Whole Brain Mapping of Neurons Innervating Extraorbital Lacrimal Glands in Mice and Rats of Both Genders

Abstract

The extraorbital lacrimal glands (ELGs) secret tears to maintain a homeostatic environment for ocular surfaces, and pheromones to mediate social interactions. Although its distinct gender-related differences in mice and rats have been identified, its comprehensive histology together with whole-brain neuronal network remain largely unknown. The primary objective of the present study was to investigate whether sex-specific differences take place in histological and physiological perspectives. Morphological and histological data were obtained via magnetic resonance imaging (MRI), hematoxylin-eosin (HE) staining in mice and rats of both genders. The innervating network was visualized by a pseudorabies virus (PRV) mediated retrograde trans-multi-synaptic tracing system for adult C57BL6/J mice of both genders. In terms of ELGs' anatomy, mice and rats across genders both have 7 main lobes, with one exception observed in female rats which have only 5 lobes. Both female rats and mice generally have relatively smaller shape size, absolute weight, and cell size than males. Our viral tracing revealed a similar trend of innervating patterns antero-posteriorly, but significant gender differences were also observed in the hypothalamus (HY), olfactory areas (OLF), and striatum (STR). Brain regions including piriform area (Pir), post-piriform transition area (TR), central amygdalar nucleus (CEA), medial amygdalar nucleus (MEA), lateral hypothalamic area (LHA), parasubthalamic nucleus (PSTN), pontin reticular nucleus (caudal part) (PRNc), and parabrachial nucleus, (PB) were commonly labeled. In addition, chemical isotope labeling-assisted liquid chromatography-mass spectrometry (CIL-LC-MS) and nuclear magnetic resonance spectroscopy (NMR spectroscopy) were performed to reveal the fatty acids and metabolism of the ELGs, reflecting the relationship between pheromone secretion and brain network. Overall, our results revealed basic properties and the input neural networks for ELGs in both genders of mice, providing a structural basis to analyze the diverse functions of ELGs.

Keywords: CIL-LC-MS; anatomy; extraorbital lacrimal gland; neural circuits; neural tracing.

Figure 1

Experimental procedures for the cell-type-specific retrograde trans-multi-synaptic tracing of whole brain. (A) Simplified drawing of ELG's geographic location on mice, and the general principle of PRV tracing from peripheral ELGs to the central nervous system. (B) Experimental design showing the injection site for C57BL6/J (example injection sites combined lobe No. 2 and lobe No. 4, with PRV531-GFP and PRV724-dsRed injected respectively).

Figure 2

Distribution patterns of whole-brain inputs to the ELGs. (A) Whole-brain distribution of all labeled neurons along the AP axis. Colored lines, input distribution for the individual mouse; colored line with a shaded area under it, average labeled distribution (left, male; right, female). M1–3, male mouse 1–3; F1 – 3, female mouse 1–3. (B) Whole-brain distribution of the labeled neurons within nine major brain regions from bilateral hemispheres, quantified with all slices. (C) The proportion of GFP+ and dsRed+ in nine major brain regions from bilateral hemispheres, quantified with all slices (left, GFP+; right, dsRed+). (D) Whole-brain distribution of all labeled neurons within 45 subregions in bilateral hemispheres (only significant differences have been labeled on the corresponding top of the bars, otherwise n.s.). GFP+, dsRed+, n.s., no significant difference; *p < 0.05 and **p < 0.01.

Figure 3

Distribution patterns of labeled neurons in HY. (A,B) Representative images showing labeled neurons from the mouse ipsilateral HY to the ELGs. Scale bar: 1 mm (left lower magnification images) and 200 μm (for right higher magnification images); (A, male; B, female). (C) Numbers of the labeled neurons (GFP+ and dsRed+) in different subregions of HY (left, male; right, female). (D) Distribution of the labeled neurons within HY (significant difference labeled on the bar top, otherwise n.s.). (E) Distribution patterns of the labeled neurons along the AP axis in different subregions of HY. (F) Distribution of GFP+ and dsRed+ within subregions of HY (left, male; right, female). GFP+, PRV-GFP positive neurons; dsRed+, PRV-dsRed positive neurons., n.s., no significant difference; *p < 0.05.

Figure 4

Distribution patterns of labeled neurons in OLF. (A,B) Representative images showing labeled neurons from the mouse ipsilateral OLF to the ELGs. Scale bar: 1 mm (left lower magnification images) and 200 μm (right higher magnification image); (A, male; B, female). (C) Number of labeled neurons (GFP+ and dsRed+) in different subregions of OLF (left, male; right, female). (D) Distribution of the labeled neurons within OLF (significant difference labeled on the bar top, otherwise n.s.). (E) Distribution patterns of the labeled neurons along the AP axis in different subregions of OLF. (F) Distribution of GFP+ and dsRed+ within subregions of OLF (left, male; right, female). GFP+, PRV-GFP positive neurons; dsRed+, PRV-dsRed positive neurons. n.s., no significant difference.

Figure 5

Distribution patterns of labeled neurons in the STR. (A,B) Representative images showing labeled neurons from mouse the ipsilateral STR to the ELGs. Scale bar: 1 mm (left lower magnification images), 200 μm (right higher magnification image for male), and 500 μm (right higher magnification image for female); (A, male; B, female). (C) Numbers of the labeled neurons (GFP+ and dsRed+) in different subregions of STR (left, male; right, female). (D) Distribution of the labeled neurons within STR (significant difference labeled on the bar top, otherwise n.s.). (E) Distribution patterns of the labeled neurons along the AP axis in different subregions of STR. (F) Distribution of GFP+ and dsRed+ within subregions of STR (left, male; right, female). GFP+, PRV-GFP positive neurons; dsRed+, PRV-dsRed positive neurons. n.s., no significant difference.

Figure 6

Distribution patterns of labeled neurons in the Pons. (A,B) Representative images showing labeled neurons from the mouse ipsilateral pons to the ELGs. Scale bar: 1 mm (left lower magnification images) and 200 μm (right higher magnification images); (A, male; B, female). (C) Number of labeled neurons (GFP+ and dsRed+) in different subregions of Pons (left, male; right, female). (D) Distribution of the labeled neurons within Pons (significant difference labeled on the bar top, otherwise n.s.). (E) Distribution patterns of the labeled neurons along the AP axis in different subregions of Pons. (F) Distribution of GFP+ and dsRed+ within subregions of Pons (left, male; right, female). GFP+, PRV-GFP positive neurons; dsRed+, PRV-dsRed positive neurons. n.s., no significant difference.

Figure 7

Anatomy of ELGs. Entire appearance on ELGs on the face of (A) Male mice (scale bar = 5 mm), (B) Female mice (scale bar = 5 mm), (E) Male rat (scale bar = 10 mm), (F) Female rat (scale bar = 10 mm). ELGs locations are labeled by an arrow and blank circle in mice and rats, respectively. Fine dissection of ELG on the face of (C) Male mice (scale bar = 2 mm), (D) Female mice (scale bar = 2 mm), (G) Male rat (scale bar = 10 mm), (H) Female rat (scale bar = 10 mm). (Figure is labeled on each lobe of ELG). (I) T2 anatomical MRI of rat's ELG (horizontal section). The red arrows point to the blood vessels, and the green arrows point to the ducts. (J) Weight of ELGs isolated from animals. The values are presented as the means ± SEM, **p < 0.01, n.s., no significant difference.

Figure 8

Hematoxylin-eosin stain of ELG. (A,C) Representative images of a whole ELG (50 × microscope, scale bar = 400 μm) from mice (A, male; C, female). (B,D) Local amplification (400X microscope, scale bar = 40 μm) of panels on the (A,C), respectively. (E,G) Representative images of a whole ELG (20 × microscope, scale bar = 1000 μm) from rats (A, male; C, female). (F,H) Local amplification (400 × microscope, scale bar = 40 μm) of panels on the (E,G), respectively. Average cell size in Hematoxylin-eosin stain images of ELG sections from mice (I) and rat (J). The values are presented as the means ± SEM, **p < 0.01.

Figure 9

The average NMR spectra for total metabolites (¹²C+¹³C) from the POCE (¹H observed/¹³C edited) for the rat's ELGs. The blue line represents the average concentration of metabolites, with the red shaded part being the SEM.