Quantitative Analysis of Mouse Dural Afferent Neurons Expressing TRPM8, VGLUT3, and NF200

Abstract

Objective: To quantify the abundance of dural afferent neurons expressing transient receptor potential channel melastatin 8 (TRPM8), vesicular glutamate transporter 3 (VGLUT3), and neurofilament 200 (NF200) in adult mice.

Background: With the increasing use of mice as a model system to study headache mechanisms, it is important to understand the composition of dural afferent neurons in mice. In a previous study, we have measured the abundance of mouse dural afferent neurons that express neuropeptide calcitonin gene-related peptide as well as two TRP channels TRPV1 and TRPA1, respectively. Here, we conducted quantitative analysis of three other dural afferent subpopulations in adult mice.

Methods: We used the fluorescent tracer Fluoro-Gold to retrogradely label dural afferent neurons in adult mice expressing enhanced green fluorescent protein in discrete subpopulations of trigeminal ganglion (TG) neurons. Mechanoreceptors with myelinated fibers were identified by NF200 immunoreactivity. We also conducted Ca²⁺ -imaging experiments to test the overlap between TRPM8 and VGLUT3 expression in mouse primary afferent neurons (PANs).

Results: The abundance of TRPM8-expressing neurons in dural afferent neurons was significantly lower than that in total TG neurons. The percentages of dural afferent neurons expressing VGLUT3 and NF200 were comparable to those of total TG neurons, respectively. TRPM8 agonist menthol evoked Ca²⁺ influx in less than 7% VGLUT3-expressing PANs in adult mice.

Conclusions: TG neurons expressing TRPM8, VGLUT3, and NF200 all innervate adult mouse dura. TRPM8 and VGLUT3 are expressed in distinct subpopulations of PANs in adult mice. These results provide an anatomical basis to investigate headache mechanisms in mouse models.

Keywords: NF200; TRPM8; VGlut3; dural afferent neurons; headache; migraine.

Figure 1. FG labels more dura afferent neurons than DiI

Representative images of two TG sections showing DiI- and FG-labeled dural afferent neurons. The arrows indicate double-labeled neurons. The thick and thin arrowheads indicate neurons with only FG and DiI labeling, respectively.

Figure 2. The distribution of neurons expressing TRPM8 channels in TG and dural afferent neurons

(A) Representative images of TG sections from a wild-type mouse with dural FG application and TRPM8^EGFPf/+ mice with and without dural FG labeling, respectively. The arrows indicate a FG⁺ dural afferent neuron that is also EGFP⁺. (B) The percentages of EGFP⁺ neurons in total TG population and in FG⁺ dural afferent neurons in TRPM8^EGFPf/+ mice receiving dural FG application (n = 2 male and 2 female mice; on average, 2380 TG neurons and 100 FG⁺ neurons were counted from each mouse; *p < 0.05, two-tailed t-test). (C) Cumulative distributions of the cross-sectional areas of EGFP⁺, FG⁺ and EGFP⁺FG⁺ neurons (n = 577, 395 and 27 neurons pooled from 4 mice, respectively; same neurons as in B). The sizes of EGFP⁺ and EGFP⁺FG⁺ neurons are significantly smaller than that of the FG⁺ dural afferent neurons (p < 0.001 and p < 0.01, respectively, Kruskal-Wallis ANOVA with Dunn's post hoc test). The sizes of EGFP⁺ and EGFP⁺FG⁺ neurons are comparable (p = 0.8).

Figure 3. Overlap between EGFP signal and EGFP-ir in TG and dural afferent neurons from TRPM8^EGFPf/+ mice

Representative images of TG sections from wild-type and TRPM8^EGFPf/+ mice with dural FG application, respectively. The EGFP signal and EGFP-ir overlap well in the TRPM8^EGFPf/+ section. Neither is present in the wild-type section, validating the specificity of the EGFP antibody. The arrows indicate a FG⁺ dural afferent neuron that exhibits both EGFP signal and EGFP-ir.

Figure 4. VGLUT3-expressing TG neurons detected by EGFP signal and EGFP-ir

Representative images of TG sections from a wild-type mouse with dural FG application and a VGLUT3^EGFP mouse without dural FG labeling, respectively. The EGFP signal and EGFP-ir overlap well in the VGLUT3^EGFP section. Neither is present in the wild-type TG section. EGFP signal does not bleed into the FG channel; and FG fluorescence does not interfere with the EGFP or EGFP-ir signals.

Figure 5. The distribution of VGLUT3-expressing neurons in TG and dural afferent neurons

(A) Representative images of two TG sections from VGLUT3^EGFP mice with dural application of FG. The arrowheads indicate FG⁺ dural afferent neurons that are also EGFP⁺. (B) The percentages of TG neurons and FG⁺ dural afferent neurons that are EGFP⁺ (n = 2 male and 2 femalemice; on average, 1360 TG neurons and 127 FG⁺ neurons were counted from each mouse; p = 0.44, two-tailed t-test). (C) Cumulative distributions of the cross-sectional areas of EGFP⁺, FG⁺ and EGFP⁺FG⁺ neurons (n = 479, 511 and 54 neurons pooled from 4 mice, respectively; same neurons as in B). The sizes of EGFP⁺ and EGFP⁺FG⁺ neurons are significantly smaller than that of the FG⁺ dural afferent neurons (p < 0.001, Kruskal-Wallis ANOVA with Dunn's post hoc test).

Figure 6. The response to TRPM8 agonist menthol in TG neurons from VGLUT3^EGFP mice

(A) Representative images of dissociated TG neurons from VGLUT3^EGFP mice. Upper panel: EGFP signal. Middle and lower panels: representative Fura-2 images before and after application of 100 μM menthol, respectively. The blue to green color transition reflects increase in intracellular Ca²⁺. The arrows and arrowheads indicate EGFP⁺ and EGFP-negative (EGFP^–) neurons, respectively. (B) The percentages of menthol-responsive neurons in 55 EGFP⁺ TG neurons and 58 EGFP⁺ DRG neurons cultured from 10 VGLUT3^EGFP mice (5 male and 5 female). An additional 10 EGFP⁺ neurons were cultured from 3 (2 male, 1 female) TRPM8^EGFPf/+ mice (***p < 0.001, Fisher's exact test).

Figure 7. The distribution of NF200⁺ neurons in total TG and dural afferent populations

(A) Representative images of a TG section containing FG⁺ dural afferent neurons and NF200-labeled neurons. Arrowheads indicate neurons that are both FG⁺ and NF200⁺. (B) The percentages of TG neurons and FG⁺ dural afferent neurons that are NF200⁺ (n = 1 male and 2 female mice; on average, 1700 TG neurons and 120 FG⁺ neurons were counted from each mouse; p = 0.11, two-tailed t-test). (C) Cumulative distributions of the cross-sectional areas of NF200⁺, FG⁺ and NF200⁺FG⁺ neurons (n = 890, 240 and 84 neurons pooled from 3 mice, respectively; same neurons as in B). The sizes distributions of NF200⁺, FG⁺ and NF200⁺FG⁺ neurons are similar (p = 0.11, Kruskal-Wallis ANOVA).