Serotonin type 1D receptors (5HTR) are differentially distributed in nerve fibres innervating craniofacial tissues

Abstract

We tested the hypothesis that the 5HT(1D)R, the primary antinociceptive target of triptans, is differentially distributed in tissues responsible for migraine pain. The density of 5HT(1D)R was quantified in tissues obtained from adult female rats with Western blot analysis. Receptor location was assessed with immunohistochemistry. The density of 5HT(1D)R was significantly greater in tissues known to produce migraine-like pain (i.e. circle of Willis and dura) than in structures in which triptans have no antinociceptive efficacy (i.e. temporalis muscle). 5HT(1D)R-like immunoreactivity was restricted to neuronal fibres, where it colocalized with calcitonin gene-related peptide and tyrosine hydroxylase immunoreactive fibres. These results are consistent with our hypothesis that the limited therapeutic profile of triptans could reflect its differential peripheral distribution and that the antinociceptive efficacy reflects inhibition of neuropeptide release from sensory afferents. An additional site of action at sympathetic efferents is also suggested.

Figure 1

Quantification of 5HT_1DR in migraine-associated tissues (MATs) and non-MATs. (A) Top panel: Western blot of total protein (150 μg/lane) from whole-cell lysates of dura (D), common (CC), internal (IC), external (EC) carotid arteries, circle of Willis (CW) and temporalis muscle (TM) probed for 5HT_1DR. All structures were obtained from a single rat. Middle panel: blot in A was reprobed with antibodies against PGP9.5 and β-actin. Bottom panel: Control blots were pre-incubated with the peptide used to generate the 5HT_1DR antibody. (B) Pooled data (n = 8) indicate the presence of statistically significant differences between tissues with respect to the amount of 5HT_1DR. (C) Significant differences between CC, CW and EC were also observed when data were normalized to β-actin. (D) There was a statistically significantly difference in the relative amount of PGP9.5 between target tissues. (E) Pooled data indicate the presence of statistically significant differences between tissues with respect to relative levels of 5HT_1DR normalized to PGP9.5. *Statistically significant differences from CC. +Statistically significant differences from CW. #Statistically significant differences from D where P < 0.05.

Figure 2

(A) Diaminobenzidene immunohistochemistry of 5HT_1DR in cerebrovascular tissues. 5HT_1DR LI is found in nerve fibres innervating dura mater and run parallel (arrowheads) and perpendicular (arrow) to the middle meningeal artery (MMA). (B) There is staining of both larger nerve trunks and smaller fibres (arrow) in the dura. 5HT_1DR LI is found in nerve fibres innervating the external carotid arteries (C) and branches of the circle of Willis (D) also. There appears to be staining of fine terminal nerve fibres (arrowheads).

Figure 3

5HT_1DR LI was detected in both peptidergic and non-peptidergic fibres in the dura mater. (A,B) Examples of fibres in which both 5HT_1DR LI and calcitonin gene-related peptide (CGRP) LI were detected. There are also CGRP LI fibres that do not overlap with 5HT_1DR LI (arrowhead). (C) Examples of fibres in the dura mater in which only 5HT_1DR LI was detected.

Figure 4

Calcitonin gene-related peptide (CGRP) LI was not detected in rat superior cervical ganglion (SCG). Sections of trigeminal ganglia (TG) (A) and SCG (B) from the same rat were probed in parallel for the presence of CGRP LI. Whereas CGRP-LI was present in a subpopulation of small- and medium-diameter neurons in the TG (arrowheads), CGRP-LI was undetectable in SCG neurons. (C) Omission of primary antibody resulted in a pattern of staining similar to that observed in B.

Figure 5

5HT_1DR LI was detected in fibres with tyrosine hydroxylase (TH) LI in the dura. (A) TH LI reveals dense sympathetic postganglionic innervation of the dura mater. TH LI appeared in close proximity to meningeal arteries (arrows) and in the parenchyma of the dura (arrows). (B) Example of fibres in which both 5HT_1DR LI and TH LI were detected in the dura.

Figure 6

5HT_1DR LI (arrows) was present in subpopulations of neurons in trigeminal ganglia (TG) (A) and superior cervical ganglion (SCG) (B). (C) Top panel: Western blot analysis of 5HT_1DR LI in TG and SCG protein: blot is from tissue obtained from a single rat. The high-molecular-weight band present in the dura (arrow) was present in neither TG nor SCG. Bottom panel: Pooled data (n = 5) indicate that relative levels of 5HT_1DR are greater in TG than in SCG. *P ≤ 0.05.

Figure 7

Tyrosine hydroxylase (TH) LI was present in trigeminal ganglia (TG), but not in dural afferents. (A) TH LI was present in a small subpopulation of TG neurons (right panel). None of the TG neurons with TH LI was retrogradely labelled from the dura (middle panel). (B) TH LI was undetectable in TG neurons retrogradely labelled from the dura.