The 6-Hz-induced psychomotor seizures in mice were evoked by current (6 Hz, 0.2 ms rectangular pulse width, 32 mA, 3 s duration) generated by an S48 Square Pulse Stimulator and CCU1 Constant Current Unit (Grass Technologies, West Warwick, RI, USA). The animals were administered with increasing doses of KA-228 and KA-232 , and the anticonvulsant activity of each drug was evaluated as the ED50. This experimental procedure has been described in detail in our previous studies [19 (link),35 (link)].
S48 square pulse stimulator
Manufactured by Natus
Sourced in United States
The S48 Square Pulse Stimulator is a lab equipment used to generate square-wave electrical pulses. It can be used to provide controlled electrical stimulation in various experimental and research settings.
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7 protocols using s48 square pulse stimulator
1
6-Hz-induced Seizures in Mice: Anticonvulsant Evaluation
2
Hippocampal Long-Term Potentiation Assessment
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General population measurements of field excitatory postsynaptic potentials (fEPSP) were recorded with NaCl-filled (150 mM) glass micropipettes inserted into the stratum radiatum of the CA1 hippocampal subfield using a SUPER-Z head-stage attached to a BMA-931 AC/DC Bioamplifier (CWE-inc, Ardmore, PA, USA), Digidata1200 (Molecular Devices, Sunnyvale, CA, USA), and pClamp 9.0 software (Molecular Devices). Schaffer collaterals were electrically stimulated (0.3 ms constant current pulses) with bipolar tungsten electrodes using a S48 square pulse stimulator (GRASS technologies, Warwick, RI, USA). After slice recovery in the recording chamber (approximately 30 min), input–output curves relating stimulus current intensity to fEPSP slope and amplitude were generated. Stimulus intensity required for half-maximal fEPSP slope was selected. Paired-pulse experiments were performed using an intra-pulse duration of 50 ms. Stimulation for fEPSP was performed at a rate of 1 stimulus every 30 seconds for baseline recordings. Long-term potentiation (LTP) was then induced using tetanic stimulation (100 Hz) for 1 second. One minute following LTP, test stimulation (1 stimulus every 30 seconds) resumed for a period of 1 h and afterwards input–output curves were repeated. Slices from each animal were averaged together to give a n-value of one.
3
In situ Skeletal Muscle Contractile Analysis
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Skeletal muscle contractile function was assessed in situ using a protocol adapted from Kalmar and Greensmith [21 (link)] and MacIntosh, Esau, Holash and Fletcher [22 ]. Animals were anesthetised with tribromoethanol (20 mg kg/100 g body mass i.p.) and the sciatic nerve was exposed and connected to a platinum electrode. The distal tendon of the tibialis anterioris was connected to a surgical silk suture, which was connected to a previously calibrated force transducer. The sciatic nerve was stimulated using an S48 square pulse stimulator (Grass®) and the contractile data were acquired and processed using a data acquisition system (Biopac Systems, USA; AcqKnowledge software, version 4.4). 0.5 V pulses were used to maximise motor unit recruitment. The stimulation protocol consisted of three successive steps separated by 60 s intervals: 1) Single twitch: 5 isolated pulses at 1 Hz, 200 ms duration; 2) Maximum tetanic force: one pulse train at 150 Hz, duration 300 ms; 3) Fatigue test: one pulse train per second at 40 Hz, with the duration of 333 ms for 3 min.
4
Corneal Stimulation-Induced Seizure Protocol
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Seizure activity in mice was evoked by a current (6 Hz, 0.2 ms rectangular pulse width, 32 mA, 3 s duration) generated by an S48 Square Pulse Stimulator and CCU1 Constant Current Unit (Grass Technologies, West Warwick, RI, USA). After the application of an ocular anesthetic (0.5% solution of tetracaine hydrochloride) to the mouse corneas, the animals underwent corneal stimulation and were placed separately in Plexiglas cages (25 cm × 15 cm ×10 cm) for the observation of the presence or absence of psychomotor seizures, as described previously [15 (link),78 (link),79 (link)]. When the observation of 8 mice in the respective group was finished, the animals underwent euthanasia by CO2 narcosis. To determine median effective doses (ED50 values) of ASMs, the drugs were administered i.p. at the following doses: LCM: 2–10 mg/kg; LEV: 10–20 mg/kg; PB: 2–12 mg/kg and VPA: 50–150 mg/kg.
5
Anticonvulsant Screening of Novel Compounds
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All experiments on animals were performed in accordance with EU Directive 2010/63/EU for animal experiments and complied with the ARRIVE guidelines. Ethics approval for animal experimentations was obtained from the Local Ethics Committee (Lublin, Poland, approval number: 65/2018). Psychomotor seizures in mice were evoked by current (6 Hz, 0.2 ms rectangular pulse width, 32 mA, 3 s duration) generated by an S48 Square Pulse Stimulator and CCU1 Constant Current Unit (Grass Technologies, West Warwick, RI, USA). After application of ophthalmic anaesthetic (0.5% tetracaine hydrochloride) to the mice corneas, the animals underwent corneal stimulation and were placed separately in plexiglas cages (25 × 15 × 10 cm) for the observation of the presence or absence of psychomotor seizures17 (link). The animals were administered with increasing doses of TP-10, TP-315, TP-427, and the anticonvulsant activity of each compound was evaluated as the ED50 value (median effective dose of the drug, which protects 50% of mice against convulsions) calculated using the log-probit method of Litchfield and Wilcoxon18 (link).
6
Suppressing Cortical Activity with Topical TTX
7
Acute 6-Hz Seizure Model Protocol
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The acute 6-Hz seizure model was conducted as explained previously [30 (link)–32 (link)]. Corneal stimulation (0.2 ms rectangular pulses at 6 Hz for 3 s) was delivered by a constant-current device (CCU1) connected to an S48 Square Pulse Stimulator (Grass Technologies, West Warwick, RI, USA). A fixed current intensity of 32 mA was used to allow this data to be directly compared with data obtained in the aforementioned studies. Animals were randomly divided into three groups with ten animals in each group. Before stimulation, a drop of saline was applied to each eye to ensure optimal current conductivity. Seizures were characterized by a stunned or fixed posture often accompanied by rearing, forelimb clonus, and twitching. The duration of seizure activity was measured as the evaluation criterion for the protective effect of the compound. The animal was considered to be protected if it resumed its normal exploratory behavior within 7 s of the stimulation. In contrast, the compound had no protective effect if the above typical characteristic behaviors occurred in the animal after drug administration or if the duration of the seizure was longer than 7 s.
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