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to later neural responses. We then connected the recording electrodes directly to the stimulating electrodes and changes in polarity, amplitude, frequency and duration of the stimulus were observed. The stimulator and oscilloscope were calibrated to specific settings for this measurement. See table 1 for artifact detection equipment settings. Stimulator Oscilloscope Frequency: 2/sec Timebase: 1 msec/div Delay: 1 msec Trig source: external Duration: 1 msec Trig mode: AC coupl (in) Volts: 0.2 V Slope: +(in) Mode: repeat Gain Ch 1: 0.2 V/div Stim: regular pulses Display mode: Ch 1 Polarity: monopolar AC/GND/DC: AC (in) Pulse: out Table 1. Equipment settings for electrophysiology equipment Manipulation of Extracellular Ion Concentrations A dissected frog sciatic nerve was provided for experiments. We threaded the nerve (attached to a suture) through the recording and bath chambers into the stimulating chamber so that the leading end rested upon stimulating electrodes in the main chamber and the distal end rested on the recording electrode in the first chamber (chamber C). Petroleum jelly was used to prevent seepage of solutions between the two recording chambers (chambers A & C) by injecting it into the two small holes separating the chambers. We placed frog ringers solution (control solution) into each of the small chambers and in the main chamber. In the control solution, we recorded activity from 10 successive stimulations of the nerve in order to record the following categories of data: threshold, peak amplitude and refractory period. These data collections were acquired through various fluid exchanges in which extracellular fluid types were changed out and tested. The equipment settings listed in table 1 remained the same except for the changes shown in table 2. Stimulator Oscilloscope Duration: 0.06 msec Gain: 1 mV/div Table 2. Summary of changes made in equipment settings from table 1. For the first fluid exchange, we removed the control Ringers solution from the closest chamber to the main chamber (chamber A) and discarded. We refilled Chamber A with 0.33X Na+ Ringers solution and repeated the exchange twice more to ensure full fluid exchange. Once all fluid was exchanged, the nerve was stimulated and the abovementioned data was recorded for 10 trials with at least 5 seconds between trials. This exchange procedure was repeated, replacing the solution with 0.03X Na+ Ringers solution.
03 Na+ Ringers NaCl 0. the control Ringers solution was replaced with 0.376 1.200 2. gm/L Molarity mM Physiological Ringers NaCl (54.99 MW) 0.015 CaCl2 (111.081 ChCl 15.120 1.878 .878 3.381 NaH2PO4 0.381 NaH2PO4 (137.030 36.878 NaHCO3 0.482 75.015 [K} mM 1.00 MW) 0.063 KCl (74.381 1.878 40.140 1.015 CaCl2 0. we exchanged the 3X K+ Ringers solution the previous solution and AP characteristics were recorded.878 NaHCO3 0.015 CaCl2 0.To establish a new baseline.878 NaHCO3 (84.140 1.878 [Na} mM 36.631 111.03X Na+ Ringers solution formula [Na} mM 112.88 MW) 6.417 Table 3.200 2.015 115.140 1.878 1.01 MW) 0.081 TOTALS 118.086 TOTALS 118.063 2.33X Na Ringers solution formula gm/L Molarity mM 0.33 Na+ Ringers NaCl 2. 0.015 [K} mM 1.140 1.120 1.878 [Na} mM 0.981 KCl 0.150 112. we repeated the exchange using control Ringers solution and new action potential characteristics were recorded.33X K+ Ringers solution using the same fluid exchange protocol and the same data types were recorded.486 1.981 2.091 2.381 1.081 ChCl 10.970 TOTALS 118.55 MW) 0.415 Table 5. For full solution data see tables 3-7.458 [K} mM 1. 0.005 0.381 1.140 1.120 1.381 NaH2PO4 0.421 + Table 4. After determining the new baseline.091 KCl 0.200 2.140 1. Control ringers solution formula gm/L Molarity mM 0. Finally.
015 111.620 NaHCO3 0. threshold and refractory period duration.381 1.081 ChCl 0.419 Table 7.gm/L Molarity mM 3.120 1.200 2.150 112.458 0.05) but no significant difference in the 0.634 NaHCO3 0.69)=5.0046) and a post hoc test showed a significant decrease in amplitude in the 0. In a one-way ANOVA comparing the peak amplitude of the nerve in Ringers control to the altered Na+ solutions.).03X Na+ solution (q=3. nor were there any differences between these two and the Ringers control solution (Fig 3).33X K+ solution and the 3X K+ solution.015 [K} mM 0.634 [Na} mM 112.33X K+ Ringers solution formula RESULTS [Na} mM 108.120 1. We substituted two sodium deficient solutions for the Ringers control solution to test the impact of sodium on action potential.200 2. and we used an average of all four to obtain a more accurate result in all subsequent statistical tests in which one-way ANOVA was used to compare the two alternate solutions (Na+ or K+) to the Ringers control. p<0.420 5.944 108.381 NaH2PO4 0.309 2. There were no significant differences between the 0.140 1.620 We assessed Frog sciatic nerve action potential characteristics in various extracellular mediums. two sodium deficient solutions were to test sodium’s impact on the action potential.015 CaCl2 0.33X Na+ solution (Fig 2).33X K+ Ringers NaCl 6. A standard Frog Ringers solution was used as a control.063 2. One-way ANOVA revealed no significant differences between the data recorded from the four Ringers control solutions (Fig 1.381 1. .176 1. and potassium deficient and potassium superfluous solutions were used to test potassium’s impact on action potential. The specific traits we tested were peak amplitude.046 0.416. 0.634 5.015 CaCl2 0. We made the same amplitude recordings while exposing the nerve to altered K+ solutions: one that was K+ deficient and one that was K+ superfluous.381 NaH2PO4 0. a significant difference from solution was revealed (F(2.620 115.140 1.419 Table 6.0X K+ Ringers NaCl 5.063 KCl 0.309 KCl 0.836.0X K+ Ringers solution formula gm/L Molarity mM 0.081 TOTALS 118. 3.261 TOTALS 118.704 [K} mM 5. p=0.
893. p<0. p<0.0001).03X Na+ solution.73. In the K+ solutions.80)=28. A significant increase was seen in the 0.0001).10. No significant differences were seen between ringers control solutions. Significant increases were seen in both 0. These data are shown in Fig 9. In the Na+ solutions.69)=1153. No significant differences were seen between Ringers control and K+ solutions. Data shown are MeanSEM We used the same solution variations to assess the extracellular medium’s impact on action potential threshold. Data shown are MeanSEM Fig 5.05). * * * * * Fig 4.* Fig 1. .05) as seen in Figure 6. an extremely significant effect of solution on action potential threshold was seen (F(2. p<0.0001).03X Na+ solution (q=22. Post hoc testing showed a significant increase in threshold in the 0. p<0.05).60. Data shown are MeanSEM We also used the extracellular fluid variations to record changes in refractory period duration.33X K+ solution (q=18. There was a significant difference between the control ringers solutions (F(3. A significant decrease was seen in Ringers 1.05) and a very significant threshold decrease in the 3X K+ solution (q=21. In the 0.26. A significant decrease was seen in the 0.69)=250. Data shown are MeanSEM Fig 2. p<0. Data shown are MeanSEM Fig 6.750.05) and an extremely significant increase was in the 3X K+ solution (q=7.33X and 0. Again.11. No significant changes in refractory period duration were seen as a result of the Na+ solutions (Fig 8). p<0. a significant effect of solution was seen (F(2.69)=33.0001). no significant differences were seen in the ringers control solutions (Fig 7. p<0.33X K+ solution and a very significant decrease was seen in the 3X K+ solution. p<0.05) and a significant increase in the 0. Dunnett’s multiple comparison test showed a very significant threshold increase in the 0. Fig 3.). These relationships can be seen in Figure 5. p<0.3. so we used Ringers 1 for the threshold tests (Fig 4.33X Na+ solution (q=13.).03X Na+ solutions. When testing the impact of K+ solutions on refractory period duration.10. one-way ANOVA revealed an extremely significant impact of solution (F(2. a very significant increase was seen over the control solution (q=3. Data shown are MeanSEM.33X K+ solution. p<0.
Data shown are MeanSEM Fig 8.33X and 3X K+ solutions.03X Na+ solutions. No significant differences were seen between Ringers control solutions. Significant increases were seen in both 0.33X and 0. Data shown are MeanSEM . Data shown are MeanSEM Fig 9.* * Fig 7. No significant differences were seen in 0.
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