Figure 4. PBN-SL^Tacr1 neurons respond to sustained noxious stimulation.

(A) Cartoon depicting the approach for recording population activity in PBN-SL^Tacr1 neurons: a Cre-dependent viral vector encoding the genetically encoded calcium indicator GCaMP6f was injected into the PBN of Tacr1^Cre mice and an optical fiber was placed over the injection site. Fiber photometry recording was used to monitor population calcium responses to different somatosensory stimuli. (B) After recording, posthoc staining and confocal imaging of PBN sections confirmed GCaMP6f expression (green) and fiber placement (dotted line). (C) An example photometry trace from a lightly anesthetized mouse showing a robust, time-locked calcium response to tail pinch with blunt forceps (stimulus onset at 0 s; dotted line). Note the response lasts the duration of the stimulation (shaded region) before returning to baseline. As a control for movement artifacts, 405 fluorescence (Blue trace; Isosbestic) was also monitored and showed no changes. (D–H) Average population calcium responses in awake mice to sustained (D, E, and G) and acute (F, H) noxious stimuli. Dark lines are means of responses from multiple animals aligned to the start of stimulation (time 0) with the standard error shown in light grey. X-axis shows time in seconds aligned to the start of stimulation; F indicates ΔF/F scaling (D) Pinching (hindpaw) with a blunt forcep (n = 6 mice). (E) Clip assay applied to hindpaw (n = 5 mice). (F) von Frey stimulation (0.6 g) aligned to paw withdrawal (n = 4 mice) (G) 55°C Hot plate test aligned to when mice are placed in a chamber and lasting 25 s (n = 3 mice). (H) Radiant heat test (Hargeaves) aligned to paw withdrawal (n = 7 mice). For all traces, time is in seconds, 0 is the start of the trial, change in fluorescence/total fluorescence is shown (black line; F).

Figure 4—figure supplement 1. Population calcium responses in PBN-SL^Tacr1 neurons to pinch are not somatotopically restricted.

(A) Example traces from two separate lightly anesthetized mice showing robust responses to a series of sustained pinches with blunt forceps (blue arrowheads) of different body parts (tail, left hindpaw, right hindpaw, belly, cheek, whisker pad, ear, and neck). (B) Noxious pinprick does not evoke a population calcium response although does cause a rapid nocifensive withdrawal behavior (red dashed line = stimulation). For all traces, time is in seconds, 0 is the start of the trail, change in fluorescence/total fluorescence is shown (black line; F).

Figure 4—figure supplement 2. The chemical irritant allyl isothiocyanate (AITC) evokes large and sustained increases in calcium signaling in PBN-SL^Tacr1 neurons.

Example traces from an awake mouse to topical application of saline (A; black traces) and AITC (B; blue traces) on the hindpaw. AITC evokes a very robust response followed by long-lasting increases in spontaneous calcium transients. Note that despite the increased baseline activity and behavioral hyperalgesia after application, AITC pre-treatment (>5 min) does not change the sensitivity of these neurons to acute noxious stimulation. Both acute noxious mechanical (C, D) or thermal (E, F) stimuli fail to activate PBN-SL^Tacr1 neurons either in control or AITC treated conditions. Red arrows indicate behavioral responses (paw withdrawals). Time is in seconds, 0 is the start of the trail, change in fluorescence/total fluorescence is shown (black line; F).