Before stimulation, the fiber optic cannulas were connected with a 473 nm blue laser or 589 nm yellow laser diode49 (link),56 (link). Light pulse trains were generated by a stimulator (SEN-7103, Nihon Kohden, Japan) and output by an isolator (ss-102J, Nihon Kohden). For instantaneous photostimulation, each trial was applied 20 s after a stable NREM or REM sleep event by observing the online EEG/EMG display. For prolonged photostimulation, programmed light pulse trains (5 ms pulses at 20 Hz for 30 s and at 30 s intervals for 1 h) were used from 9:00 to 10:00. The recorded EEG/EMG during the same period of PSTN-mCherry mice served as the control. Animals were perfused after receiving 1 h-prolonged photostimulation for c-Fos staining. The light intensity at the tip of the optical fiber was tested by a power meter (PM10, Coherent) before each experiment time and calibrated to emit 20–30 mW/mm2. For the open-field test, programmed light pulse trains (5 ms pulses at 20 Hz for 30 s and at 30 s intervals for 10 min) were used after the mice had adapted to the field environment for 5 min.
Ss 102j
Manufactured by Nihon Kohden
Sourced in Japan
The Ss-102J is a compact, lightweight, and portable patient monitoring device designed for use in hospital and clinical environments. It provides continuous monitoring of a patient's vital signs, including heart rate, respiratory rate, and peripheral oxygen saturation (SpO2). The device features a clear, easy-to-read display and is designed for simple and intuitive operation.
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7 protocols using ss 102j
1
Optogenetic Modulation of Sleep-Wake Cycles
2
In Vivo Light Stimulation Protocol
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For in vivo light stimulation, light-pulse trains were generated via a laser stimulator (SEN-7103, Nihon Kohden, Japan) and output through an isolator (ss-102J, Nihon Kohden, Japan). A rotating optical joint (FRJ_FC-FC, Doric Lenses, Canada) was used to relieve torque and was attached to the external end of the optical fiber. For acute photostimulation, each stimulation epoch was applied 20 s after identifying a stable NREM or REM sleep event by real-time online EEG/EMG analysis. Light-pulse trains (5-ms duration each) were programmed and conducted during the light period, when mice are inactive. The cut-off line for stage transitions was 60 s after the laser was turned on. For chronic photostimulation, programmed light-pulse trains (5-ms pulses at 20 Hz, with 10-s on/ 20-s off for 120 cycles) were used from 09:00 to 10:00. EEG/EMG recordings during the same period on the previous day served as a baseline control. Power intensities of blue or yellow light at the tip of the optical fiber were calibrated to emit 3–7 mW [51 (link)].
3
Contractile Force of Fast and Slow Muscles
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We assessed the contractile force of the fast and slow muscles using sciatic nerve electrical stimulation. The EDL muscle and SOL muscle were selected as examples of fast and slow muscles, respectively. A fast muscle is mainly composed of type 2 fibers, while a slow muscle is composed of type 1 fibers. The experimental method was as described by Shilveira and Fortes [29 (link), 30 (link)].
The animals were anesthetized by the intraperitoneal injection of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg). The level of anesthesia was judged by the loss of the pedal withdrawal reflex. Thereafter, their limbs were fixed on a perfusion platform and we incised the right hind limb skin and subcutaneous tissue to expose the sciatic nerve. Two platinum electrodes were attached to the hemi-lateral sciatic nerve. The insertions of the ipsilateral EDL and SOL muscle were cut and connected to a force transducer (45196A; NEC Sanei Instruments Inc., Tokyo, Japan). The resting length and stimulation voltage were adjusted to induce maximum contraction. The electrical stimulator (SEN-3301; NIHON KOHDEN, Tokyo, Japan) and isolator (SS-102 J; NIHON KOHDEN, Tokyo, Japan) were set with an impulse frequency of 1 Hz and duration every 0.5 ms. Five contractions were performed to determine the mean force (mN).
The animals were anesthetized by the intraperitoneal injection of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg). The level of anesthesia was judged by the loss of the pedal withdrawal reflex. Thereafter, their limbs were fixed on a perfusion platform and we incised the right hind limb skin and subcutaneous tissue to expose the sciatic nerve. Two platinum electrodes were attached to the hemi-lateral sciatic nerve. The insertions of the ipsilateral EDL and SOL muscle were cut and connected to a force transducer (45196A; NEC Sanei Instruments Inc., Tokyo, Japan). The resting length and stimulation voltage were adjusted to induce maximum contraction. The electrical stimulator (SEN-3301; NIHON KOHDEN, Tokyo, Japan) and isolator (SS-102 J; NIHON KOHDEN, Tokyo, Japan) were set with an impulse frequency of 1 Hz and duration every 0.5 ms. Five contractions were performed to determine the mean force (mN).
4
Optogenetic Control of Sleep-Wake States
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Before the testing day, mice were given one day to adapt to optical fiber cables (0.8 m long, 200 μm diameter; RWD) that were placed inside the implanted fiber cannulae. On the testing day, 473 nm laser pulses (10 ms, 20 Hz) were delivered via an optic cable (Newton Inc, Hangzhou, China) using a pulse generator. Light pulse trains were generated via a stimulator (SEN-7103, Nihon Kohden, Japan) and delivered through an isolator (SS-102J, Nihon Kohden). For acute photostimulation, each stimulation epoch was applied at 20 s after identifying a stable NREM or REM sleep event via real-time online EEG/EMG analysis. Light pulse trains (5 ms pulses of various frequencies and durations) were programmed and conducted during the inactive period. For chronic photostimulation, programmed light pulse trains ( 10 ms blue-light pulses at 20 Hz for 25 s, every 60 s for 1 hr) were used. The 473 nm laser stimulation was performed from 09:00 to 10:00. Baseline EEG/EMG recordings were acquired at the same time of day on the previous day prior to laser stimulation. Sleep–wake cycle parameters (e.g. durations of NREM sleep, REM sleep, and wakefulness, as well as sleep–wake transitions) were scored over an entire hour for each mouse. After receiving photostimulation, mice were sacrificed at 30 min after the final stimulation for subsequent c-fos staining.
5
Sciatic Nerve Denervation and EMG Analysis
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TA muscle denervation was confirmed by electromyography (EMG) testing of nerve-to-muscle transmission at 8 weeks of age in the denervated rat groups (n=4-8/group). Under anesthesia, each of the left and right sciatic nerves was stimulated with bipolar hook electrodes connected to the stimulator and isolator (SEM-4201, SS-102J, Nihon Kohden) using supramaximal (~10 V) square wave pulses, 0.1 msec in duration[24 (link)]. On the denervated hindlimb, the stimulation points lay proximal and distal to the lesion site, while on the contralateral hindlimb, they were at mid-thigh level. Surface EMG electrodes (3-mm-diameter) were attached to the shaved anterior surface on the TA muscle and used to check whether muscle action potentials were induced by nerve stimulation (Figure 1D ).
6
Photostimulation of Neuronal Activity
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The optical fiber cannula was attached to a rotating joint (FRJ_FC-FC, Doric Lenses, Canada) to relieve torque. The joint was connected via a fiber to a 473 nm blue laser diode (Newton Inc., Hangzhou, China). Light pulses were generated through a stimulator (SEM-7103 Nihon Kohden, Japan) and output via an isolator (ss-102J, Nihon Kohden). For 1 hr photostimulation, we used programmed light pulse trains (5 ms pulses at 20 Hz for 50 s with 40 s intervals for 1 hr). Light stimulation was conducted from 9 p.m. to 10 p.m. EEG/EMG recorded during the same period on the previous day served as baseline. Light intensity was tested by a power meter (PM10, Coherent) before each experiment and calibrated to emit 20–30 mW/mm2 from the tip of the optical fiber cannula.
7
Optical Fiber Cannula Photostimulation Protocol
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The optical fiber cannula was attached to a rotating joint (FRJ_FC-FC, Doric Lenses, Canada) to relieve torque. The joint was connected via a fiber to a 473nm blue laser diode (Newton Inc., China). Light pulses were generated through a stimulator (SEM-7103, Nihon Kohden, Japan) and output via an isolator (ss-102J, Nihon Kohden). For photostimulation, we used programed light pulse trains (5-ms pulses at 30 Hz for 30 s) and recorded electroencephalogram/electromyogram during the experiments. Light intensity was tested by a power meter (PM10, Coherent, USA) before each experiment and calibrated to emit 20 to 30 mW/mm 2 from the tip of the optical fiber cannula. 19 No a priori statistical power calculation was conducted. In this part of the experiment, we used sample sizes that indicated as sufficient to identify biologically meaningful differences in the previous studies. 31
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