The Effect of Tactile Stimulation Within

Different Crayfish Tail Regions on

Extracellular Recordings from Third Nerves
By: Sarah Cooper and Lindsey Sydnor
NBIO 301
Introduction
Crayfish Lab Part 2 Tasks:

● record spontaneous activity of the third nerve

● record activity during sensory stimulation of the side of the tail and of hairs
both ipsilateral and contralateral to the nerve

Presumable takeaways:

● Difference between spontaneous and stim firing rates

● Difference between ipsilateral and contralateral stim
● Difference between tail fin and flank stim
● Identify distinct axons (as many possible out of 6 total per segment)
Furthering Crayfish Lab Part 2
Categorically stimulate specific regions

● increases ability to identify specific significant stimuli

● thoroughly tests ipsilateral vs contralateral

Record from more third nerves

● Repeatable results

● Is it possible to classify all 6 of the axons across the different segments?

Method: Extension of Part 2 of the Crayfish Lab
1. Preparing the Sample
● Using procedures from
the Crayfish Lab, we
dissected the tail
section to isolate third
nerves
● Isolated five third-
nerves
 Method: Extension of Part 2 of the Crayfish Lab
2. Data Collection

● Used a suction electrode to get extracellular recording of a third nerve. Firing

qualitatively assessed in lab via audio monitor.
● After getting a stable recording of spontaneous nerve activity, stimulated tail
in the following ways:

● Repeated Data Collection procedure for all 5 isolated third nerves

Method: Extension of Part 2 of the Crayfish Lab
3. Data Analysis

● Used the Spike Discriminator Tool to identify different axons by visual

inspection of the data and manual annotation of clusters
● For each defined axon, used Rate Meter Analysis to quantify the frequency of
action potentials over time
● Performed two-tailed t-test for unequal variance comparing spontaneous vs.
stimulated conditions
● Performed two-tailed t-test for equal variance comparing ipsilateral vs.
contralateral stimulation
 Spontaneous Firing

Firing of Third
Nerve
Example of
Significant
Difference
Spiking During Contralateral Flank Stimulation
This example from segment one
produced a p value of 0.00048.

Beginning of Stim
 Contralateral Flank
Stimulation

Example of
Significant
Difference
Continued…
Comparative Time (s)
Rate Meters
Spontaneous
Recording
Results: Segment 1 Spontaneous
vs. Stimulated Firing

P-Values for Segment 1 Spontaneous Activity vs Stim per Axon

Amplitude Contralateral Contralateral Ipsilateral Ipsilateral Contralateral
Range (mV) Flank Run 1 Flank Run 2 Flank Run 1 Flank Run 2 Endopod Hairs

R1: 3.1-6.3 0.24177 0.24177 0.56586 0.34976 0.74261

R2: 7.4-11 0.00048*** 0.00313*** 0.00057*** 0.00056*** 0.03906*

R3: 14.8-18.2 0.02097* 0.05266 0.00397** 0.01368* 0.33880

R4: 21-23 0.08186 No Change 0.16609 No Change No Change

*** p < 0.001 ** p < 0.01

* p < 0.05
Highlights of Data from P-Value Table
● Ipsi/Contra flank stimulation produced a significant change in firing rate for the
2 different axons (7.4-11mV and 14.8-18.2mV amplitudes).

● Contralateral Endopod Hairs produced a significant change for one axon (7.4-
11mV).

● No significant change from flank stim for the 3.1-6.3 mV axon.

● Excluded from table b/c no change or no significant effect:
○ Contra/Ipsi Exopod Body and Hairs
○ Contra/Ipsi Endopod Body
○ Telson

● Note: Based on Segment 1 results, we focused on flank stimulation for our

analysis of the remaining segments.
Segment 2 Spontaneous vs.
Stimulated Firing

P-Values for Segment 2 Spontaneous Activity vs Stim per Axon

Amplitude Contralateral Contralateral Ipsilateral Ipsilateral
Range (mV) Flank Run 1 Flank Run 2 Flank Run 1 Flank Run 2

3.1-6.3 0.02708* 0.07712 0.03636* 0.32941

4.4-7.1 0.00053*** 0.13883 0.02066* 0.00053***

11.8-20 0.01389* 0.01363* 0.00317*** 0.03337*

58-62.4 0.03679* 0.03679* 0.01496* 0.16785

*** p < 0.001 ** p < 0.01

* p < 0.05
Segment 3 Spontaneous vs.
Stimulated Firing
P-Values for Segment 3 Spontaneous Activity vs Stim per Axon
Amplitude Range Contralateral Ipsilateral Flank Ipsilateral Flank
(mV) Flank Run 1 Run 1 Run 2

3-10.5 0.3354 0.0147* 0.0260*

20-22.2 0.0663 0.0424* 0.0032***

*** p < 0.001 ** p < 0.01

* p < 0.05
Note:
● Difference between Contralateral Flank and Ipsilateral Flank is not significant (using
two-tailed, equal variance t-test)
● Because of time constraints and because we thought it most fruitful to focus on 3
consecutive ipsilateral segments, we did not analyze data for segments 4 and 5.
Ipsilateral Vs Contralateral Flank Stim
● Used a two-tailed, equal variance T-Test to compare Ipsi vs Contra within
Segment 1, 2, and 3
● P-values show that for each visible axon, there was no significance difference
between ipsilateral and contralateral flank stimulation (p>0.05 for all axons)
Contralateral Flank Stimulus Spiking Ipsilateral Flank Stimulus Spiking

stim stim
 Inability to Compare Across-Segment Axons
1) Suctioning
Segment 2 Differences
Suction

Third nerve

2) Different Axon
Properties

Segment 3
Discussion
Not all types of stimulation produced a significant response

Contralateral and ipsilateral flanks produced almost identical responses

Unable to classify all axons within third nerves of each segment or across
segments
Discussion
● This serves more as a preliminary investigation. Previous lab difficulties made
experimental questions uninformed. Data informs future experimenters on
efficient stimulation techniques.
● Using these third nerves to test crayfish sensory responses may be an ill-
informed decision (extrapolating the data from this one crayfish to others).
● Why is response to flank stronger than response to tail fin stim?
○ Possibly to do with role of third nerve in postural muscles rather than escape response
○ Possibly predation response to a side stimulation; automatic curling
Considerations for Future Experiments
● Use consistent method of stimulation (brushing vs pressing)
○ Rule out lack of response in tail fin due to insufficient stimulation
● Stimulate for longer
○ Easier to compute significance
● Repeat calculations of significance of all stimulation types across all
segments
○ Verify that response to flank stim is stronger than tail fin stims universally
● Repeat refined experiment over multiple crayfish
○ To relate axons across segments, need samples with all 6 axons present
in multiple segments
Thank you!