Lab 3_Neurophysiology_2023

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Biology

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Dec 6, 2023

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Procedure ZOO3610 Lab Studies in Animal Physiology I Lab 3: Investigation of the Neurophysiological Properties of the cockroach leg ( Gromphadorhina portentosa ) In this lab, you will record and measure extracellular action potentials generated from various stimuli using a cockroach leg. Adapted from protocol written by staff at ADInstruments Background Cockroaches have a number of sensory organs similar to those found in humans. While these organs are sensitive to different types of stimuli, the basic cellular function is the same. Sensory neurons in these organs are responsible for detecting external stimuli from the organism's environment. These specialized cells convert the stimulus into electrical and chemical signals for transmission to the rest of the organism’s nervous system (Figure 1). Figure 1. Sensory Neuron Different types of neurons respond to different stimuli, including pressure, stretch, chemical transmitters, and electrical currents passing across the cell. If the stimulus is sufficient the cell will reach its threshold voltage. The cell membrane in the axon and cell body contains voltage- gated ion channels, which allow the neuron to generate and propagate an electrical impulse called an action potential. Cells generate this potential by using adenosine tri-phosphate (ATP)

to power pumps that move positively charged ions out of the cell, thereby creating a difference in charge on each side of the insulating cell membrane. By inserting electrodes into the cell, measurements of this difference in charge across the membrane can be made. This is often referred to as the resting membrane potential. The cockroach leg is studded with stiff hairs and spines. At the base of each spine is a single sensory neuron. The cell body of the neuron lies just under the cuticle of the spine, with the un-branched dendrite of the neuron projecting up through in the overlying cuticle (Figure 2). Figure 2. Sensory Spine from a Common Cockroach The cockroach spines sit in a flexible membrane to allow for movement. When the spine moves, the dendrite of the neuron is distorted, opening mechanically gated ion channels in the dendrite. This creates a receptor potential, which in turn triggers action potentials. Most of the small hairs projecting from the surface of the cuticle are sensory, responding to air puffs and other gentle stimuli. These sensory neurons function to allow the animal to sense vibrations, such as sound, in their environment. Equipment Setup Equipment should be setup as depicted (Figure 3). Three leads with hooks attached to the Bio Amp cable connected to the PowerLab unit. A USB cable is connected to the computer. If any of these components are not connected, ask the instructors for assistance.

Figure 3. Equipment Setup for PowerLab 26T Cockroach Dissection A cockroach will be anesthetized and leg removed by the instructor. Gently transfer the leg to a dissection pan with the forceps. Allow the leg to sit in the dish in its natural position.  Place three stainless steel pins into the isolated leg as shown in Figure 4a.  Once the pins are inserted, gently lift the leg up from the dish by partly withdrawing the pins from the wax base. This is important so that the spines on the underside of the leg are not stimulated by contact with the surface of the pan.

Figure 4a. Pin and Electrode Placement (not to scale) Figure 4b. Cockroach leg anatomy from: www.entomologa.ru/figures/22.htm  Attach the Micro-Hooks from the three recording leads to the pins as shown in Figure 4a. Secure these with modeling clay or adhesive tape if necessary, making sure you have access to the spines. Coil the Bio Amp Cable so it is out of the way of the experiment.  Launch LabChart and open the settings file “Sensory Nerve Settings” from the Experiments Tab in the Welcome Center. It will be located in the folder for this experiment.  Start recording. Make sure you can see activity in the Chart View. If not, check to make sure all electrodes are attached properly and the leg is not damaged. If you cannot see a response, the leg may be damaged and you will need to dissect a new leg.  Stop recording and proceed to Exercise 1. Exercise 1: Exploring Different Stimuli In this exercise, you will examine the effects of different stimuli applied to the hairs and spines of the cockroach leg. When you press on a leg spine, you are evoking neural activity. This evoked response will likely coexist with spontaneous activity. The spontaneous activity may come from the same cell as the evoked responses, different cells, or both. Following this

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