Male Sprague-Dawley rats weighing 150–170 g were briefly anesthetized (2% isoflurane) and injected with BoNT-A (onabotulinumtoxinA; final dose = 5 units) using two different injection paradigms. In the first paradigm, four injections of BoNT-A (each containing 1.25 units diluted in 5 μl saline) were made along the lambdoid (two injection sites) and sagittal (two injection sites) sutures (Figure 1(a)). In the second paradigm, eight BoNT-A injections (each containing 0.625 units) were made: two along the sagittal suture, two in the temporalis muscle and four in the trapezius muscle (Figure 1(b)). All injections were given at the animal facility between 1 p.m. and 4 p.m. Seven days later, injected rats (by now weighing 250–300 g—housed in a specific pathogen-free facility equipped with solid bottom cages of 1800 cm2 of floor space and hardwood chip bedding; kept at 12 hours light/dark cycle, 22–26℃, and inspected twice daily for signs of stress or discomfort) were anesthetized with urethane (1.8 g/kg intraperitoneally (i.p.)) and prepared for single-unit recording of C- and Aδ-meningeal nociceptors. All electrophysiological experiments were carried out between 9 a.m. and 6 p.m. in a room especially fitted for electrophysiological recording in deeply anesthetized rats. To test for effects of extracranial injections of BoNT-A on responses of meningeal nociceptors to stimulation of their intracranial (dural) receptive fields, testing determined mechanical response threshold and responses to topical application of the TRPV1 agonist capsaicin and the TRPA1 agonist MO. For comparisons, similar experiments were carried out in 47 naïve and 25 sham rats (i.e. rats in which recordings were made seven days after they were anesthetized with 2% isoflurane and injected with vehicle at the same sites as the muscle-plus-suture group). As the results from the two control groups were very similar (e.g. scatterplots in Figure 2(b) and (c)) and not statistically different, they were combined for statistical comparisons.

Injection paradigm. (a) The suture paradigm consisted of four injections of BoNT-A, each containing 1.25 units, along the superior sagittal and transverse sinuses. (b) The suture-plus-muscle paradigm consisted of eight injections of BoNT-A, each containing 0.625 units. As depicted by the red dots, four injections were made in the clavicotrapezius muscle, two in the temporalis muscle, and two along the superior sagittal suture. BoNT-A: onabotulinumtoxinA.

Mechanical threshold for activation of C- and Aδ-meningeal nociceptors from their dural receptive fields are not affected by extracranial BoNT-A injections. (a) Mechanical response threshold of three different C-type meningeal nociceptors recorded in control rat (top), and in rats treated seven days earlier with BoNT-A injections into the sutures (middle) or the suture plus muscles (bottom). Boxed inset in each plot shows the shock artifact, the spike waveform, and the response latency. (b), (c) Mechanical thresholds for all C-units (b) and Aδ-units (c), shown as scattergraphs (top) and boxplots (bottom). Blue, control rats; red, suture-injected rats; green, suture-plus-muscle injected rats. In the scatterplots for the control rats, the filled and open circles represent values recorded in naïve and saline-injected rats, respectively. Boxplots illustrate median (thick horizontal line), interquartile range (25th–75th percentile; lower and upper box boundaries) and observations below and above the 25th and 75th percentile (whiskers) of the mechanical response threshold. Note that mechanical threshold values represent the smallest mechanical force capable of activating the neurons (i.e. innocuous force). BoNT-A: onabotulinumtoxinA.