BOLD fMRI of C-Fiber Mediated Nociceptive Processing in Mouse Brain in Response to Thermal Stimulation of the Forepaws

Abstract

Functional magnetic resonance imaging (fMRI) in rodents enables non-invasive studies of brain function in response to peripheral input or at rest. In this study we describe a thermal stimulation paradigm using infrared laser diodes to apply noxious heat to the forepaw of mice in order to study nociceptive processing. Stimulation at 45 and 46°C led to robust BOLD signal changes in various brain structures including the somatosensory cortices and the thalamus. The BOLD signal amplitude scaled with the temperature applied but not with the area irradiated by the laser beam. To demonstrate the specificity of the paradigm for assessing nociceptive signaling we administered the quaternary lidocaine derivative QX-314 to the forepaws, which due to its positive charge cannot readily cross biological membranes. However, upon activation of TRPV1 channels following the administration of capsaicin the BOLD signal was largely abolished, indicative of a selective block of the C-fiber nociceptors due to QX-314 having entered the cells via the now open TRPV1 channels. This demonstrates that the cerebral BOLD response to thermal noxious paw stimulation is specifically mediated by C-fibers.

Fig 1. Thermal stimulation setup.

(a) Scheme of setup of the laser stimulation with feedback loop for temperature control. (b) Close-up view of the cube for paw fixation made from black Perspex. Two hole diameters were used, enabling irradiation at spot sizes of 1 and 2mm, respectively. The thermocouple used for temperature monitoring was positioned immediately adjacent to the irradiated area of the paw. (c) Temperature profiles during stimulation, recorded at the mouse forepaw for three different animals. Target temperature of 45 ± 0.5°C was reached for at least 30 s. d) Experimental protocol for fMRI studies.

Fig 2. BOLD signal changes induced by thermal stimulation.

(a) Anatomical MR image (top panel) with overlay of respective section from mouse brain atlas (IAL +3.7 mm). Regions relevant for pain processing are indicated (SI: primary somatosensory cortex, forepaw region; MI: primary motor cortex; CGC: cingulated cortex) and representative EPI image (second panel). Combined group activation maps after left and right thermal forepaw stimulation of 45 (n_scans = 19, third panel) and 46°C (n_scans = 12, bottom panel) show activated regions derived from GLM analysis (p = 0.0001, cluster size 15 voxels) for all animals overlaid on the mouse brain atlas. The scale bar indicates the percentage of animals showing significant BOLD activation at the given threshold. (b) Mean temporal BOLD profile of the somatosensory cortex (S1; red with error bars) and thalamus (dashed gray; without error bars) contralateral to the stimulated paw (n_scans = 12, orange). Stimulation parameters: 46°C, 1 mm. Grey shaded blocks indicate stimulation periods. Arrow indicates amplitude measure for quantitative analysis (for somatosensory cortex S1). (c) Maximum BOLD signal amplitude of first stimulation period for S1 and thalamus for T = 45°C/2 mm, T = 46°C/2 mm, and T = 46°C/1 mm. (d) Decay rate of BOLD signal as a function of heat dissipated. There is a linear correlation between the decay rate and the amount of ‘noxious`heat (T_thresh = 42°C, R² = 0.988, open symbols) and (T_thresh = 43°C, R² = 0.974, filled symbols) deposited in the tissue. All values are given as mean ± SEM.

Fig 3. Pretreatment with capsaicin and QX-314 abolishes BOLD response.

Activation maps and BOLD signal profiles after left and right thermal forepaw stimulation at 45°C. (a) Control condition, thermal stimulation of naïve animals. The white outline indicates the area used for extracting BOLD signal profiles. (b) After pretreatment with QX-314 (n_scans = 6); (c) after pretreatment with capsaicin (Cap, n_scans = 6,); and (d) after pretreatment with QX-314 and capsaicin (n_scans = 14). Images show activated regions derived from GLM analysis (p = 0.0001, cluster size 15 voxels) for all animals overlaid on the mouse brain atlas. The scale bar indicates the percentage of animals showing significant BOLD activation at the given threshold. Profiles show BOLD response for individual treatments (red). For reference, the profiles of control (naïve) animals are indicated in dark grey. (b-d). All values are given as mean ± SEM.

Fig 4. Pretreatment with capsaicin and QX-314 abolishes BOLD response.

(a) Maximum BOLD signal changes of the S1 contralateral to the stimulated paw and the thalamus. Pretreatment with QX-314 and capsaicin (Cap) led to an abolishment of the BOLD signal, while treatment with either substance alone did not decrease the BOLD signal change. All values are given as mean ± SEM. For clarity, only p-values for the S1 region are displayed. All p-values are given in the text.