Khloe Frank NBio 301 AC Figure 1: Extracellular Measurement of Spontaneous Activity of Axons in Crayfish Third Nerve.

Extracellular recording of a crayfish third nerve, made with a sucking electrode. Voltage (in mV) is shown on the y axis and has been amplified by a factor of 100. Time (in seconds) is shown on the x-axis. Using the Spike Histogram Discriminator tool of LabChart, I categorized the action potentials into four distinct groups: Unit 7, Unit 6, Unit 8, and Unit 2. The amplitudes in Unit 7 were 0.0035 0.0005 mV. The amplitudes in Unit 6 were 0.005 0.001 mV. The amplitudes in Unit 8 are 0.008 0.001 mV. The amplitudes in Unit 2 were .015 0.001 mV. Rate meters for each unit are shown on the following page; the y-axis is number of action potentials per bin of 0.01 seconds, and the x axis is time in seconds.

Khloe Frank NBio 301 AC Time (s) Frequency (Hz) 0 9 10 8 15 8.5 20 5 30 1 These data were measured using the rate meter tool in LabChart.

Time (s) Frequency (Hz) 0 7 10 7.5 15 9.5 20 6 30 5 These data were measured using the rate meter tool in LabChart.

Time (s) Frequency (Hz) 0 0 10 0 15 0 20 0 30 1 These data were measured using the rate meter tool in LabChart.

Time (s) Frequency (Hz) 0 0 10 1 15 2.5 20 0.5 30 0 These data were measured using the rate meter tool in LabChart.

Khloe Frank NBio 301 AC Figure 2.1: Extracellular Measurement of Axon Activity in Crayfish Third Nerve in Response to Ipsilateral Tail Stimulation. Extracellular recording of a crayfish third nerve, made with a sucking electrode. Voltage (in mV) is shown on the y-axis and has been amplified by a factor of 100. Time (in seconds) is shown on the x-axis. Using the Spike Histogram Discriminator tool of LabChart, I categorized the action potentials into four distinct groups: Unit 1, Unit 2, Unit 3, and Unit 4. The amplitudes in Unit 1 are 0.0025 0.0005 mV. The amplitudes in Unit 2 are 0.0055 0.001 mV. The amplitudes in Unit 3 are 0.0075 0.001 mV. The amplitudes in Unit 4 are 0.0265 0.002 mV. Rate meters for each unit are shown on the following page; the y-axis is number of action potentials per bin of 0.01 seconds, and the x axis is time in seconds. The ipsilateral tail stimulation was done with an instantaneous Q-tip poke and thus is marked by one downward arrow at the moment of prodding.

Khloe Frank NBio 301 AC

Time Frequency (s) (Hz) 0 2 5 0.5 10 4 12.5 1 15 0 These data were measured using the rate meter tool in LabChart.

Time Frequency (s) (Hz) 0 3 5 0 10 2 12.5 3 15 1 These data were measured using the rate meter tool in LabChart.

Time Frequency (s) (Hz) 0 3 5 2.5 10 0.5 12.5 12 15 4 These data were measured using the rate meter tool in LabChart.

Time Frequency (s) (Hz) 0 0 5 0 10 0 12.5 7 15 0 These data were measured using the rate meter tool in LabChart.

Khloe Frank NBio 301 AC Figure 2.2: Extracellular Measurement of Axon Activity in Crayfish Third Nerve in Response to Contralateral Tail Stimulation. Extracellular recording of a crayfish third nerve, made with a sucking electrode. Voltage (in mV) is shown on the y-axis and has been amplified by a factor of 100. Time (in seconds) is shown on the x-axis. Using the Spike Histogram Discriminator tool of LabChart, I categorized the action potentials into four distinct groups: Unit 3, Unit 1, Unit 2, and Unit 4. The amplitudes in Unit 3 were 0.0026 0.0006 mV. The amplitudes in Unit 1 were 0.005 0.001 mV. The amplitudes in Unit 2 were 0.007 0.001 mV. The amplitudes in Unit 4 were 0.0275 0.0005 mV. Rate meters for each unit are shown on the following page; the y-axis is number of action potentials per bin of 0.01 seconds, and the x axis is time in seconds. The contralateral tail stimulation was done with an instantaneous Q-tip poke and thus is marked by one downward arrow at the moment of prodding.

Khloe Frank NBio 301 AC

Time Frequency (s) (Hz) 0 4 5 1 10 9 12.5 5 15 8 These data were measured using the rate meter tool in LabChart.

Time Frequency (s) (Hz) 0 1 5 1 10 1 12.5 7 15 1 These data were measured using the rate meter tool in LabChart.

Time Frequency (s) (Hz) 0 2 5 3.5 10 3 12.5 5 15 3 These data were measured using the rate meter tool in LabChart.

Time Frequency (s) (Hz) 0 0 5 0 10 0 12.5 3 15 0 These data were measured using the rate meter tool in LabChart.

Khloe Frank NBio 301 AC Figure 3.1: Spontaneous Action Potentials from Third Nerve Cause Post Synaptic Potentials in Crayfish SFM Cells. Simultaneous extracellular recording of spontaneous action potentials in a crayfish third nerve (using a suction electrode) and intracellular recording of the resulting post synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis, and has been amplified by a factor of 100 for the extracellular recording (upper channel). Time (in seconds) is shown on the x-axis. Using the Spike Histogram Discriminator tool of LabChart, I categorized the action potentials into three distinct groups: Unit 1, Unit 5, and Unit 4. The amplitudes in Unit 1 were 0.0025 0.0005 mV. The amplitudes in Unit 5 were 0.0046 0.0016 mV. The amplitudes in Unit 4 were 0.0158 0.0015 mV. Rate meters for each unit are shown on the following page; the y-axis is number of action potentials per bin of 0.002 seconds, and the x axis is time in seconds. (Data from Courtney Roberts and Samantha Iverson.)

Khloe Frank NBio 301 AC

Khloe Frank NBio 301 AC Figure 3.2: Process for Measuring EPSP Magnitude and Delay Time for Table 1. Simultaneous extracellular recording of spontaneous action potentials in a crayfish third nerve (using a suction electrode) and intracellular recording of the resulting post synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis. Time (in seconds) is shown on the xaxis. For each action potential-EPSP pair, the downward arrow marks the start of the delay time (the onset of the action potential), and the upward arrow marks the end of the delay time (the onset of the EPSP).The sideways arrow pointing right marks the original membrane potential, and the sideways arrow pointing left marks the membrane potential at the peak of the EPSP; the difference between these two potentials was calculated with the Marker tool of Labchart to determine the magnitude of the EPSP.

Khloe Frank NBio 301 AC Table 1: Raw Data for Figure 3.

EPSP 1 Triggered by Spike of . . . Delay Time Unit 4 (Green) 0.004125s -0.0140 mV 2 5.65 mV Unit 5 (Blue) 0.00575s (-54.54 -48.89) -0.0030 mV 3 1.26 mV Unit 5 (Blue) 0.005675s (-54.64 -53.08) -0.0051mV 4* 8.54 mV Unit 4 (Green) 0.00525 (-53.82 -45.28) -0.0152 mV 5 4.6 mV Unit 5 (Blue) 0.0057s (-55.02 -50.42) -0.0032 mV 6 10.07 mV Unit 4 (Green) 0.00515s (-55.42 -45.35) -0.0130 mV 7* 0.89 mV Unit 5 (Blue) 0.00595s (-51.99 -51.10) -0.0045 mV 8 5.08 mV Unit 5 (Blue) 0.0058s (-54.37 -49.29) -0.0045 mV 9 10.69 mV Unit 4 (Green) 0.00475s (-55.42 -44.73) -0.0172 mV 10* 5.19 mV Unit 5 (Blue) 0.006s (-53.97 -48.78) -0.0033 mV 11 1.74 mV Unit 5 (Blue) 0.0046s (-54.73 -52.99) -0.0061 mV 12* 5.13 mV Unit 5 (Blue) 0.0055s (-53.83 -48.70) -0.0031 mV 13* 3.48 mV Unit 4 (Green) 0.004875s (-48.70 -45.22) -0.0155 mV *Each of these EPSPs occurred before the membrane potential had completely repolarized from the previous EPSP Magnitude 9.6 mV (-55.32 -45.72)

Khloe Frank NBio 301 AC Figure 4.1: Measuring Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at a frequency of 1Hz for periods of 5-6 seconds, and recorded the resulting EPSPs (Box A). We then averaged data from 2 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time; the first EPSP peak was 1.125 mV. Note that the data points appear to be linear at this frequency of stimulation, with a constant facilitation index of approximately 1.0 0.3.

3 2.5 2 1.5 1 0.5 0 0 1 2 3 4 5 6

Khloe Frank NBio 301 AC Figure 4.2: Measuring Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at a frequency of 2Hz for periods of 4 seconds, and recorded the resulting EPSPs (Box A). We then averaged data from 2 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time; the first EPSP peak was 1.03 mV. Note that the data points do not appear to be linear at this frequency of stimulation until 1.5 seconds after the onset of the current pulses.

3 2.5 2 1.5 1 0.5 0 0 1 2 3 4 5

Khloe Frank NBio 301 AC Figure 4.3: Measuring Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at 5Hz for periods of 3 seconds, and recorded the resulting EPSPs (Box A). We then averaged data from 3 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time; the first EPSP peak was 0.927 mV. Note that the data points do not appear to be linear at this frequency of stimulation until approximately 1.5 seconds after the onset of the current pulses.

4 3.5 3 2.5 2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5

Khloe Frank NBio 301 AC Figure 4.4: Measuring Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at 10Hz for periods of 3-4 seconds, and recorded the resulting EPSPs (Box A). We then averaged data from 3 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time; the first EPSP peak was 1.367 mV. Note that the data points do not appear to be linear at this frequency of stimulation until approximately 1.9 seconds after the onset of the current pulses.

4 3.5 3 2.5 2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5

Khloe Frank NBio 301 AC Figure 4.5: Measuring Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the y-axis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at 20Hz for periods of 3-7 seconds, and recorded the resulting EPSPs (Box A). We then averaged data from 3 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time; the first EPSP peak was 1.63 mV. Note that the data points do not appear to be linear at this frequency of stimulation until approximately 2.7 seconds after the onset of the current pulses.

6 5 4 3 2 1 0 0 0.5 1 1.5 2 2.5 3 3.5

Khloe Frank NBio 301 AC Figure 5.1: Time Course of Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the yaxis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at a frequency of 10Hz for periods of 2.2 seconds while simultaneously using a function generator to continuously apply pulses of current at a frequency of 0.787Hz. The resulting EPSPs that were recorded are shown in Box A. We then averaged data from 2 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time. The downward arrow marks the time at which we stopped applying pulses of current from the pulse stimulator. Note that the pattern of the data changes at this point from increasing to decreasing facilitation index over time; the first EPSP peak was 1.367 mV.

Khloe Frank NBio 301 AC Figure 5.2: Time Course of Facilitation in Crayfish Neuromuscular Junction. Intracellular recording of post-synaptic potentials in a crayfish SFM cell. Voltage (in mV) is shown on the yaxis. Time (in seconds) is shown on the x-axis. In this preparation, the crayfish nerve had been crushed between the nerve cord and the extracellular suction electrode. We used a pulse stimulator to apply pulses of current at a frequency of 20Hz for periods of 1.5 seconds while simultaneously using a function generator to continuously apply pulses of current at a frequency of 0.787Hz. The resulting EPSPs that were recorded are shown in Box A. We then averaged data from 3 trials to produce a facilitation plot for this frequency of stimulation (Box B). The plot shows the facilitation index of each EPSP as a function of time. The downward arrow marks the time at which we stopped applying pulses of current from the pulse stimulator. Note that the pattern of the data changes at this point from increasing to decreasing facilitation index over time; the first EPSP peak was 1.63 mV.