other panels, scale bars equal 20 μm. n.s. indicates not significant (p > 0.05, Mann-Whitney U test). n = 5 mice per sex (3 sections/mouse, bilateral counts).