Effect of Nicotine and MSG on Neurons
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Effect of Nicotine and MSG on Neurons

We now move to the cricket cercal preparation to investigate neuropharmacology.


Overview

In this lab you will:
  1. Learn about how neuromodulating agents work and which ones are found in common household products
  2. Learn how to extract neuromodulating agents from common products.
  3. Test how these agents affect neuronal spiking patterns.
  4. Investigate how different classes of neuromodulators affect various populations of neurons in the cricket.

Objectives

Before doing this lab you should understand:

  • The basics of cricket anatomy
  • How neuromodulators affect a synapse
  • How the differential expression of receptors can alter neuronal responsiveness

After doing this lab you should be able to:

  • Explain neuropharmacology and neuromodulation
  • Describe how alcohol, glutamate, and nicotine affect neuronal firing rates
  • Design an experiment to test how a substance may affect the nervous system

Equipment

Crickets Ice water or Freezer Dissection Scissors Toothpick 3" Petri Dish Corrugated Cardboard (cut to fit into petri dish) Plastic Pipettes Distilled H20, NaCl, Baking Soda, MSG, Ethanol, Tobacco 1 cc (1 ml) Syringes

Educational Standards

This lesson plan is designed to meet the Next Generation Science Standards and the Common Core Standards for Science and Technology. Click the grade level below to see which standards apply.

    Next Generation Science Standards.
    Standard Definition
    Life Sciences
    MS-LS1-2 Develop and use a model to describe the function of a cell as a whole and ways parts of cells contribute to the function
    MS-LS1-3 Use argument supported by evidence for how the body is a system of interacting subsystems composed of groups of cells.
    MS-LS1-8 Gather and synthesize information that sensory receptors respond to stimuli by sending messages to the brain for immediate behavior or storage as memories.
    Common Core Standards for Science and Technology (Grades 6-8)
    Standard Definition
    Key Ideas and Details
    CCSS.ELA-Literacy.RST.6-8.1 Cite specific textual evidence to support analysis of science and technical texts.
    CCSS.ELA-Literacy.RST.6-8.2 Determine the central ideas or conclusions of a text; provide an accurate summary of the text distinct from prior knowledge or opinions.
    CCSS.ELA-Literacy.RST.6-8.3 Follow precisely a multistep procedure when carrying out experiments, taking measurements, or performing technical tasks.
    Craft and Structure
    CCSS.ELA-Literacy.RST.6-8.4 Determine the meaning of symbols, key terms, and other domain-specific words and phrases as they are used in a specific scientific or technical context relevant to grades 6-8
    CCSS.ELA-Literacy.RST.6-8.6 Analyze the author’s purpose in providing an explanation, describing a procedure, or discussing an experiment in a text.
    Integration of Knowledge and Ideas
    CCSS.ELA-Literacy.RST.6-8.7 Integrate quantitative or technical information expressed in words in a text with a version of that information expressed visually (e.g., in a flowchart, diagram, model, graph, or table).
    CCSS.ELA-Literacy.RST.6-8.9 Compare and contrast the information gained from experiments, simulations, video, or multimedia sources with that gained from reading a text on the same topic.
    Range of Reading and Level of Text Complexity
    CCSS.ELA-Literacy.RST.6-8.10 By the end of grade 8, read and comprehend science/technical texts in the grades 6-8 text complexity band independently and proficiently.

    Next Generation Science Standards
    Standard Definition
    Life Sciences
    HS-LS1-2 Develop and use a model to illustrate the hierarchical organization of interacting systems that provide specific functions within multicellular organisms
    Common Core Standards - Science and Technology
    High School (Grades 9-10)
    Standard Definition
    CCSS.ELA-Literacy.RST.9-10.4 Determine the meaning of symbols, key terms, and other domain-specific words and phrases as they are used in a specific scientific or technical context relevant to grades 9-10 texts and topics.
    CCSS.ELA-Literacy.RST.9-10.5 Analyze the structure of the relationships among concepts in a text, including relationships among key terms (e.g., force, friction, reaction force, energy).
    CCSS.ELA-Literacy.RST.9-10.6 Analyze the author's purpose in providing an explanation, describing a procedure, or discussing an experiment in a text, defining the question the author seeks to address.
    CCSS.ELA-Literacy.RST.9-10.7 Translate quantitative or technical information expressed in words in a text into visual form (e.g., a table or chart) and translate information expressed visually or mathematically (e.g., in an equation) into words.
    CCSS.ELA-Literacy.RST.9-10.9 Compare and contrast findings presented in a text to those from other sources (including their own experiments), noting when the findings support or contradict previous explanations or accounts.
    CCSS.ELA-Literacy.RST.9-10.10 By the end of grade 10, read and comprehend science/technical texts in the grades 9-10 text complexity band independently and proficiently.
    Common Core Standards - Science and Technology
    High School (Grades 11-12)
    Standard Definition
    CCSS.ELA-Literacy.RST.11-12.1 Cite specific textual evidence to support analysis of science and technical texts, attending to important distinctions the author makes and to any gaps or inconsistencies in the account.
    CCSS.ELA-Literacy.RST.11-12.2 Determine the central ideas or conclusions of a text; summarize complex concepts, processes, or information presented in a text by paraphrasing them in simpler but still accurate terms.
    CCSS.ELA-Literacy.RST.11-12.3 Follow precisely a complex multistep procedure when carrying out experiments, taking measurements, or performing technical tasks; analyze the specific results based on explanations in the text.
    CCSS.ELA-Literacy.RST.11-12.4 Determine the meaning of symbols, key terms, and other domain-specific words and phrases as they are used in a specific scientific or technical context relevant to grades 11-12 texts and topics.
    CCSS.ELA-Literacy.RST.11-12.6 Analyze the author's purpose in providing an explanation, describing a procedure, or discussing an experiment in a text, identifying important issues that remain unresolved.
    CCSS.ELA-Literacy.RST.11-12.7 Integrate and evaluate multiple sources of information presented in diverse formats and media (e.g., quantitative data, video, multimedia) in order to address a question or solve a problem.
    CCSS.ELA-Literacy.RST.11-12.8 Evaluate the hypotheses, data, analysis, and conclusions in a science or technical text, verifying the data when possible and corroborating or challenging conclusions with other sources of information.
    CCSS.ELA-Literacy.RST.11-12.9 Synthesize information from a range of sources (e.g., texts, experiments, simulations) into a coherent understanding of a process, phenomenon, or concept, resolving conflicting information when possible.
    CCSS.ELA-Literacy.RST.11-12.10 By the end of grade 12, read and comprehend science/technical texts in the grades 11-CCR text complexity band independently and proficiently.

Introduction Activity: An Illustrated Guide to Neurotransmitters, Agonists, and their Effects

In this section students will create their own illustrated guide to the neurotransmitters used in this experiment.

Nicotine

Nicotine

Nicotine comes from the tobacco plant. Tobacco evolved nicotine to prevent insects from eating its leaves. Nicotine is a powerful acetylcholine receptor agonist. An agonist is not a neurotransmitter, it is a chemical that amplifies the effect of other neurotransmitters by binding to synapses. Nicotine amplifies the effect of acetylcholine (ACh) binding to its receptors in synapses, causing a neuron to fire more (due to increased sodium ion influx).

1a. Use the diagram below to illustrate how nicotine affects the neuron

Make sure your diagram includes:

  • Acetylcholine Receptors
  • Acetocholine molecules
  • Nicotine molecues
  • Changes in neuron firing
Synapse Nicotine

1b. Based on the information on nicotine and how it affects neurons in humans, describe how you think it will affect neuron firing.

Nicotine is an agonist for acetylcholine, so it should amplify the effect of acetylcholine, causing neurons to fire more rapidly.

1c. Draw what you think the spiking activity will look like when nicotine is introduced. Show what the signal will look like before and after the introduction. Nicotine

Ethanol

Ethanol (alcohol) is a drug that also is an agonist to neurotransmission (like nicotine it binds to receptors and increases the activity of other neurotransmitters). Ethanol increases the effect of GABA binding channels. Unlike ACh receptors, stimulation of GABA channels often leads to inhibition of action potentials. In other words, GABA channel stimulation will slow down the firing of a neuron.

2a. Make sure your diagram includes:

  • Ethanol
  • GABA receptors
  • GABA molecules
  • Changes in neuron activity
Synapse Ethanol

2b. Based on the information you have on ethanol and how it affects neuron firing in humans, describe how you think it will affect neuron firing in an insect:

Ethanol is an agonist for GABA, which reduces neuron firing. Therefore it will probably reduce neuron firing in insects as well.

2c. Draw what you think the spiking activity will look like when ethanol is introduced. Draw what you think the signal will look like before and after introducing ethanol. Ethanol

(Note: Ethanol actually does not have a detectable effect on neural firing in insects, however, students should predict a decrease in neural activity given what they know about GABA)

Introduction: Teacher's Guide

    An Anuradha Rao Memorial Experiment:
    This experiment is dedicated to Anuradha Rao, a neuroscientist who studied pharmacology and enjoyed educational outreach. Her memorial fund generously allowed Backyard Brains to present experiments and prototypes at the 2010 Society for Neuroscience Conference in San Diego, CA.
  1. Begin by telling students that in this experiment they will test the effect of neuroactive compounds on the central nervous system using commonly found chemicals and cricket neurons.
  2. Access students' prior knowledge by asking them to list some examples of drugs that affect the brain (you can list their suggestions on the chalkboard).
  3. Explain that many dangerous drugs that affect the brain are found naturally in wild species like the batrachotoxins of poison dart frogs or the tetrodotoxins of fugu puffer fish, both of which block sodium channels. Others are drugs of abuse (like cocaine, which allows dopamine to stay in synapses longer than normal).
  4. Explain that the blood-brain barrier (BBB) prevents the passive diffusion of many substances into the brain. The BBB is composed of brain endothelial cells that are packed closely together around all capillaries, with tight junctions and a higher than normal electrical resistance. This barrier ensures brain function in vertebrates and insects by maintaining the ionic integrity inside the brain. It also prevents many toxic substances and bacteria from entering the cerebrospinal fluid(CSF), the fluid surrounding the brain and spinal cord. The BBB also protects the brain from neurotransmitters that circulate in the blood stream; for example noradrenaline is released into the blood stream during the fight or flight response, but the BBB keeps the brain from being flooded with this chemical.
  5. However, the brain also needs some substances to be able to pass the blood brain barrier. Ask students what things the brain might need to pass through the barrier in order to function. If students don't mention it, explain that oxygen has to be able to pass the blood brain barrier.
    Anuradha Rao
    The blood brain barrier (above) is composed of cells that line capillaries and have very tight junctions between cells and high electrical resistance that prevents chemicals and bacteria from diffusing into the brain.
    There are also specialized barrier cells actively transport some metabolic products such as glucose across the BBB with specific proteins. The practical consequence of the BBB on our experiments is that only certain drugs with specific characteristics can be used to influence neurons.
    Discussion Question:Ask students how the blood brain barrier might make it difficult for pharmaceutical companies to design drugs that treat brain function.
  6. Tell students that despite the difficulty in obtaining neuromodulating drugs we do have access to some neuroactive compounds that we can use on our insects: nicotine, monosodium glutamate, calcium chloride, and ethanol.
  7. However, before we learn about how each of the pharmacological agents affect the brain, we should first review how neurons and neurotransmitters work. As you have previously learned in your introduction to the SpikerBox experiment, neurons are electrochemical machines. Electrical impulses travel down the axons to encode information, and in-between every neuron is a synapse. When an action potential reaches the end of the axon (the synapse), the axon releases neurotransmittors that bind to the "post-synaptic" neuron and change the neuron's electrical or chemical properties.

    Laboratory Activities

    Part 1: Preparation of Neuroactive Solutions

    Saline Solution

      Create a saline solution by cominbing 1.5g of table salt(NaCl) and 1.25g of baking soda in 250mL of distilled water.

    Nicotine

      To create a nicotine solution, take a cigarette or small cigar, remove all the shredded tobacco leaves, and place them in a small container (a clear pill bottle, for example). Fill the container with saline solution, put the cap on, shake up the mixture, and allow it to sit for a couple days to extract the nicotine. Over time the solution should turn yellowish-brown. (teachers may want to pre-prepare the solution for students).

    Ethyl Alcohol

      Mix 196 mL of saline with 4 mL ethanol to make a 2% EtOh solution. Take 12.5 mL of the 2% EtOh solution, add 87.5 mL of saline to make a 0.25% EtOh solution.

    Part 2: Cricket Cercal Ganglion Preparation

    For this exercise, you will be using measurements from the cricket nervous system to determine the effect of a drug on spiking patterns and rates.

      Troubleshooting Your Cricket Recording:
      If students do not observe movement or spiking in response to stimulation, encourage them to be patient. It may take several minutes for the cricket's VNC to warms to an optimal recording temperature. In this time period, activity measured from the cereal ganglion may seem quite low. If, after 5 minutes of warming, you are still unable to observe any spikes, adjust the electrodes and repeat stimulation. If your students do not observe spikes after another few minutes of stimulation, you may need to use a new cricket.
    1. Have students place crickets in ice water for 5 minutes. When the cricket has stopped moving and has been anesthetized, place it on the lab bench.
    2. Students should place the cricket on the cork bed of the SpikerBox, ventral (belly) side up.
    3. Have students insert the SpikerBox recording electrode through the abdomen of the cricket into the cereal ganglion. The cereal ganglion is the most posterior ganglion in the ventral nerve cord (VNC). Try to place the electrode into the center of the ganglion. Stick the ground electrode into middle of the abodem. Now, using a small syringe (you can buy these at your local pharmacy), have students inject a few drops of saline solution into the abdomen of the cricket.
    4. Using a toothpick or by blowing, students should stimulate the cerci of the cricket. This should generate a strong spiking pattern.
    5. Turn on the SpikerBox and Audacity (or, if using a mobile device, our Android or iOS app). Connect the SpikerBox to the computer / mobile device and begin recording on the software.
    6. Once students establish consistent recordings, you are ready to begin observing the normal firing pattern of the cricket cereal ganglion.

    Part 3: Testing Cercal Response

    1. Students should stimulate the cerci of the cricket by blowing from either the right or left side of the cricket. Begin recording neural data as before.
    2. As one function of the cereal system is to differentiate directionality of a wind stimulus, you should be able to see differences in response to the blowing from one side versus the other. Students should record the time and duration of stimulus (blowing) and the side the stimulus was delivered (right or left). Have students repeat this twice for each side of the cricket.
    3. In Table 1, students should diagram the spiking pattern of each trace and give a brief description of any movements generated in response to the stimulation.
    Table 1: Control Cercal Responses
    Table 1. Control Cercal Responses

    Part 4: Testing Nicotine's Effect on Neuron Firing

    1. Beginning with the nicotine solution, students will now test the effect of pharmacological agents on the cricket cereal response. Using a plastic pipette, remove any excess saline from the recording electrode. Have students begin recording the neural data from the cricket.
    2. Using the small syringe students should inject a few drops of nicotine solution into the abdomen of the cricket.
    3. After a minute (to let the solution reach the cereal ganglion), have students stimulate the cerci as they did in Exercise 3. This may take several attempts. Have them record in Table 2 the time for two successful right and left cereal stimulations. Stop recording.
    4. Using the same procedure as Exercise 3, have students amplify and sketch the traces of the successful stimulations. They should include a brief description of any movements that appear to have been generated in response to stimulation.
    5. After finishing with the nicotine solution, wash out the recording electrode with saline.
    Table 2: Effects of Nicotine on the Cricket Cercal Response.
    Table 2: Effects of Nicotine on the Cricket Cercal Response.

    Part 5: Testing the effect of ethanol on neuron activity

    Have students repeat steps 1-5 with the ethanol solutions taking care to wash out residual solutions with saline in between trials. Record the results in the two tables below.

    Table 3: Effects of 0.25% Ethanol on the Cricket Cercal Response.
    Table 4: Effects of 0.25% Ethanol on the Cricket Cercal Response.
    Table 4: Effects of 2% Ethanol on the Cricket Cercal Response.
    Table 5: Effects of 2% Ethanol on the Cricket Cercal Response.

    Discussion Questions

    1. Based upon your results, which pharmacological agents led to an increase in cercal activity? Was this effect consistent on both sides of the cricket? Explain.
      Nicotine increases neural firing, you can see there is an increase in the size of the spikes recorded after nicotine is introduced .
    2. What is happening in the synapse when compounds that increase neural firing are introduced? What interactions are occurring between the compounds and receptors? Do all of the chemicals work in the same way to increase neural firing, or do they have different mechanisms of action?
      Nicotine is an agonist for acetylcholine, so it binds to acetylcholine receptors and increases the effect of the acetylcholine in the synapse. This causes the neuron to fire more frequently.
    3. Based upon your results, did any pharmacological agents led to a decrease in cercal activity? Did this match your predictions?
      I expected to see ethanol decrease the rate of firing, but we did not see any strong effects of ethanol.
    4. Do you think it is easier to detect decreases in neuron activity or increases? Why do you think this? How could this affect what you observed in your experiments?
      It might be more difficult to detect decreases in neuron firing, because if the neuron is firing at a very slow rate, you might only detect firing infrequently. Decreases in spiking would make it hard to observe an already infrequent event. Ethanol might decrease firing rate, but we could not detect it because the neuron wasn’t firing very much in the first place.
    5. Were there any differences in the movements observed between your control and experimental conditions? What might explain this?
    6. Extension:

      Imagine you are hired by a pharmaceutical company to test the effects of a new drug on the central nervous system. They are particularly interested in whether the drug is an agonist. Design an experiment using the spiker box to determine if and how the drug affects neuron activity. Is there a situation in which the drug cold be an agonist, but you might still fail to detect an effect?
      A drug is an agonist if it increases neural firing, by binding to the receptor for another neurotransmitter. Therefore if the new drug increased neural firing, it could be an agonist, and you could tell by the increase in spikes (like Nicotine in the lab). However, a compound that increases neural firing could also be a neurotransmitter itself, like Glutamate.