Sex Differences in Estradiol Secretion by Trigeminal Brainstem Neurons

Abstract

Estrogen status is a significant risk factor in the development of temporomandibular joint disorders (TMD). Classically, estrogen status is thought to derive mainly from ovarian sources; however, it is well known that estradiol (E2) also is synthesized by neurons in the brain. This study tested the hypothesis that E2 is produced by neurons in trigeminal subnucleus caudalis (Vc), the principal site of termination for sensory afferents that supply the temporomandibular joint (TMJ), to modify evoked responses in a model of TMJ nociception in male and female rats. Intra-TMJ injection of the small fiber excitant, allyl isothiocyanate (AIC), increased the levels of E2 collected from microdialysis probes sites at Vc of ovariectomized (OvX) female rats, ipsilateral to the stimulus, whereas males displayed no change. Dialysate levels of E2 collected from probe sites in the contralateral Vc or cerebellum in OvX rats were not affected by TMJ stimulation. Reverse dialysis of anastrozole, an aromatase (ARO) inhibitor, via the probe reduced perfusate levels of E2 in Vc. Systemic administration of letrozole, a non-steroid ARO inhibitor, for 4 days prevented TMJ-evoked increases in masseter muscle electromyography (MMemg) activity. ARO-positive neurons were distributed mainly in superficial laminae (I-III) at Vc and cell counts revealed no significant difference between OvX and male rats. Intra-TMJ injection of AIC revealed similar numbers of ARO/Fos dual-labeled neurons in OvX and male rats. By contrast, the percentage of ARO neurons co-labeled for glutamic acid decarboxylase (GAD), the biosynthetic enzyme for GABA, was greater in OvX (35%) than male rats (14%). Few ARO-positive neurons were co-labeled for estrogen receptor alpha. These data indicate that E2 is secreted continuously by Vc neurons and that acute stimulation of TMJ nociceptors evokes further secretion in a sex-dependent manner. Reduced TMJ-evoked MMemg activity after ARO inhibition suggests that locally produced E2 by Vc neurons acts via paracrine mechanisms to modify TMJ nociception in female rats.

Keywords: animal models; aromatase; nociception; temporomandibular disorders; trigeminal nucleus caudalis.

FIGURE 1

Estradiol (E2) values measured in microdialysis samples were reduced in OvX female rats. (A) Example of probe placement for collecting microdialysis samples in caudal Vc (coronal section; I-III and IV-V = laminae). (B) Effect of gonadectomy in males and females on E2 values measured in microdialysis samples; ^∗p < 0.05 versus intact female.

FIGURE 2

Reverse dialysis of the non-steroidal ARO inhibitor, anastrozole (100 μm, 50 μl) through probes positioned in the Vc and simultaneously topical application on the dorsal Vc surface caused a transient decrease in E2 levels in male and OvX female rats; ^∗∗p < 0.01 and ^∗p < 0.05 versus pre drug, a = p < 0.05 and b = p < 0.01 versus male.

FIGURE 3

Intra-TMJ injection of 20% AIC (also known as mustard oil, MO, 20 μl) evoked a prompt increase E2 secretion in OvX but not male rats; ^∗∗p < 0.01 versus pre, a = p < 0.05 and b = p < 0.01 versus Male + MO (M-MO) and OvX + Mineral oil (OvX-Min).

FIGURE 4

Systemic administration of letrozole (Ltz, 10 mg/kg, sc) for 4 days reduced ATP-evoked MMemg responses in OvX rats. (A) Example of MMemg activity evoked by intra-TMJ injection of ATP (1 mM, 20 μl) in OvX (upper panel) and OvX + Ltz rats (lower panel). (B) Summary of integrated MMemg activity evoked by ATP in OvX and OvX + Ltz rats. Arrow indicates the time of intra-TMJ ATP stimulation; ^∗∗p < 0.01 versus vehicle, b = p < 0.01 versus OvX + letrozole (OvX + Ltz).

FIGURE 5

Immunofluorescent examples of ARO and ERα staining in laminae I-III of caudal Vc in an OvX rat. Note that few neurons were co-labeled. Scale = 40 μm.

FIGURE 6

Immunofluorescent staining in laminae I-III of caudal Vc for ARO and NeuN, a neuronal marker, in an OvX rat. Since NeuN is specific for neuronal nuclei but is an incomplete marker for all neurons, the chromatin stain DAPI was added to the mounting medium in some cases. Symbols: ^∗NeuN only; ^#ARO/NeuN co-staining. Scale = 40 μm.

FIGURE 7

Co-labeling of ARO/Fos neurons in caudal Vc. (A) Example of co-labeled ARO/Fos neurons after TMJ stimulation in an OvX rat. Fos-positive neurons appeared as dark-stained nuclei (^∗), while dual labeled ARO/Fos neurons appeared as dark nuclei surrounded by a lighter cytoplasmic compartment (#). (B) Summary of the percentage of ARO-positive neurons co-labeled for Fos or GAD, the biosynthetic enzyme for GABA, in male and OvX rats. b = p < 0.01 versus male. Scale bar = 30 μm.