Sen 3301

Isolated cardiomyocytes were loaded with 10 µM Indo-1 AM and electrically stimulated at 1 Hz using a two-platinum electrode insert connected to a bipolar stimulator (SEN-3301; Nihon Kohden) on the stage of an inverted microscope (IX71; Olympus) with a 20× water immersion objective lens (UApo N340; Olympus). Calcium transients were measured as the ratio of fluorescence emitted at 405/480 nm, after excitation at 340 nm, using a high-performance Evolve EMCCD camera (Photometrics). Cardiomyocytes were maintained under continuous flow in standard Tyrode’s solution, exchanged using a microperfusion system. The experiments were recorded and analyzed using MetaMorph software (version 7.7.1.0; Molecular Devices). To estimate the effect of colchicine on cell contraction, we calculated the efficiency of converting elevated Ca²⁺ levels to cell contraction by dividing the percentage of cell shortening by the Ca²⁺ amplitude.

Spontaneous and evoked action potentials of tissue preparations were recorded with glass microelectrodes filled with 3 M KCl. In the case of quiescent preparations, rectangular current pulses (1 Hz, 3 msec, 1.5 × threshold voltage) were applied through a pair of platinum plate electrodes generated from an electronic stimulator (SEN-3301, Nihon Kohden) to evoke the action potentials. The action potential parameters: frequency, maximum diastolic potential (MDP); maximum rate of rise (dV/dt)_max; action potential duration at 90% repolarization (APD₉₀) and the slope of the diastolic depolarization phase were measured.

A custom-made mouse ankle joint motor function analysis system (Bio Research Center, Nagoya, Japan) was used as previously described (Itoh et al., 2017 (link)). Briefly, the mice were anesthetized with isoflurane inhalation, and the plantar was attached to the pressure sensor. Two electrodes were attached to the shaved hind limb with viscous electrical conductive gel (CR-S; Sekisui Plastics, Osaka, Japan) in between. One was fixed to the myotendinous junction and the other was fixed 5 mm above it with adhesive tape. Electrical stimulation was applied to the skin surface of the triceps surae muscle using an electric stimulator (SEN-3301; Nihon Kohden, Tokyo, Japan) to induce muscle contraction. Isometric plantarflexion torque was calculated from the pressure applied to the sensor and the distance from the ankle joint to the sensor. The measurement was performed twice for each foot, and the average value was adopted.

After the baseline PWT measurement, the implanted electrodes were connected to an electrical stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan) via an isolator (SS-201J, Nihon Kohden) with wire leads, allowing the animals to move freely in the cage. Monopolar electrical stimuli with a frequency of 50 Hz and pulse width of 0.2 ms were applied. The amplitude was individually determined as 70–80% of the intensity required to produce slight twitching in the lower trunk muscles or leg stretching (i.e., motor threshold), as reported in previous studies [24 (link), 48 (link)]. We defined a response as increased PWT (>130% of the initial value) on the ipsilateral side after 1-h SCS.

A segment of the distal colon of 3-4 cm length was mounted in a Magnus tube (20 mL in capacity) filled with modified Tyrode's solution (pH 7.4). The solution was continuously bubbled with 95% O₂ + 5% CO₂ gas mixture and maintained at 37°C. The distal end of each segment was tied to organ holders and the proximal end was secured with a silk thread to an isometric force transducer. The preparation was stimulated electrically by means of two platinum electrodes, one of which was placed in the lumen of the preparation and the other in the bathing solution (coaxial stimulation). Supramaximal rectangular pulses of 80 V in intensity and 0.5 msec in duration were delivered by using an electrical stimulator (model SEN-3301, Nihon Kohden, Tokyo, Japan) with frequency spectra of 20 Hz for 1 sec. Longitudinal contractile activity was recorded isometrically with a force transducer (T7-8-240, Orientec, Tokyo, Japan). An initial tension of 1.0 g was applied to the colonic preparations, which were subsequently allowed to equilibrate for 30 min. At the end of this period, the tension created by the segment was considered as the resting tension and no further mechanical adjustment was made during experimentation. Isometric responses were filtered and amplified by an amplifier (NEC, AS1202, Tokyo, Japan) and recorded using a PowerLab system (AD Instruments, Bella Vista, NSW, Australia).