Engagement in Marine Neurobiology, Electrophysiology, and Microscopy Lesson Plan

Overview

Purpose: In most K-12 schools, neurobiology is not regularly taught, in part due to the complexity of the topic and the technology needed to have hands-on engagement. Oftentimes, students are only exposed to neurobiology if they take science courses in higher education, so this leaves a significant knowledge gap in people’s understanding of how the nervous system works. The primary goal of these activities is to expose young people to topics and technology in neurobiology that they might not otherwise have access to. The two lesson plans outlined below aim to introduce students to 1) the electrical properties of neurons through electrophysiology and 2) the structure of nervous tissue through microscopy and histology.

Audience: Middle school and older

Number of students: 16

Time: Day 1 Electrophysiology: <30 minutes to setup, ~1 hour to complete the activity. Day 2 Microscopy: <30 minutes to setup, ~1 hour to complete the activity.

Day 1 Electrophysiology

Brief description: This activity uses Neuron Spiker Boxes (cost-effective devices that amplify bioelectric signals) to measure the electrical activity of the nervous system in fiddler crab legs. Students will see and hear “action potentials” in response to touch sensation, and they will also introduce electricity into the system to induce motor movement.

Learning goals: Students will…

  • Explain the function of the nervous system
  • Observe the electrical properties of neurons and their role in detecting touch
  • Demonstrate the effect of electrical stimulation on motor control

Materials:

  • Backyard Brains Neuron Spiker Box kits, which includes…
    • Spiker box
    • 9V battery
    • Yellow electrodes
    • Green smartphone cable
    • Blue cable (not used)
    • Red stimulation cable
    • Apple Lightning to headphone jack adapter
  • Backyard Brains Spiker Recorder app
  • Smartphone or computer
  • Cooler and ice (preferably ice chips to increase surface area of contact with ice)
  • Plastic cups
  • Fiddler crabs
  • Toothpick or other poking tool
  • Scissors (optional)
  • Gloves (optional)

Procedure:

  • Obtaining crabs before the day of the activity
    • We collected fiddler crabs at low tide in the marsh on the intracoastal waterway in Wilmington, NC
    • You can also buy fiddler crabs from bait and tackle shops
    • Caring for fiddler crabs
      • Place wet sand or marsh mud in the bottom of a plastic container, making a gradual slope that covers about two-thirds of the bottom of the container. Fill the rest of the container bottom with saltwater.
        • Important note: make sure you have extra saltwater on hand to replenish the pool of water as it dries out. If the water level is not maintained, the fiddler crabs will die from dehydration.
      • Feed the fiddler crabs using pellet or flake fish food twice a week
  • Pre-activity questions
    • What is the nervous system?
    • How does the nervous system communicate?
    • What can we use to measure electricity of neurons/what is electrophysiology?
  • Neurobiology background information (see infographic in the Dropbox folder linked at the end of the document).
    • The nervous system is the command center of the body. It senses the environment, communicates information to different parts of the body, and controls our actions.
    • Neurons are one of the important cells that make up the nervous system. They are very special cells because they use a super speedy communication system to pass information from one part of the body to another. They use electricity!
    • Neurons generate electricity by selectively allowing positive and negatively charged elements (like sodium, potassium, and calcium) to move in and out of the neuron.
    • This movement of charge generate characteristic “spikes”, also known as action potentials, which we will see today when we perform electrophysiology.
    • Electrophysiology is a method that scientists use to detect and amplify small changes in electrical fields within a biological sample.
  • Preparing the specimen
    • Each group will get one fiddler crab
    • We will need to remove one leg from each of your fiddler crabs
      • Don’t worry! Like lots of invertebrates, they lose their legs often and can actually regrow them! They drop their legs as a defense mechanism to escape the clutches of predators.
    • Fill a plastic cup with ice and bury the fiddler crab in the ice
      • This will anaesthetize the crab so that it is dormant and numbed when we remove one of the legs
        • In simpler terms, we can say that the crabs are “asleep” for our mini surgery
    • After a couple of minutes, remove the crab from the ice and gently pull off one of its legs from the base of the carapace/shell
    • Place the leg on the corkboard of the Spiker Box with the part of the leg below the “knee” hanging off the edge of the cork
    • Find the electrodes in your kit. Insert the yellow plug into the port at the front of the box. Stick the two electrodes into the leg as specified below, pressing into the corkboard just enough to secure the specimen.
      • One electrode will go at the top of the “thigh” (i.e., femur) and the other electrode will go in at the bottom of the femur right before the “knee” joint
      • Electrodes measure changes in electrical charge due to the activity of communicating neurons
  • Recording electrical activity
    • Find the green cable in your kit. Plug the “Spikerbox”-labeled end into the green port on the left side of the box, and plug the “headphone”-labeled end into the headphone jack on your smartphone or computer.
      • If you are using a computer, you will have to change a few settings:
        • Set the band-pass filter cutoff frequencies to 1 for “Low” and 5000 for “High”. You need to press ENTER when you enter the number for it to save.
        • Check the 60 Hz box for the attenuate frequency (notch filter setting).
        • Make sure that there is a green color next to “Jack Mic Left” input so that the Spiker Box is connected to the computer.
      • You do not have to change any settings on the iPhone app.
    • Open the Spike Recorder app. Flip the “ON” switch on the front of the box.
    • Watch and hear neurons firing action potentials in real time!
    • Poke the leg with the toothpick to see how electrical activity changes when given a touch stimulus
      • Try different stimuli! Examples: harder taps, gentle pull or push of the leg, blowing air onto the leg, etc.
      • Neurons communicate differences in stimuli with different frequency/rates of spikes
    • Make sure you take at least one recording of the spontaneous activity and one recording when you give a stimulus
  • Stimulating the leg
    • Remove the green cable from the setup. Plug the red cable into your phone/computer and hook the red and black leads to the electrodes.
      • Hook the black lead to the electrode on the end of the leg and the red lead to the electrode near the “knee”.
    • Play music at a high volume. The leg will start to move to the beat as electrical current is passed from the phone to the leg.
      • I recommend playing a song with lots of bass (“Another One Bites the Dust” by Queen is a tried-and-true option)
      • Make sure the Spiker Box is OFF! If it’s on, it will make a very displeasing sound because of the interfering sounds of the music and spike noises.
      • Therefore, if we want to hear the music that the leg is “dancing” to, we will need two devices – one to deliver the current to the legs, and the other to actually play the music out loud à start the song at the same time on both devices
  • Recording electrical activity after stimulation
    • Remove the red stimulation cable and return to the original recording setup using the green cable
    • What happened to the number of action potentials after we stimulated the leg?
      • If we stimulated long enough, there should be fewer action potentials because the leg used up all of its energy to produce the muscle contractions. Firing action potentials uses a lot of energy!
    • Make sure you take at least one recording of the spontaneous activity and one recording when you give a stimulus
  • Data analysis
    • Open a recording file
      • Computer: Press the 3 parallel line (or “sandwich”) icon and select the file of interest
      • Smartphone: Select the “Recordings” tab in the bottom right of the screen. Choose the recording of interest and press “Play”.
    • Zoom in on one spike and identify the different phases of the action potential (depolarization/rising phase, repolarization/falling phase, and hyperpolarization/overshoot phase).
    • Perform spike counting following the data analysis sheet (see sheet in the Dropbox folder linked at the end of the document).
      • Do you expect more action potentials to occur spontaneously or when the leg is touched?
      • Do you expect more action potentials to occur before or after stimulation?
  • Post-activity questions
    • What is the nervous system?
    • How does the nervous system communicate? Why?
    • What can we use to measure electricity of neurons?
  • This activity was adapted from two Backyard Brains experiments

Day 2 Microscopy and Histology

Brief description: This activity uses microscopes to examine cellular structures of the nervous system. Students will identify the characteristic features of different nerve tissue types and compare the brain morphologies of various animals.

Learning goals: Students will…

  • Observe various structures of the nervous system with the help of diagrams
  • Compare the nervous systems of different animals
  • Understand the utility of using histological stains to better visualize tissues
  • Learn how to use microscopes and capture images of the slides

Materials:

  • Plastic cups
  • Hard-boiled eggs
  • White candle wax
  • Cotton swabs (Q tips)
  • Food coloring
  • Water
  • Vinegar
  • Spoon
  • Compound microscopes
  • Microscope cameras
  • Laptops
  • Amscope camera software
  • Histology slides
  • Printer
  • Photo paper

Procedure:

  • Pre-activity questions
    • How do microscopes work?
    • How many structures of the nervous system can we name?
    • What does histology mean?
  • Microscope theory
    • Microscopes use lenses, just like glasses, to focus light to our eyes. Microscope lenses magnify the image so that we can see small objects.
    • To understand how the nervous system works, we can look at structures under the microscope to infer their function.
  • Histology theory
    • Histology means using stains and dyes to reveal the structure of biological tissue.
    • Staining demo – dyeing eggs
      • Before the activity, light a white candle and dip a Q tip into the hot wax. Place dots of the hot wax onto the hard-boiled eggs.
      • Show the egg to students from a distance and point out that it is hard to see the white candle wax dots on the white egg. Mix food coloring and vinegar in a plastic cup of water. Place the hard-boiled egg with dots into the cup. Let sit.
      • Remove the eggs using the spoon. The egg will be stained with the color, which will make the white dots easier to see.
    • Cells and tissues in our bodies are transparent à we can use stains and dyes that will stick to certain things in order to more easily see small structures with a microscope
  • “Search and Find” activity (see activity and answer key PowerPoints in the Dropbox folder linked at the end of the document)
    • Each microscope station will have a set of slides for one type of neural tissue. Students will rotate between the stations to see the different types. Tissue types include salmon brain, nerve fibers, spinal cord, cerebellum, and cerebral cortex.
    • There will be diagrams of the tissue types to go along with the slides at each station. Students will use the diagrams to orient themselves and find the important structures/characteristics in the real tissue samples.
  • Comparative brain activity (see PowerPoint in the Dropbox folder linked at the end of the document)
    • Animals have differently structured brains depending on what actions they do the most often. Therefore, we can predict the behaviors/functions of animals based on their brain morphology.
      • For example, the rat brain has large olfactory bulbs because rodents rely on smell to forage for food. The dolphin brain lacks olfactory bulbs because they do not rely on smell much at all.
    • Look at diagrams of different animal brains. What is different about them? What is similar about them?
    • We can also roughly predict the intelligence of an animal based on the degree of folding on the surface of the brain.
      • Interestingly, dolphin brains have more folding than human brains. Does that mean that they are smarter than us? Intelligence can be hard to measure in other animals, so it is difficult to make the comparison.
  • Printing micrographs
    • At the end, students will be given the opportunity to take a picture of their favorite slide using the microscope camera.
      • Directions for how to take a picture on the microscope:
        • Screw the camera mounting tube onto the top of the microscope. Assemble the camera and then slide the lens into the tube. Plug the USB into the computer.
        • Open the Amscope software. Under “Camera List”, click on the camera model listed.
        • You should now see a live preview of the camera. You can play around with the different settings:
          • Exposure & Gain: Auto Exposure usually works pretty well, but if it is too bright or dim, uncheck auto and try manually changing the light intensity of the LED using the dial on the side of the scope, or play around with Exposure Time or Gain.
          • White Balance: If the color of the picture is tinted, move the white balance square to an area that is completely white and click the “White Balance” button.
          • Flip: I usually check both boxes so that the image on my computer screen looks the same as when I look through the oculars/eyepieces. You can also just rotate the camera itself until the image on the computer looks the same as through the oculars.
          • Lots of other settings to play around with if you want!
        • When you are happy with your image, click “Snap”, which creates a new, colored tab above the image. This is your picture!
        • To save the image, do File à Save As… and save it as a JPEG (or TIF if you want really high quality images)
        • To return to live mode, click on the first tab. Continue taking pictures!
    • As a take-home souvenir, we printed the student micrographs out on photo paper.
  • Post-activity questions
    • How many people now feel comfortable with how to use a microscope?
    • How many structures of the nervous system can we name?
    • Who could tell me what histology means? Why do we use stains?

Associated Materials

I have created a Dropbox folder with associated materials that anyone can view.

https://www.dropbox.com/sh/bv47nve978p4lnn/AAAok0pE4Q8YwQ2qtWEm8bVFa?dl=0

Below are the descriptions for each of the files.

“1. Neurobiology Infographic”: This is a graphic summary of some key background information on neurobiology.

“2. Electrophysiology Data Analysis Sheet”: Students can open the recording files from their electrophysiology experiments and perform data analysis. The recordings are analyzed by counting the number of spikes/action potentials that occur within a specified time interval (e.g., 5 seconds). The spikes are typically sorted by size because each different amplitude corresponds to a different nerve cell, so we can track the firing rates of specific cells. The amplitude of the spike typically indicates the size of the cell: tall spikes are associated with larger cells and short spikes are associated with smaller cells. In the example, I arbitrarily sort the spikes into two categories: “big” and “small”. It is impossible to analyze the firing rate of every single neuron in the leg, so I kept it simple by looking at the activity of big versus small cells. It is up to you to decide how to classify/sort the spikes, or you can just count all of the spikes and not worry about sorting at all.

Sometimes it can be difficult to pick out the spikes from the rest of the background noise, or you might also want help categorizing which spikes should be considered “big” or “small”. When you choose a recording, click either the “flask” symbol on the computer screen or select “Find Spikes” in the smartphone app. You can use the colored line to set a window, and spikes within that window will be labeled with the corresponding color in your playback recording! Add more colored windows to create more categories of spike amplitudes.

 “3. Nervous System Histology Search and Find Activity”: Students will use these PowerPoint slides to guide their observations of the different types of nervous tissue slides under the microscope.

“4. Nervous System Histology Search and Find Answer Key”: Educators can use these PowerPoint slides to help students find the answers to the questions.

“5. Comparative Brain Activity”: Students will examine images of brains from different animals to learn how the structure of brains can correlate to the adapted behaviors/functions of animals.