Differential ascending projections of temporomandibular joint-responsive brainstem neurons to periaqueductal gray and posterior thalamus of male and female rats

Abstract

Several craniofacial pain conditions, including temporomandibular joint disorders (TMJDs), are more prevalent in women than men. The basis for sex differences in deep craniofacial pain is not known. The present study compared the magnitude of ascending projections from temporomandibular joint (TMJ)-responsive neurons in trigeminal brainstem with the ventrolateral periaqueductal gray (vlPAG) or posterior nucleus of the thalamus (Po) in males and female rats. Fluorogold (FG) was injected into vlPAG or Po, and TMJ-responsive neurons were identified by Fos-like immunoreactivity (Fos-LI) after mustard oil injection. TMJ-evoked Fos-LI was similar in males and females; however, significant differences in cell counts were seen for FG single-labeled and Fos/FG double-labeled neurons in trigeminal brainstem. After vlPAG injections, the number of FG-labeled neurons in trigeminal subnucleus interpolaris (Vi), ventral interpolaris/caudalis transition (vl-Vi/Vc), and dorsal paratrigeminal region (dPa5) was greater in females than males. The percentage of Fos/FG double-labeled neurons in vl-Vi/Vc and dPa5 after vlPAG injection also was greater in females than males. In contrast, after Po injections, males displayed a greater number of FG-labeled neurons in superficial laminae (Lam I/II) of trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C(1-2)) and deeper laminae (Lam III/V) at C(1-2) than females. The percentage of Fos/FG double-labeled neurons in Lam I/II of Vc after Po injection also was greater in males than females. These data revealed significant sex differences in ascending projections from TMJ-responsive neurons in trigeminal brainstem. Such differences may influence the ability of males and females to recruit autonomic reflexes and endogenous pain control circuits relevant for TMJ nociception.

Figure 1

Average number of Fos-positive neurons produced in the caudal trigeminal brainstem nuclear complex (TBNC) and upper cervical dorsal horn after unilateral injection of the small fiber excitant, mustard oil (20% solution, 25 µl), into the temporomandibular joint (TMJ) region of male and female rats. Results expressed as average total number of cells per mm (± SEM) as a function of distance from the obex. *P < 0.05, **P < 0.01 ipsilateral versus contralateral. N = 8–10 rats per group.

Figure 2

Fluorogold (FG) injection sites in the periaqueductal gray (PAG). A. Drawings display the spread of FG (shaded regions) for injections in males (PAGM1–6) and female rats (PAGF1–4). Each vertical column represents the results for one experimental animal. Numbers to the left of the first column indicate the rostrocaudal distance from bregma according to the rat atlas of Paxinos and Watson (1997). B. Micrograph example of an injection site in the PAG. Abbreviations: AQ, aqueduct; DR, dorsal raphe; DLPAG, dorsolateral PAG; LPAG, lateral PAG; SCP, superior cerebellar peduncle; VLPAG, ventrolateral PAG. Calibration = 200 µm.

Figure 3

Average total number of FG-positive neurons per mm within the TBNC and upper cervical dorsal horn of male and female rats. A. Total number of FG neurons after vlPAG injections of FG. B. Total number of FG neurons after PO injections of FG. Abbreviations: contra, contralateral to injection; ipsi, ipsilateral to injection. *P < 0.05, **P < 0.01 ipsilateral versus contralateral; a = P < 0.05, b = P < 0.01 male versus female. N = 8–10 rats per group.

Figure 4

Examples of Fos, FG and Fos/FG double-labeled neurons from a male rat after vlPAG injection of FG and TMJ stimulation with mustard oil. A. Field view of the dorsal paratrigeminal region (dPa5). A1–3: enlarged images of numbered regions in A. B. Field view of the commissural nucleus tractus solitarii (NTS). B1–3: enlarged images of numbered regions in B. C. Field view of the caudal ventrolateral medulla (CVLM). C1–3: enlarged images of numbered regions in C. Symbols: + = Fos, # = FG, and *= Fos/FG double-labeled neuron. All examples are from the side ipsilateral to the TMJ stimulus. Calibration: low magnification (A, B, C) = 100 µm; high magnification (A1, A2, and A3) = 50 µm.

Figure 5

Examples of Fos, FG and Fos/FG double-labeled neurons from a male rat after vlPAG injection of FG and TMJ stimulation with mustard oil. A. Field view of the ventral interpolaris/caudalis transition (vl-Vi/Vc) transition region. A1–3: enlarged images of numbered regions in A. B. Field view of superficial laminae of Vc. B1–3: enlarged images of numbered regions in B. C. Field view of the upper cervical dorsal horn. C1–3: enlarged images of numbered regions in C. Symbols: + = Fos, # = FG, and *= Fos/FG double-labeled neuron. All examples are from the side ipsilateral to the TMJ stimulus. Calibration: low magnification (A, B, C) = 100 µm; high magnification (A1, A2, and A3) = 50 µm.

Figure 6

Camera lucida drawing of an example of Fos, FG and Fos/FG double-labeled neurons in the caudal brainstem and upper cervical spinal cord after FG injection into the vlPAG. A. Example from a male rat. A1, Fos (●) and FG (Δ) labeling; A2, Fos/FG double-labeled neurons (*). B. Example from a female rat. B1, Fos (●) and FG (Δ) labeling; B2, Fos/FG double-labeled neurons (*). Numbers to the side of the right most column refer to the distance in mm relative to obex. All drawings are from the side ipsilateral to the TMJ stimulus.

Figure 7

Double-labeled cells after vlPAG injection and TMJ stimulation in males (M) and females (F). A. Percentage of FG-labeled neurons also labeled for Fos in vl-Vi/Vc and dPa5. B. Percentage of FG-labeled neurons also labeled for Fos in superficial laminae of Vc and C_1–2. C. Percentage of Fos-positive neurons also labeled for FG in vl-Vi/Vc and dPa5. D. Percentage of Fos-positive neurons also labeled for FG in superficial laminae of Vc and C_1–2. *P < 0.05, **P < 0.01 ipsilateral versus contralateral; a = P < 0.05 male versus female. Ipsi and contra refer to ipsilateral and contralateral to the TMJ stimulus.

Figure 8

Fluorogold injection sites in posterior nucleus of the thalamus (Po). A. Drawings display the spread of FG (shaded regions) for injections in males (PoM1–4) and E2-treated female rats (PoF1–4). Each vertical column represents the results for one experimental animal. Numbers to the left of the first column indicate the rostrocaudal distance from bregma according to the rat atlas of Paxinos and Watson (1997). B. Micrograph example of an injection site in the Po. Abbreviations: CL, centrolateral thalamic n.; LD, lateral dorsal thalamic n.; Po, posterior thalamic n.; VAL, ventral anterolateral n.; VPM, ventral posterior medial thalamic n.; VPL, ventral posterior lateral thalamic n. Calibration = 200 µm.

Figure 9

Camera lucida drawing of an example of Fos, FG and Fos/FG double-labeled neurons in the caudal brainstem and upper cervical spinal cord after FG injection into the PO. A. Example from a male rat. A1, Fos (●) and FG (Δ) labeling; A2, Fos/FG double-labeled neurons (*). B. Example from a female rat. B1, Fos (●) and FG (Δ) labeling; B2, Fos/FG double-labeled neurons (*). Numbers to the side of the right most column refer to the distance in mm relative to obex. All drawings are from the side ipsilateral to the TMJ stimulus.

Figure 10

Double-labeled cells after Po injection and TMJ stimulation in males (M) and females (F). A. Percentage of FG-labeled neurons also labeled for Fos in vl-Vi/Vc and dPa5. B. Percentage of FG-labeled neurons also labeled for Fos in superficial laminae of Vc and C_1–2. C. Percentage of Fos-positive neurons also labeled for FG in vl-Vi/Vc and dPa5. D. Percentage of Fos-positive neurons also labeled for FG in superficial laminae of Vc and C_1–2. *P < 0.05, **P < 0.01 ipsilateral versus contralateral. Ipsi and contra refer to ipsilateral and contralateral to the TMJ stimulus.