1. Electrophysiological Frequency Analysis of Blaptica dubia
Desiree Griego, Maria Orozco, Andres Romero
Northern New Mexico College
Department of Biology
Abstract
Data Collection and Methods
Data Analysis and Findings
Discussion
B. dubia roaches, the SpikerBox apparatus in conjunction with the free downloadable software Audacity, and MATLAB provide an inexpensive way to study neurophysiology. The purpose of our study is to analyze the electrophysiological frequency response of Blaptica dubia roaches after injection of two types of solutions: saline and a nicotine solutions. We recorded the action potential signals generated by electrical microstimulation of spines on B. dubia roach legs. Locally generated MATLAB codes are then used to analyze the frequency response and generate frequency distributions.
We designed a distance, d, to measure the difference between two distributions.
The distance is computed by subtracting the percentages at each of the frequencies between 10 Hz and 250 Hz and summing the absolute value of the distances at each of the frequencies.
A lab of approximately 100 Blaptica dubia roaches is maintained at Northern New Mexico College.
Procedure
1. Place male B. dubia cockroaches in freezer (approximately 5 minutes). The males have full wings while the female roaches have stunted wings.
2. Inject control cockroaches with .05 mL of 6.2 millimolar solution of NaCl in ventral abdomen
3. Inject experimental cockroaches with .05 mL 6.2 millimolar solution of nicotine in ventral abdomen
4. Allow solutions to be absorbed by cockroaches for 5 minutes
5. Sever a hind leg below the trochanter on the femur using scissors. Attach leg to SpikerBox leads. Record activity using Audacity while stimulating spines on leg with metal rod.
Electrophysical frequency analysis using MATLAB
1. Eliminate basal response of signal by filtering small amplitude signals.
2. Calculate the time difference in seconds between all high amplitude spikes.
3. Compute the reciprocal of the time difference to find a frequency in Hertz (Hz).
4. Create a relative frequency distribution of all frequencies between 10 Hz and 200Hz.
Table 1: Difference between frequency distributions computed using distance.
Specimen
Control 1
Control 2
Exp 1
Exp 2
0.0
76.1
95.1
51.1
43.1
40.6
58.9
Raw data collected from SpikerBox
Introduction
Neuroscience programs typically require expensive equipment, Institutional Research Board clearance, and protocol training if vertebrates are used in laboratories. Therefore, there is a need for alternatives to study neuroscience. One alternative is to use invertebrates which typically have a smaller number of identifiable neurons and can remain viable for long periods. Thus, learning neurophysiology in a lab setting can be done inexpensively using the SpikerBox apparatus and invertebrates. In this study we investigate electrophysiological signals when stimulating the leg of Blaptica dubia or orange-spotted cockroach as a model organism.
Conclusions
By removing a leg from the cockroach's mesothorax, the last and largest leg closest to the abdomen, we were able to gather electrophysiological data via action potentials and analyze the response using MATLAB. Incidentally, the leg will grow back. We did notice that a large percentage of frequency events occur at approximately 60 Hertz for both the control and nicotine roaches.
While Table 1 shows differences between the frequency distributions of saline and nicotine injected B. Dubia, large differences exist within the control and nicotine trials themselves. We believe additional tests need to be conducted, and our procedure needs to be refined before conclusions can be made. The major benefit of this approach is that it is inexpensive. In addition, our approach merges principles from both biology and mathematics.
Control 1: Saline solution
Theory
The SpikerBox produces vast amounts of data per recording event. This is an excellent opportunity to integrate mathematical and coding lessons with biological principles. Many science disciplines are increasingly reliant on a computer science. Any future scientist will have an advantage in their field if they understand, from beginning to end, where their data is produced and how it is organized and analyzed.
Our research required cooperation between our math and biology departments. We gather hands-on experience in generating as well as processing the data.
It is well known that the body has a complex reaction to nicotine. Clinical and experimental studies have shown both depressant and stimulant effects, depending on dosage. Acetylcholine is the neurotransmitter used by insects and nicotine can bind to acetylcholine receptors (Marzullo and Gage 2012).
For this reason, nicotine was chosen in our study to see if differences will be observed in neural activity, via the SpikerBox, Audacity, and MATLAB software.
Control 2: Saline solution
References
Dagda RK, Thalhauser RM, Dagda R, Marzullo TC, Gage GJ (2013) Using crickets to introduce neurophysiology to early undergraduate students. J Undergrad Neurosci Educ 12 (1): A66-A74.
Experimental 1: Nicotine solution
Marzullo TC, Gage GJ (2012) The SpikerBox: a low cost, open-source bioamplifier for increasing public participation in neuroscience inquiry. PLoS One 7:e30837. 
Acknowledgements
We would like to acknowledge the New Mexico Alliance for Minority Participation (NM AMP) program and the NIH BUILD SEED grant for their support. We also wish to thank Dr. Ricoy for the use of his lab and Cathy Pacheco for her assistance.
Experimental 2: Nicotine solution