Mechanical sensitivity was evaluated using von Frey filaments (Stoelting) that ranged from 3.63 to 125.89 g as previously reported (37). The choice of the standard von Frey test instead of the electronic von Frey test or Hargreaves test was based on the ease with which comparisons can be made with our previous studies (8, 38, 39). Briefly, the series of von Frey filaments was applied from below the wire mesh to the central region of the plantar surface of the left and right hindpaws in ascending order, beginning with the lowest filament (3.63 g) after 10 min of habituation to the testing environment. A withdrawal response was considered valid only if the hindpaw was completely removed from the platform, indicating a positive response. The stimulus was incrementally increased until a positive response was observed and then decreased until a negative response was observed. The ascending and descending testing protocol was used to determine the paw withdrawal threshold as previously reported (39). Paw withdrawal thresholds were recorded before self-administration (serving as an individual baseline), before the initiation of escalation (pre-escalation), and after 14 days of escalation (post-escalation). The data are expressed as grams of applied force. In a separate cohort of animals, we performed the same experiment, with the only difference that after a single injection of NicA2-J1, hyperalgesia was measured after 2, 10, and 21 hours of nicotine self-administration and then 24 and 48 hours into withdrawal, corresponding to 3, 11, 22, 46, and 70 hours after NicA2-J1 treatment.

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