Nbio 302 Manual-2020

NEUSCI 302, University of Washington
Autumn Quarter, 2020

“Introduction to Systems and Behavioral Neurobiology”
Instructor: Dr. Marti Bosma

Course Associate: Dr. Michael Kennedy
The NEUSCI 302 Laboratory Exercises were developed and written in collaboration with:
Jim Canfield • Charlie Hass • Jordanna Henry • Michael Kennedy

Melissa Rosenberger • Allison Rudkin • Andrew Straw • Scott Votaw
Additional modifications by Dennis Dever and David Perkel
This manual was produced by:
Marti Bosma • David J. Perkel • Michael Kennedy

Jim Canfield • Charlie Hass • Joseph Sisneros
Not for duplication without authors’ permission.

University of Washington, Seattle, Washington 98195
NEUSCI 302 Autumn 2020
Table of Contents
Syllabus for NEUSCI 302, Autumn 2020 ..................................................................................... 3
Instructors and contact information: .......................................................................................................................... 3
Class schedule .......................................................................................................................................................................... 3
Course Objectives................................................................................................................................................................... 4
Course Structure ..................................................................................................................................................................... 4
Grades .......................................................................................................................................................................................... 5
Lecture and Exam Schedule .......................................................................................................... 6
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NEUSCI 302 Autumn 2020 Syllabus
Syllabus for NEUSCI 302, Autumn 2020

The course has a web site: https://canvas.uw.edu/courses/1400919
Updates and extra information will be published there.

Instructors and contact information:
Marti Bosma
• Bio: Ph.D. UCLA (1986). Professor, Biology Department, UW. My research laboratory
concentrates on understanding the mechanisms by which serotonin neurons regulate
spontaneous activity in the embryonic hindbrain and how the invertebrate Hydra is able
to perform unusual behaviors using only a nerve net.
• Office: LSB 261. Office hours, via Zoom, by appointment.
• Email: martibee@uw.edu
Course Associate :
Dr. Michael Kennedy
• Bio: Ph.D. Physiology, Arizona State University (1994); M.S. Biology, Central
Washington University (1985). Teaching Professor, Biology Department, UW.
• Phone: 206-221-5375
• Email: kennem@u.washington.edu
Teaching Interns (TIs) (extra office hours and contact information)
Madison Bravo (Maddie)(she/her) Monday 9:00-11:00 mabravo3@uw.edu

Madelyn Gray (she/her) Monday 2:30-4:30 mmgray@uw.edu
Holly Hake (she/her) Monday 3:20-5:20 hakehs@uw.edu
Russell Marx (they/them) Thursday 1:00-3:00 marxr@uw.edu
Class schedule
1. Lectures: MWThF 11:30AM–12:20PM via Zoom.
2. Lab sections: M, 1:30-4 :30; Tu, 9:30-12 :30 ; Tu,W, 1:30–4:30PM. HSB T-366
3. Office Hours: Wednesday 9:00 – 11:00 AM; Friday 1:00 – 3:00 PM, via Zoom
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Course Objectives
To provide a basic understanding of systems neurobiology. We will use both 'top down'
and 'bottom up' approaches to practical and theoretical aspects of nervous system function.
Topics covered include the structure and function of sensory systems, development of specific
neural systems, sensory-motor integration, theories of information processing, and the relation of
behavior to underlying neural mechanisms. The emphasis will be on depth, rather than breadth,
in key systems that illustrate both the problems for generating behavior from neural circuits and
their solutions.
Required Texts
• Principles of Neural Science (Kandel, Schwartz & Jessell, 5th edition;
ISBN-13 978-0071390118) (called 'KSJ' in notes)
Material may be used from other texts – unfortunately, the libraries are not available so we
cannot put these texts on reserve for the class. We may choose to make relevant portions of the
books available on the Canvas site. Availability of UW library resources are here:
https://www.lib.washington.edu/coronavirus
Course Structure
Lectures. All lectures will be virtual via Zoom. Although they will be recorded, the number of
courses being handled by Zoom on the web is very high, and it is likely that some recordings will
fail. For this reason, in addition to below, it is important that you attend Zoom lectures. The
course will consist of four 50-minute lectures per week (MWThF), and will cover basic
information pertaining to systems and behavioral neurobiology. Data strongly shows that
students who attend Zoom lectures synchronously do better on the assessments for the class.
Important note: Most of the material presented in lectures does not correspond closely to
specific readings in the textbooks. The primary source of information is the lectures themselves.
It is lecture and laboratory materials that will form the basis for examination questions. For this
reason it is very important that you attend all lectures. In the event that you are ill, or have other
university-sanctioned activities, please contact one of your instructors with appropriate
documentation, and we will make alternative arrangements to the materials you missed.
If you wish to request disability accommodations, please contact the instructors as early as
possible, no later than the first week of class.
Exams. There will be four exams, all in-class: Friday, October 9; Monday, October 26; Monday,
Nov 23; Friday, Dec 11. The day prior to the in-class exams may include a portion of a lecture,
but may also include a question and answer session regarding exam material. Exams will include
material covered in class, handouts, laboratories and supplementary reading. Exam questions will
be primarily short answer format. There will be no cumulative final exam during Finals week.
Laboratory exercises. Labs will be substantially different than in Neusci 301 due to COVID-19
in King County. Because the lab space is too small to allow all students to be present for each lab
section, and because some students will be unable to attend lecture/lab, labs will be a hybrid of
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in-person and remote. Each section will be broken into lab groups of 2 or 3 students by Dr.
Kennedy before the quarter begins, and will be further subdivided into Group A and Group B.
Groups with 3 persons will have one person attending remotely for the entire quarter. Members
of Group A and Group B will be in-person alternate weeks, as shown on the Schedule of
Laboratory Exercises, below. When Group A students are doing the lab exercises in person,
members of Group B (and remote students, if part of the group) will Zoom in as virtual lab
partners. Virtual lab partners will have remote control of the rig computer while their partner
does the physical lab manipulations. If there are two virtual lab partners you will need to
communicate and figure out controls. You may need to have a separate Zoom meeting for each
lab, making sure to record the lab. Virtual and in-person roles will be reversed in alternate
weeks. Each lab exercise will be repeated for 2 weeks so each in-person student gets the chance
to physically “drive” the experiments. (The exception to this is the electric fish labs, where each
fish preparation will be used for one week only, see schedule.)
Religious accommodation. Washington state law requires that UW develop a policy for
accommodation of student absences or significant hardship due to reasons of faith or conscience,
or for organized religious activities. The UW’s policy, including more information about how to
request an accommodation, is available at Religious Accommodations Policy
(https://registrar.washington.edu/staffandfaculty/religious-accommodations-policy/).
Accommodations must be requested within the first two weeks of this course using the Religious
Accommodations Request form (https://registrar.washington.edu/students/religious-
accommodations-request/).
Grades
Points will be distributed as follows, and grades will be assigned on the 4.0 scale:
In-class exam I: 50 Points
In-class exam II: 60 Points
In-class exam III: 70 Points
In-class exam IV: 60 Points
Lab write-ups 100 Points

Lab performance 30 Points
Oral Presentation: 30 Points
Total: 400 Points
We reserve the right to adjust this point distribution.
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NEUSCI 302 Autumn 2020 Overview of Labs
Lecture and Exam Schedule

Note: The schedule is designed to link concepts and material covered in lab work to theoretical
material covered in lectures. Some sections may take more or less time than indicated, but we
will make every attempt to keep things 'in synch'
Weeks 1-2 Introduction/Auditory/Sound localization (September 30 – October 9)

Transition from neurons to circuits. Simple neural circuits. Basic properties of sensory
circuits - topography, modality, coding, receptive fields, lateral inhibition.
Auditory system. Information content of sound, organization of the cochlea, sensory
neurons as filters. Structure and physiology of the auditory system, neural coding in the
auditory system, bat echolocation and neural basis of echolocation.
Friday, October 9 In-class Exam 1
Week 3 Visual system (October 12 – October 16)

The optics and evolution of different eyes. Overview of visual perception of form, color
and motion. Visual illusions. Phototransduction and lateral inhibition. Structure of the
vertebrate retina. Retinal pre-processing. Central pathways. Maps in primary visual
cortex. Visual processing of form and motion.
Week 4 Somatosensory system (October 19 - October 23)

Structure and physiology of the somatosensory system, discussion of sensory space,
comparative somatosensory systems.
Monday, October 26 In-class Exam 2
Weeks 5-7 Electrosensory and electromotor systems (October 26 – ~November 13)

Introduction to electroreception: elasmobranch (shark, skates and rays) and weakly
electric fish.
Biology and behavior of weakly electric fish. Electric communication. Behavior and
neural basis of the jamming avoidance response (JAR). Three-dimensional analysis of
objects by electric fish. Neuromodulation of electrosensory and auditory systems.
Wednesday, November 11 No class/lab - Veteran’s Day
Week 8 Neuromodulation. (~November 16 – November 20)

Central pattern generators and control of behavior.
Effects of hormones on behavior and physiology. Song control system of birds as a
model system to study brains and behavior. Aplysia as a model for sensory modulation.
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Monday, November 23 In-class Exam 3
Thursday, Friday, November 26,27 No classes - Thanksgiving Holiday
Weeks 9-10 Development of the spinal cord and cortex (November 23 – December 4)
Development of the cortex/layering. Development of the auditory system. Hair cell
maturation and tonotopic mapping. Modification of maps by sensory input/loss.
Week 11 Development of the visual system (December 7 – December 11)

Development of the eye and pathfinding by retinal ganglion cell axons. Spontaneous
activity in developing retina and the correspondence to layers within the LGN. Activity-
dependent mechanisms of synaptic stabilization and competition.
Friday, December 11 In-class Exam 4
Laboratory Exercises
As in NEUSCI 301, our aim this quarter is to focus on depth, rather than breadth. We will have
increased expectations from NEUSCI 302. For example, you will wire your own equipment and
be expected to explain how signals travel between the preparation and the computer.
Additionally, you will write an abstract for each lab report. We have selected a small number of
experimental preparations that can be used to illustrate a variety of basic principles common to
many neural circuits. All involve sensory or motor systems. You will perform extracellular
recordings to characterize these systems and will learn quantitative techniques for analyzing the
response tuning of graded and spiking neurons in both the time and frequency domains.
Schedule of laboratory activities
Days Exercise Points

Re-familiarize with laboratory equipment –
Sept 30, Oct 5, 6 -
Group A 1:00-2:00; Group B 2:30-3:30
Group A
Oct 7, 12, 13 Lab 1. Mechanoreceptors in the cockroach leg: principles of
Group B neural coding as applied to processing of auditory 25
Oct 14, 19, 20 information.
Oct. 9, Exam 1
Group A Lab 2. Photoreceptor physiology: recording the
Oct 21, 26, 27 electroretinogram from fly photoreceptors and temporal 25
Group B coding in light and dark.
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Oct 28, Nov 2, 3

Oct 26, Exam 2
Group A Lab 3 – Group A: Electromotor behavior and electro-
Nov 4, 9, 10 communication in Gnathonemus petersii. (Veterans Day 10
observed November 11)
Group B Lab 3 – Group B: Sensory-motor integration: the jamming
Nov 16-18 avoidance response in Eigenmannia sp. or Apteronotus 15
albifrons
Group A
Nov 23-25
Lab 4: Electromyographs of cockroach flight muscles:
Group B 25
behavioral evidence for central pattern generators.
Nov 30, Dec 1,2
Nov 23, Exam 3
Dec 7-9
Oral presentations of lab work, via Zoom. 30
Dec 11, Exam 4
throughout Lab Performance points. 30
Lab reports and grading
We will assess the laboratory work in 3 ways, designed to integrate practical and theoretical
aspects of scientific reasoning:
1. In-lab performance will be assessed on the basis of lab reports for each lab. For most
labs, we ask you to provide a detailed and formal report in a similar format like a
scientific paper (i.e. with Abstract, Introduction, Methods, Results, Discussion and
Literature cited). In these, you will outline and discuss the questions investigated,
describe your experimental methods, and present & describe the results of your own
experiments (in the form of figures and graphs made from data that you collect in class).
Remember to include statistics to the level discussed in Neuroscience 301.
2. Up to 30 performance points can be EARNED by how an individual performs in the
laboratory. Laboratory performance points are separate from exam scores, laboratory
reports and oral presentation points. How a student performs in laboratory will be key to
earning points. Many components go into laboratory performance points. Some obvious
criteria are being well-prepared and coming to class on time. This by itself does not
guarantee full points. Being focused on the laboratory at hand, working independently
and cooperatively with ones' partner, finishing laboratories in a timely manner, a student's
attitude in laboratory, and much more go into earning ones' laboratory performance
points.
3. An oral presentation [joint by you and your lab partner(s)] in the last week of the quarter
allows you to present results and ideas derived from one of your lab exercises. Possible
points: 30.
Please note: In grading both written lab reports and oral presentations, we will place more
emphasis on your depth of understanding of concepts and effort made to place results in the
context of material from lectures and additional reading than on the results themselves. You are
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strongly encouraged to discuss your results and ideas with your lab partners, TAs, and other
colleagues.
As in NEUSCI 301, however, your written work should be exclusively yours.

Unfortunately, it is still necessary to point out that using others’ words as your own is not
consistent with academic integrity. For the University of Washington policy, please see:
http://depts.washington.edu/grading/issue1/honesty.htm. Although we very much hope that no
problems arise, we reserve the right to deal with any such issues, by sending a complaint to
the relevant disciplinary committee.
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NEUSCI 302 Autumn 2020 Lab 1: Cockroach mechanosensation
Lab 1: A quantitative analysis of neural coding using the cockroach

mechanosensory system
Aims
• Become comfortable setting up and using equipment that makes physiological recordings
of neural activity. Also, learn how to design experiments using such equipment.
• Understand the concept of neural coding and how to experimentally explore information
coding in a sensory system.
• Learn to use quantitative experimental approaches and analytical techniques to probe the
function of the nervous system.
New Tools
• Assorted pieces of electrophysiology equipment and related wires/connectors
• A quantifiable stimulus–Yamaha speaker with attached stimulator probe
• Peri-Stimulus Time Histogram (PSTH)
• Tuning Curve
Introduction
The cockroach leg is covered with sensory spines. A single mechanosensory neuron at the base
of each spine responds to mechanical deflection. Mechanically gated channels lead to
depolarization of the sensory terminal and this induces action potentials in its afferent axon. We
can record these spikes by placing electrodes at opposite ends of the leg and measuring the
difference in extracellular voltage between the electrodes.
A major problem in studying sensory systems is determining exactly what information is carried
in the neural signal. This brings us to a central concept in sensory physiology–the idea of neural
coding. The nervous system is used to carry information about something in the world and this
information is "coded" as a neural signal. The goal of the neurophysiologist is to decode these
neural signals and to understand how they are generated, processed through the nervous system,
and used to generate behavior.
You can learn a lot about a sensory system by qualitatively exploring the range of effective
stimuli. In NEUSCI 301, you observed that most of the cockroach spine sensory afferents are
quiet when the spines are relaxed. When you displace the spine, they emit strong bursts of spikes
and generally respond more strongly (with a higher rate of spikes per unit time) when the spine is
displaced in one direction vs. the other. Note: In this lab we will equate "strength of response"
with the rate of spike production, but as you will learn later in the course, this does not hold for
all systems.
This qualitative approach leaves many questions unanswered. In this lab, you will take care to
exercise fine control over the experimental conditions to gain a more detailed and thorough
understanding of mechanosensory coding. You will study the response of the system to periodic
(back and forth) mechanical stimulation and see that the system acts as a "filter," relaying certain
frequencies well while attenuating others. You will also measure the reliability of the system as a
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signal detector. That is, you will measure the variability of the response to repeated presentations
of identical stimuli.
The basic stimulus arrangement that we will use today allows us to drive the spine back and forth
at a variety of frequencies. We will use a mechanical probe connected to a loudspeaker
diaphragm. Usually, when the diaphragm of a loudspeaker oscillates back and forth, it produces
sound pressure waves, which we can detect via auditory mechanoreceptors of the cochlea in our
inner ear. In this lab we will connect the vibrating diaphragm to the cockroach leg spine with a
probe, thus, imitating the mechanical linkage between the oval window and the tympanic
membrane of our ear. Since the hair cells of the cochlea use a similar mechanotransduction
mechanism to the cockroach mechanoreceptors, we are working with a preparation that is strictly
analogous to that of the mammalian auditory system. We will use it to investigate the ways in
which this system encodes information about strength (volume or amplitude), frequency (pitch)
and timing (phase) of an auditory stimulus. We could drive the loudspeaker with the computer
using any arbitrary waveform, but we will be using sinusoidal waves (pure tones) today.
Key Concepts
Threshold
Neurons have a threshold for response and sensory neurons are no exception. Stimuli with
amplitudes below this threshold may elicit no response at all. The threshold may be directly
related to the generator potential required for action potential initiation, but not always.
Sometimes the nature of stimulus transduction may limit the response. For example, there may
be a mechanical limit to how small a deflection of a sensory spine is required for channels to
open.
Dynamic Range
Once threshold is exceeded, there is a limit to the range of stimuli that can be encoded. Once the
response reaches a maximum (again often set by the underlying biophysical machinery that
generates the response), further increases in stimulus strength produce no further increase in
response. The dynamic range of a response is comprised of the stimulus values from threshold to
the stimulus value that produces a maximum response.
Response Tuning
Different stimuli may evoke threshold or supra-threshold responses at different absolute levels.
For example, if we record from a photoreceptor that is most sensitive to green light (i.e., it is
"tuned" to green light), but stimulate it with blue light, we might need to make that light very
intense to get a response. On the other hand, if we use green light, we might require only a very
dim intensity to get a response. Measuring the threshold for a given stimulus provides a very
reliable way to plot the sensitivity of the system against a parameter (e.g., wavelength of light or
frequency of sound) to examine response tuning.
Simple Tuning Curves
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A simpler experiment than estimating threshold for each condition is to simply plot response
against the investigated parameter, while using a constant strength of stimulation. Using the
photoreceptor example again, you could choose a constant brightness of light at different
wavelengths and measure the different amplitudes of response as a function of wavelength. But
what level do you choose? If you choose too high a level, you may saturate the response at many
wavelengths thus giving the impression that the system is more broadly tuned than it is (i.e.,
responses may be near maximal even away from the optimum wavelength). On the other hand, if
you choose too low a level, you may find that you can only measure responses at close to the
optimal wavelength.
Spiking Responses Are Noisy

By this we mean that each time an identical stimulus is applied the response of the neuron is
slightly different. The features of the response that are consistent from trial to trial give us a good
idea of what the neuron is encoding.
Noise Influences Experimental Design

We can design experiments to reduce the effects of this noise. Sampling spikes in larger "bins" or
over repeated trials averages out the effect of noise on our estimate of the response.
Thinking in Frequencies
Engineers have developed many powerful tools to dissect the processes governing the behavior
of a physical system. Many of these tools can be applied to studying sensory systems. Among the
most powerful approaches is analyzing the frequency response of a system. We present the
system with a periodic stimulus and measure the properties of the output. For example, a typical
input stimulus might be a sinusoid with a particular frequency, amplitude, and phase. In this
case, we would expect a sinusoidal output from the system and would measure its frequency,
amplitude, and phase relative to the stimulus. In general, we present the system with different
stimuli and measure how the output changes. It is one of the best ways of characterizing the
properties of a neural filter.
What factors might determine the filter properties? In NEUSCI 301, you looked in some detail at
the RC properties of cell membranes. If you inject a current which modulates rapidly (i.e., high
frequency) into a cell, the slow charging of membrane capacitance will smooth most of the
bumps out to produce a Vm which varies little. If you modulate the current slowly, the membrane
capacitance has a chance to charge and the modulations are able to produce a modulation in
response from the cell. This kind of RC circuit is an example of a low-pass filter, because low
frequencies are "passed" and high frequencies are not. Other circuits can be high pass filters
(only transmitting high frequencies) or even band-pass filters (transmit frequencies in a narrow
range). The RC properties of sensory neurons are one way in which systems like
mechanoreceptors are frequency tuned (band-pass filters).
Why bother measuring the frequency response? Most real biological signals would not be simple
sinusoidal waveforms like those that we are using today. Natural signals would present the
sensory system with complex modulations. Only a subset of these complex waveforms may
represent information relevant to the behavior of the animal. Measuring the frequency response
of a sensory system thus provides us with important cues not only about the limitations of the
system, but also its likely role in the real world.
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Equipment Setup
Wiring
Your setup has not been fully wired, so your first task is to connect the key components. We
want to encourage you to think of yourselves as the investigators and approach this equipment as
your tools for answering the questions you’re interested in.
Look at the equipment that we have put out for you. You will see a speaker assembly that you
will use to move the spines back and forth. A probe at the center of the loudspeaker cone will
allow you to contact the spine. You’ll also see a cork with pins in it similar to the one you used
last quarter for recording from the cockroach leg nerve. This time it’s been attached by an
aluminum platform to the micromanipulator so you can precisely position the spine against the
probe on the loudspeaker.
Recording Wiring
Think about where the nerve signals are coming from in the recording set up and where they
need to go. You have a special wire with a pair of female connectors at the end that connect onto
male connectors that are soldered to the pins in the cork (the recording electrodes). This is where
the neural signal from the prep begins.
• Connect these leads to the pins in the cork in order to measure extracellular spikes in
the leg and note which channel of the extracellular amplifier it connects to (this may
vary across setups). Consider why it’s necessary to connect both leads. That is, why
the active one alone won’t do and what would happen if the leads were touching after
you connected them. Now, think about where the signal goes from here. After the
signal is amplified, you will want it to be cleaned of electrical "noise."
• Use a BNC cable to connect the output of the amplifier to the input of the Humbug.
Once the signal is clean, you will want to do two things with it: 1) see it on the
computer and 2) hear it on the audio monitor.
• Split the output of the Humbug by placing a BNC "T" connector on the Humbug
output.
• Connect a BNC cable to one end of the "T" connector and connect the other end of
this cable to input channel 1 on the PowerLab box. The PowerLab box turns the
analog signal you have been working with thus far into a digital signal that the
computer can understand. There is a cable connecting the PowerLab to the computer.
You will never need to touch the connection between the PowerLab and the
computer, but you should know that this connection exists.
• Connect the special audio cable to the other end of the "T" connector and plug this
wire into the Grass Audio Monitor if this has not already been done.
• Finally, set the gain on the amplifier to 1000X. Set the low cut-off to 10Hz. Set the
high cut-off to 20000Hz. Turn the notch filter off.
Stimulus Wiring
You will use the computer to drive the loudspeaker that you are using as a stimulus. So, there
needs to be a connection between the computer and the stimulus speaker. You also want to see
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the stimulus waveform on the computer. No, this doesn't happen automatically–you need to
make an additional connection that sends the stimulus back into the Powerlab.
• Place a BNC "T" connector on output channel 1 of the PowerLab.
• Connect the special phono-BNC cable to one end of the "T" connector and plug the cable
into the speaker input on the stand-alone speaker.
• Connect a BNC cable to the other end of the "T" connector and send this signal to input
channel 2 of the PowerLab. This will allow you to see the same waveform that you send
to the speaker.
• Set the speaker volume low (about 1/5 turn above the minimum setting) and check that
the tone control is turned all the way down (anti-clockwise). This maximizes the low-
frequency response of the speaker. Now you can use the PowerLab to dictate what
amplitude and frequency of vibration you will use to drive the spine.
Computer Setup
You will be using Labchart to record the signal from the cockroach mechanoreceptors and there
is some software setup required before you start.
• Open LabChart and go to the "Setup" menu and modify the "Channel Settings." Set the
number of channels to 2. Make sure that both channels are on (checked). The sample rate
is important for getting an accurate representation of your spikes. Set the sampling rate to
10K samples/second. Set the input amplifier voltage range to 50 mV for channel 1 and to
500mV for channel 2. You may need to adjust these settings later, but this should give
you a good start.
• Now, go to the "Setup" menu and open the "Stimulator."
• Set the stimulator to deliver sinusoidal pulses ("Mode" = Sine). Check "Continuous." Set
the Range to 200Hz and set the Frequency slider to 20Hz. Set the Stimulator Range to 1
volt.
You have now set up LabChart to produce a sinusoidal waveform on the output channel just as if
it was a function generator. You can use the Amplitude control to set the strength of the stimulus.
Set this to somewhere around 0.1 volts for now. Close the Stimulator window.
• Set the duration of recording to 1 sec. This means that when you press the Start button in
the LabChart window, it will sample the data at the same time as it starts output of the
sinusoidal stimulus. It will stop after 1 second of data collection.
Animal Preparation
• Get a cockroach leg and impale it on the pins as you did last quarter. Handle the leg
gently to minimize damage to the delicate sensory spines.
• Turn the extracellular amplifier power on. Turn from Stim to Record, and then turn on
the audio monitor (not too loud). If everything is okay, you should hear neural activity
that modulates when you stimulate spines or blow on the leg. Try evoking spikes by
touching a spine with a toothpick (gently!). Find a spine that gives you a good,
consistent response of a distinct amplitude.
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• Before you attach the spine to the probe, you need to choose a reasonable stimulus
amplitude. Start LabChart. Begin with small stimulus amplitudes (~0.1 V), so that you
don’t blow the speaker. You can select "Stimulator Panel" from the "Setup" menu to
display the current output amplitude and adjust it conveniently between trials. Administer
a stimulus and watch the movement of the speaker probe under the microscope and figure
out what voltage ranges will work to get the desired range of displacements relative to the
size of the spine. Set an initial amplitude to try for the experiment.
• Attach your chosen sensory spine to the probe: Put a little ball of dental wax on the tip of
the speaker probe, this will allow you to embed the spine in the wax, so that the spine
moves in perfect conjunction with the speaker. Move the speaker so that the probe tip is
close to the leg and use the micromanipulator controls to position the leg so that the probe
touches a suitable spine at the best angle for moving it through its "normal" range of
motion. Move the spine away from the probe using the X-axis motor. Now, put a bit of
dental wax at the end of the speaker probe. Move the spine back so that it embeds into the
wax. Now you are ready to experiment.
Data Collection
Measuring the Frequency Response of a Spine

• Begin with a low frequency stimulus (20 Hz). Start LabChart. If there is no response
from the spine, first check to be sure the spine is still connected. If it is, perhaps you
should increase the amplitude of the oscillations (by increasing the stimulus amplitude in
the "Stimulator Panel"). Adjust the display settings so that you can see your spikes
clearly. You need to get a nice clean train of spikes whose total size and peak amplitude
are very similar. You also want the spike size to far exceed the baseline noise level. This
is important for later analysis–you want to make sure that you can isolate one unit from
all other signals in the trace.
Question: What are the characteristics of the response? Are spikes generated
randomly or rhythmically?
• You get a fair sense of the response by looking at the raw data that LabChart obtains, but
we can quantify the response by constructing a Peri-Stimulus Time Histogram (PSTH) of
the spikes. Before we try to do this, run your stimulus 10 times. Notice that each trial is
separated by a block marker in the LabChart window. Now you have recorded the
response to the same stimulus presented 10 times. We can use LabChart’s histogram
extension to view the resulting spike trains and see how they differ from each other.
• Use the mouse to select a number of spikes that you just recorded. Set up a new “Spike
train” by choosing Spike Histrogram -> Spike Train Setup. Click on “New”. With the
“General” radio button pressed, give the new train a name (for example, “20 Hz”). Next
press the “Data Source” radio button and choose “Selection” so that only the selected
data will be analyzed. Next, press the “Detector” radio button and set an appropriate
noise threshold so that noise will be ignored. Click “OK”.
• Open the "Spike Discriminator" (Spike Histogram > Discriminator). Hopefully you have
a distinct "cluster" of points corresponding to spikes from a single unit (neuron), as well
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as many other "events" of random amplitude and duration. If you can’t pick out a distinct
cluster, you should start over with a new spine (consult your TA). Now, slide the
horizontal and vertical lines (some may initially be hidden by the axes) that control the
discriminator width and amplitude (height) parameters to isolate your single unit from the
rest of the events present in the recording. If there is more than one cluster, choose the
spikes with the largest amplitude. Once you set these parameters, they will apply to any
further selections you make in the LabChart window. Close the box. Remember to check
the "Discriminator" occasionally as the recording progresses, so that you know the
"Discriminator" settings remain appropriate.
• Next, drag across the 10 "blocks" of data that you have obtained and then go into (Spike
Histogram > Peristimulus Time). Using the “Page” icon at the bottom of the window, set
Sweep Source to "Block Start." and Sweeps Per Page to 1 for now. You can now view
the blocks one at a time. You’ll see a bar plot with a bunch of tic marks above it (if you
don’t see the tic marks, hit the little arrow at the top right of the plot). The tic marks are a
raster plot, which shows when each spike occurred in time during that trial. The bar plot
is a histogram, the PSTH, that divides the trial into time bins and gives the response in
each bin, measured in spikes/sec. You can switch between the histograms for each trial
by using the Page control at the bottom of the window.
Question: Does anything show up clearly from trial to trial as being similar?
• If you go back to (Spike Histogram > Peristimulus Time) and (from the Page icon at the
bottom of the PSTH window) change Blocks Per Page to 10, you can analyze all 10
blocks of data at once. Now there are 10 raster plots, one for each trial. It is now much
easier to see the similarities and differences between trials. The histogram now is the sum
of the number of spikes in each bin. You can change the bin size for the histogram—
plotting the response at higher or lower time resolution—by using the two buttons or the
pull-down menu, located at the lower left of the window (in the example below, the bin
size is 5ms). Notice that because you set Blocks Per Page to 10, the histogram averages
all 10 trials together (all 10 trials now constitute 1 page). You should see that the spikes
occur at a similar time in each trial.
Question: Why? How many spike "burst" are there in each "train" (per trial) and how does this
relate to the frequency of stimulation?
16
Figure 2. Raster display and PSTH of neural response to periodic stimulation.
The advantage of a PSTH is that it removes some of the variability of the response by averaging
spikes into bins thus allowing an estimate of spike rate as a function of time. The disadvantage,
however, is the loss of information about the time course of the response. Notice how if you
increase the bin size, the plot becomes smoother, but increasing it too much obscures the
temporal fluctuations in the response.
Choose a bin size that is a compromise between the two extremes, and print your results. Note:
set the color scheme to "black and white" rather than "grayscale." Make sure to write a lot about
the stimulus parameters—everything that you feel might be relevant—on the PSTH, for later
comparison. Print one or two "typical" histograms for single trials, so that you can use them to
illustrate the effect of repeating the experiment in your write-up.
You can get a single number to quantify your result for each experiment by setting the bin size
for the histogram to be the same as the duration of the experiment (1 second). Note that in any
histogram, when you point the mouse at the border of the data point plotted on the histogram, it
displays the firing rate (in spikes per second) for that "bin." You should keep notes about this
value for each experiment.
• Now you have a record of the response to your stimulus. But how can you convince
someone that the response and the stimulus are related? You need to do a control
experiment to show that the neuron doesn't behave that way spontaneously. This sounds
obvious, but you’d be surprised how easy it is to fool yourselves in these kinds of
experiments if you haven’t done the proper controls. To do this, set the stimulus
amplitude to 0 and record 10 more 1-second records with no amplitude on the stimulus
and construct a PSTH. Print this for your records.
Your control experiment serves a second function of giving you a measure of the
"baseline" or "resting" firing rate of the neuron. Most likely this will be zero for these
cells, but many neurons show spontaneous activity. In this case, it is common to express
the response of the neuron as the change in firing rate (response minus spontaneous rate)
rather than the absolute number.
17
• Use the stimulator panel to increase the stimulus frequency to 50Hz.

Question: What is the effect on the firing rate? Do spikes still occur in regular bursts at the
stimulus frequency?
Designing Your Own Experiments
The Effect of Varying Stimulus Frequency on Firing Rate

Design a simple experiment to quantify the effect of varying the stimulus frequency on the
neuron’s firing rate. Don’t go to frequencies much lower than 20Hz, because our loudspeakers
are not capable of reproducing these faithfully. Depending on the amount of time available, you
should test the effect of varying stimulus frequencies in the range of 20-200 Hz (e.g., at 10
different frequencies). Note that the response (in spikes per second) is not the same at all
stimulus frequencies. Plot a graph of stimulus frequency against neuronal response (spikes per
second). This is an example of a frequency tuning curve. You may find a distinct "best
frequency" (optimum frequency), which the cell responds to with the greatest number of spikes.
In your lab report, briefly describe your experiment, then report and discuss your results.
The Effect of Varying Stimulus Amplitude on Firing Rate

Using the "best" frequency for the spine, design another experiment to test the effect of varying
the stimulus amplitude in appropriate sized steps. You will need to keep an eye on time and
design your experiment so that you obtain as many amplitudes as possible. Make sure to perform
several measurements for each stimulus frequency that you choose (5 is probably enough, but
more is better!). Plot another graph, this time showing the effect of amplitude. In your lab report,
briefly describe your experiment and report and discuss your results. Does the response
saturate? What amplitude is the absolute threshold at this frequency?
Other Possible Experiments

If you have time, you might want to try a different stimulus frequency and again look for the
threshold in the response. Is it the same as at the "best" frequency? Alternatively, you might want
to repeat one or more of the experiments with a different spine. Do all spines behave the same
way? The choice and experimental design is up to you. In your lab report, briefly describe the
experiment and report and discuss your results.
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Assignment
DUE: Normal lab report write-up (see instructions on page 8) . Due at the beginning of class on
Friday Oct 23, via Canvas, to avoid conflict with the exam on Oct 26.
Be sure to include the data and figures requested in the directions, and refer to the questions
below.
The following points should be addressed somewhere in your write-up:

• Show the raw data trace of one of your responses as well as a PSTH of the response.
Describe the features of the response. Is there any relationship between the timing of the
spikes and the cycling of the stimulus? When do the spikes appear on successive cycles?
At which phase of the cycle do they appear (i.e., do they always occur at the beginning of
the cycle (phase = 0) or at the peak of the cycle (phase = p/2 = 90°)? Is this phase
relationship reliable from one cycle to the next? How many spikes appear in each cycle?
Is there much variability?
• Graph the frequency response that you derived from one of the experiments you
designed. You want to show the neurons firing rate (its response in spikes per second) as
a function of the stimulus frequency. What might be responsible for the response
characteristics you see (hint: think about membrane properties of single neurons)? What
other factors influence the frequency response that you measure (hint: think about the
frequency response properties of the equipment)?
• Report briefly on any other experiments you conducted.
Project Ideas
• Deliver a noisy stimulus and carry out a reverse correlation analysis. For each spike that
occurred, what was the average stimulus in the 100 ms leading up to that spike?
• Carry out a detailed analysis of amplitude and frequency tuning for a single spine. Can
you fit the two–dimensional data with a simple function that could parameterize the
response?
19
NEUSCI 302 Autumn 2020 Lab 2: Fly vision
Lab 2: Photoreceptor Physiology: Recording the ERG from fly

photoreceptors and temporal coding in light and dark.
Background reading
1. Delcomyn chapter 11, pages 280-282: Please read this section before coming to lab.
Additional reading
• Hornstein EP, O'Carroll DC, Anderson JC, Laughlin SB (2000) Sexual dimorphism
matches photoreceptor performance to behavioral requirements. Proc R Soc Lond B Biol
Sci 267:2111-2117.
• Laughlin SB, Weckstrom M (1993) Fast and slow photoreceptors—a comparative study
of the functional diversity of coding and conductances in the Diptera. J Comp. Physiol. A
172:593-609.
• Laughlin SB (1996) Matched filtering by a photoreceptor membrane. Vision Research
36:1529-1541.
• Tatler B, O'Carroll DC, Laughlin SB (2000) Temperature and temporal resolving power
of fly photoreceptors. J. Comp. Physiol. A 186:399-407.
Aims
• To become familiar with the general organization of the insect eye, and record
extracellular field-potentials (ERGs) from the retina under light and dark-adapted
conditions.
• To measure "receptive field" properties of photoreceptors in the time domain (as opposed
to space) using a two-point discrimination task.
New Tools
• Light Emitting Diode (LED)
• LED driver
• TDS210 Oscilloscope
Introduction
Visual processing by the peripheral visual system provides a unique opportunity to study the
ways in which behavior is constrained by the basic limitations of signal transduction and
transmission. This week we will use recordings of insect photoreceptor impulse responses
(responses to brief flashes of light) and step responses (responses to longer flashes of light) and
responses to periodic stimulation (flicker) to analyze the low-pass filtering properties of the
visual system at different light levels. This introduces the concept that receptive fields can be in
many dimensions – space, time, color, etc. For example, a neuron might not respond well to a
single brief stimulus in a given location, but might respond very well to two or more identical
stimuli separated in time by 50 ms. In this example, the cell has a receptive field in space and
also in time.
20
To investigate the receptor characteristics of the eye of the flesh fly, Sarcophagus carnaria, you
will record an electroretinogram (ERG). The electroretinogram is an extracellular recording of
the activity of neurons in the earliest stages of visual processing. As a flash of light is presented
to the eye, individual photoreceptors and their underlying circuitry are stimulated. Their
electrical activity can be recorded by placing an extracellular recording electrode into the eye.
Many factors can influence the responses recorded including the duration and intensity of the
stimulus, the size and type of recording electrode, the position of the electrode, and the
adaptation state of the eye.
Most animals are active under a large range of light intensities, and many changes take place
between the light and dark-adapted states of the photoreceptors. In insects, circadian influences
cause the synthesis of additional photoreceptor membrane at night and the membrane resistance
rises. Coupled to this are changes in the rates of critical parts of the transduction cascade, so the
impulse response slows down. All of these factors increase the gain of the photoreceptors. The
dark-adapted receptors respond at lower threshold intensity, and saturate readily at higher
luminance. We can quantify these changes in the impulse response and observe their effect on
the ability of the eye to code rapid changes in light intensity (its temporal resolution) in an
experiment analogous to the two-point discrimination test that we used on ourselves in the first
week. In this case, the two points will be separated in time rather than space. Sensory neurons
have a receptive field in time as well as space!
One obvious feature of the response seen in your ERGs is the latency. The latency is the lag
between the onset of a stimulus and the onset of the response to that stimulus. The latency of the
ERG you obtain looks the way it does for several reasons. First, the first stages of
phototransduction, (the conformational change of retinal from 11-cis to all trans forms) take only
a few microseconds, but subsequent stages involve a second messenger cascade, (including
activation of the G protein (transducin) and/or an IP3 cascade). This cascade amplifies the signal
massively, but is relatively slow, taking several milliseconds to open channels in the membrane.
Second, the membrane time-constant itself is added to this. In order to transduce and respond to
single photons, the membrane resistance of photoreceptors is high–typically in the range 40-200
megaohms for dark-adapted insect photoreceptors. Thus, a small current leads to a large change
in membrane potential. But this high resistance, coupled with the massive surface area of
photoreceptors (an adaptation for trapping any available light) leads to a large membrane time
constant, which further blurs (low-pass filters) the transient nature of the response. Incidentally,
the Rl-6 photoreceptors of flies such as Sarcophagus, are the "fastest" photoreceptors known.
Our own photoreceptors are almost 5 times slower in response characteristics!
Another obvious feature of the response is the duration of the response. This graded response is
surprisingly slow compared to an action potential–lasting many milliseconds or tens of
milliseconds (depending on room light level). One useful measure of duration is the response
half-width. This is the width of the response in time over which it remains at 50% or greater of
its maximal amplitude. The slower the response, the longer the half-width. These features are
illustrated in Figure 1.
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Figure 1. Illustration of the latency and half-width of a response to stimulation.
Figure 2 shows an example of a step response. The transients in this step response reflect the
contribution to the ERG of neurons post-synaptic to the photoreceptors–the lamina monopolar
cells (LMCs). If you drive your electrode past the photoreceptor layer, these are the cells you
will be recording from.
Figure 2. Trace showing the lamina cell step response to stimulation.
These cells are rapidly adapting, and invert the sign of the photoreceptor response. Whereas
photoreceptors in flies depolarize to light (mediated by light-gated Na+ channels), the lamina
cells hyperpolarize by an unusual mechanism. They have high resting Na+ and K+ conductance,
which cause the cells to be weakly depolarized at resting potential. The neurotransmitter is
histamine, which causes Cl- channels to open. Chloride entry hyperpolarizes the LMC. As Cl-
enters the cell, voltage gated ion channels (delayed rectifier K+ channels), which are normally
open at the resting potential, close. This causes the membrane to depolarize, thus "adapting" to
the incoming stream of neurotransmitter. When the light turns off, the cessation of histamine
release by the photoreceptors causes the Cl- channels to close. Since the balance of Na+ to K+
currents is now altered compared to the normal resting state (Na+ conductance dominant), the
neuron then depolarizes rapidly. This is then, at a slight delay, opposed by opening of voltage-
gated K+ channels, and the cell returns back to its resting state. In this way, the lamina cells
respond only to changes in light intensity (contrast), not to absolute intensity the way the
photoreceptors do. Depending on exactly where your electrode tip is located, the transient
responses of the LMCs may be stronger or weaker. Thus, they can dominate your ERG so that
the impulse responses appear to be depolarizing.
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General Methods
Recording
We will use high resistance quartz micropipettes filled with NaCl and connected to an
intracellular recording amplifier as the recording electrode. The very small size of the hole at the
tip of such electrodes allows us to localize the ERG to responses from nearby cells. Because
these electrodes are very small, in some cases you may also obtain intracellular responses from
single photoreceptors.
Preparation
To introduce the electrode into the eye, we will make a small hole on its exterior surface and
insert the electrode through this hole. Because an insect eye consists of a series of repeated
subunits, called ommatidia, each acting as a miniature eye, a hole made at the top of the eye
allows access to optically intact photoreceptors elsewhere in the eye (refer to Figure 3). This
provides a unique opportunity to record from a receptor with its normal sensory input intact.
Figure 3. Illustration of fly eye anatomy with pipette trajectory shown.
Stimulus
Photoreceptors will be stimulated by means of a light emitting diode (LED), controlled by an
LED "driver." The LED driver has a BNC input that allows the LED brightness to be varied by
changing the input voltage (using the MacLab system). This allows the same stimulus to be
repeated under computer control and responses to be averaged over many presentations to reduce
noise in the response. LEDs have the advantage over other light sources for these purposes in
that they are cheap, small, have low power requirements and can switch between light levels
with extremely high temporal resolution (limited typically by the hardware that drives them).
The LEDs that we have obtained for this course are a newly developed type with extremely high
intensity green light output. The wavelength of light that they emit is ideally matched to the
spectral sensitivity of the main class of typical insect photoreceptors (the short visual fibers, or
retinula cells R 1-6). Prior to the development of such high intensity LEDs, producing such high
23
intensity of light at these wavelengths would have required a Xenon arc lamp and an optical
bench with interference filters and electronic shutters costing many thousands of dollars. The
LEDs cost $7 each.
NOTE: Light emitting diodes (LEDs) have fairly strict operating requirements. Just as with an
ordinary incandescent light globe, it is important to be aware that they can be over-driven and
damaged easily. With the kind of LED that we are using, the LED will show a slight color
change from green to bluish-green if it is over-driven. Be alert to this possibility as you work
through the exercise. LEDs ought to be driven by a current limited source at a specific forward
voltage (so termed because LEDs, like other diodes, only allow current to flow in one direction).
LED drivers used in visual physiology typically achieve intensity control by regulating this
current, in the range 0-80mA. The LED drivers that we will use were expressly designed for
driving LEDs, but can still be over-driven.
We will use the PowerLab output to control the current. You will notice a "threshold" voltage
below which the LED driver produces no output. Between the minimum and maximum voltages,
the LED brightness will increase in a manner that is proportional to the stimulator voltage.
Equipment Setup
Wiring
Wiring the rig for this week is somewhat difficult, so using the wiring diagram on the following
page should prove helpful. The logic underlying the connections illustrated in the diagram is no
different from that used in wiring the rig for any other electrophysiological experiments–think
"signal-processing-output." You will need to understand this logic for future wiring that will not
be illustrated for you.
Recording wiring – The intracellular amplifier will be used for today's recordings. The
output of the intracellular amplifier will go to the Humbug input. The Humbug output
will split and go to the PowerLab input channel 1 and the oscilloscope channel 1.
Stimulus wiring – You will use LabChart to generate a stimulus pulse that drives the
LED via the LED driver. A connection needs to be made from the output channel of the
PowerLab to the input of the LED driver. You want the output of the LED driver to go to
three different places: (1) the LED, (2) PowerLab input channel 2, and (3) oscilloscope
channel 2.
24
Figure 4. Wiring diagram of the recording and stimulus setup (fly included).
Computer Setup (and stimulus testing)

Turn on the LED driver with the switch located on the front panel. Also, open Labchart.
Configure Labchart to produce a single pulse:
>Setup >Stimulator >Setup >Sampling!

• Stimulator mode = pulse • Start: Uncheck the "Trigger"
• Output = fixed # of pulses checkbox
• Voltage range (right side of • Stop: Check "Fixed duration" and set
dialog) = 5 Volts duration to 500ms
• Pulse duration = 100ms
• Delay = 40ms
• Amplitude = 2V
!
25
Administer the pulse via Labchart. Can you see the pulse on Channel 2 of both the oscilloscope
and Labchart. Is the amplitude of the pulse seen on the oscilloscope and Labchart the same as
that which you specified? If not, it is likely because the pulse you specified (2 Volts) is driving
the LED driver, which may put out a different voltage depending on the strength of the battery
that runs this device. Note this difference in voltage during your experiments.
There is a threshold voltage below which the LED will not flash. Adjust the stimulator voltage
and note what threshold value is needed to produce a visible flash and what voltage produces a
"bluish green" flash. The bluish green flash indicates the maximum at which the LED can be
driven without blowing it out. These values constitute the dynamic range of the LED. Do not
exceed the voltage that produces the bluish green flash–typically 3.5 volts.
The stimulus settings used for the dark-adapted trials will be similar to those that you have been
working with thus far in your stimulus testing. Because the room will be dark, for “light-
adapted” trials, a "background voltage" must be used. A background voltage causes a constant
dim light to be produced between flashes of the brighter light stimulus. To set the background
voltage:
>Setup menu >Stimulator

• Amplitude = 3.5V
• Duration = 2ms
Set “Baseline” slightly higher than the threshold you found for the LED output to produce
light dimmer than the stimulus (probably higher than 1.8V).
Once this is set, stimulate and observe the LED. You should see that the flash now occurs
superimposed over a steady level of LED output. Note that at any one absolute output level you
can define a contrast of the stimulus. One definition of contrast commonly used is:
Contrast = (I -I ) / (I +I )
max min max min
Imax and Imin are the maximum and minimum intensities of your pulse. As you increase the
contrast (difference) between the foreground and background intensity of the flash, the flash
becomes more distinct and easier to see (we will quantify our sensitivity to contrast in lab next
week). The "background" (or baseline) brightness allows you to determine the adaptation state of
the stimulated photoreceptor. Set the "background" to zero and dim the room lights for a dark-
adapted response. Use a higher "background" level for a light-adapted response. To calculate
contrast properly, you need to use the calibration data that we will supply for each LED driver.
26
Animal Preparation
• You will be given a fly already restrained by wax in a small plastic tube. Be very careful
not to touch the insect’s eyes. Use clay or dental wax to attach the tube holding your fly
to the washer on the magnetic ball joint. Position the fly ventral-side down with the head
pointing away from you.
• Position your ground electrode. Slip the silver wire up the tube so that it lies underneath
the thorax and abdomen of the fly. Use a small piece of Kimwipe moistened with insect
Ringer (do not fill the tube) to help hold the wire in place and make an electrical contact
with the fly. Do not use the 3M NaCl that you fill electrodes with! Don't add so much
that you drown the fly!
• Set up the animal as shown in Figure 5. (Note: depending on which side of the air table
you position your manipulator, you may want to set your animal up as the mirror image
of this diagram). Use the swivel of the insect stand to fine tune the position of the fly,
keeping in mind how you want to insert the electrode into the hole in the eye.
• Set up the dissecting microscope so you can examine the eyes. Then, use a syringe
needle to make a small hole in the cornea of the eye near the top, about 10 or 20 facet
rows below its dorsal margin. Your TA will demonstrate the technique. Think ahead!
Choose the eye most appropriate to your recording set-up orientation (see Figure 5). Try
to make the hole small, but large and deep enough that you see wet, reddish material
(visual pigment of the photoreceptors and surrounding glial cells) through the hole.
• Position the recording electrode near the opening in the eye. The electrode should
approach the hole nearly tangentially to the surface of the eye. This allows the tip of the
electrode to be advanced laterally into a region of the eye that has not been damaged by
the needle. Using a tangential approach, you can also keep the tip relatively close to the
surface and thus in the photoreceptor layer rather than deeper, into the lamina layer. If
your electrode goes too deep and enters the lamina layer, your ERG will not look as
described later in this manual, because lamina cells give inverted responses compared
with photoreceptors. Nonetheless, such sign-inverted responses can provide perfectly
acceptable data for this lab exercise, so don’t pull your electrode out just to try to
find a photoreceptor response!!
Figure 5. Illustration of recording and stimulation geometry.
27
• Position your LED about 2 cm from the eye in this equatorial position, using the coarse
knob on the micromanipulator. Approach the hole in the eye with a NaCl filled electrode.
Don't fuss about bubbles in the electrode. This is a bubble friendly lab. The tip resistance
is already 30–50 MOhms, so 1 or 2 extra MOhms won't make much difference.
• After using the coarse position controls of the manipulator to bring the electrode tip close
to the hole in the eye, use the motorized microdrive on the manipulator to slowly and
very carefully introduce the electrode through the hole. Set the "step size" of the
motorized manipulator to 10 micron steps. Advance the electrode in a series of steps by
pressing the X, Y, and Z forward controls in short presses. Caution: There is a risk of
driving the electrode forward at high speed if the button is inadvertently held down for a
second or more. To avoid this, set the "motor speed" control to minimum. In this
position, internal friction in the drive mechanism prevents the motor from rotating in the
sustained mode that the manipulator supports.
• Once your electrode is into the hole, advance it using only the X forward control,
approximately 5–10 steps farther (50–100 microns).
• You should now have a circuit made via the extracellular space so you can turn on the
intracellular amplifier. Zero out any DC offset (position knob), and clear any electrode
blockage using a short burst of "buzz" or transient injection of large DC current. If the
baseline potential oscillates wildly, try advancing another 2–5 steps (20–50 microns) and
see if this produces a stable extracellular potential.
Data Collection
Testing the Setup

• Record the ERG to a bright, brief flash of light presented to the eye.
>Setup >Stimulator
• Amplitude = 3.0V ** Remember to reset Baseline voltage to 0.
• Mode = Pulse
• Duration = 2ms
You should see a small bump in potential lasting perhaps 30ms or more. This is an "impulse
response." You may also see an artifact of the LED stimulus in your recording in synchrony with
the stimulus. This is due to electrical "crosstalk" between the LED driver circuitry and the
recording electrode. Because of its high impedance, the electrode acts as an effective antenna for
picking up all kinds of electrical interference. If you can't see any response, try adjusting the
display range (gain) on both the oscilloscope and in Labchart. Try adjusting the position of the
LED and see if the response gets larger or smaller. Note that there is a distinct region of the eye
that exhibits the largest response to stimulation. Center the LED in this region–this area of the
eye is the spatial receptive field of the cells you are recording from. This is not really the
receptive field of a single photoreceptor, but because our electrode resistance is very high, we do
get an ERG that is dominated by responses from a limited number of nearby receptors.
28
• Determine the input-output relationship of the response by giving a series of flashes of 2

ms duration with varying brightness, over the range from just detectable to the maximum
intensity when the LED turns “bluish green” (as you determined above). How many
different intensities are appropriate (there is no single right answer, but 2 is too small and
100 is too many)?
• To reduce the effect of noise in your recordings, you can average a series of responses to
stimuli. Set Labchart to record the average of 20 flashes of light. Stimulate with
"averaging" and note the effect on your recording compared to the traces you obtained
without averaging.

• Amplitude = 3.0V • Mode = Average
• Duration = 2ms • Average = 20 Sweeps
• Mode = Pulse • Source = User
• Delay = 5 sec
!
• Reduce the duration of the flash to 1 ms:

• Amplitude = 3.0V • Mode = Average
• Duration = 1ms • Average = 20 Sweeps
• Mode = Pulse • Source = User
• Delay = 5 sec
!
What is the effect of this shorter flash of light on the amplitude of the ERG? Why? (Note:
This response is termed the "impulse response," because the stimulus duration is much
shorter than the eye can resolve).
• Increase the stimulus voltage to 3.5 volts, while still using a pulse duration of 1 ms.
Record the average impulse response again. It should appear similar to the response seen
in step number 1 (2ms, 3.0 volts). Why?
• Increase the stimulus duration to 200ms to produce a "step response." Also, set Sampling
to present multiple flashes.
29

• Amplitude = 3.0 V • Mode = Average
• Duration = 200 ms • Average = 20 Sweeps
• Mode = Pulse
!
Run the experiment once. You should see a step response similar to that illustrated in Figure 2.
Yours may not look quite like this, but look for at least the presence of transients at light on and
light off, as well as a sustained level of graded response.
Temporal properties and dark adaptation of photoreceptors

• Now that you have become acquainted with the steps for data acquisition and the features
of the ERG it is time to look at how the adaptation state of the receptors affects the
response to stimulation.
You will need to reset the stimulus parameters to record a dark-adapted (DA) impulse
response. Generally, this is an impulse response to a stimulus with no background
illumination. By now your TAs will have turned the room lights down. If not, please ask
them to do so and turn off any extraneous lights in your setup. It isn't necessary for things
to be totally dark, so don't be too obsessive about this if some of your fellow students
need to use their microscope lamps.
In this period, let the eye rest in this state for 3-4 minutes without stimulus. Set your LED
to produce a dim flash of light (try 2 ms duration or less). Set "brightness" to be just
bright enough to stimulate the ERG, with a background stimulator voltage of 0 (i.e., no
background illumination).
As the eye dark-adapts, set up Labchart so that it acquires a full second of data on each
sweep and set it to record the average of 20 sweeps. Note: the reason for using such a
long sweep is that we don't want the flashes of light from each sweep to occur so close
together in time that they light adapt the eye. Once the eye is dark-adapted, record the
dark-adapted impulse response. What is the response latency? What is the approximate
“half-width” (this is common scientific jargon for width at half amplitude)?
• Measure the ability of the dark-adapted photoreceptors to discriminate flicker. You can
use the stimulator to generate a series of impulses separated by a period of time that you
specify by clicking on the “Interval” box in the Stimulator dialog and then setting the
“Interval” slider. In this case set the stimulator to produce 2 impulses. Sample 500 ms
worth of data each time. It is up to you how you configure your stimulus, but the aim is to
try and produce a curve that relates the frequency of the flicker to the modulation of the
membrane potential that the stimulus evokes.
If you make the flashes far apart, for example, you should see a discrete response to each
flash in the "pulse train." Between responses the potential falls close to the normal resting
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level. If you use pulses very close together, they will fuse into a single impulse response.
Decide how to analyze your data. For example, you could measure the difference
between the inter-pulse potential and the maximum pulse evoked level. This value will be
the same as the single impulse response amplitude when the pulses are far apart (and
perfectly resolved) and will approach zero as the impulses "fuse." You can then make a
plot relating the frequency of the pulse train to the amplitude of the response modulation.
This is the so-called temporal "modulation transfer function" of the eye. Refer to Figure 6
for an example of what the response to flicker will look like.
Figure 6. Example traces showing flicker fusion.
Temporal properties and light adaptation of photoreceptors

• After finishing the dark-adapted recording, record the light-adapted (LA) impulse
response. Now you will need to set the "background" to deliver constant, low voltage. Set
the stimulator back to deliver a single pulse. Recall the stimulator level that is the
threshold for the LED to produce light. You found this value when you were working
under the "computer settings" subheading of the "equipment setup" section of this lab.
Make a new stimulus, where the background level is set slightly above this threshold
value (this should not be much higher than 2.5 volts). Set the foreground to a level close
to maximally bright (no more than 4 volts). Let the eye light-adapt for a half minute or so
(light adaptation is much faster than dark adaptation). Now put Labchart into "Overlay"
mode and repeat your stimulus.
31
What happens to the impulse response? Ignore any absolute scale changes here. Is the
light-adapted response faster or slower than the dark-adapted response? Are there any
other differences?
• Repeat your flicker experiment in this light-adapted state. Do you see a shift in the
frequency response to higher frequencies? How do you think the impulse response is
related to the flicker response? The flicker response is a measure of the "temporal"
receptive field of the cell. Think about the somatosensory two-point discrimination lab
(week 1). Can you see the analogy?
Assignment
Normal lab write-up.

DUE: This report will be due at the start of each section November 9-12 via Canvas; each lab
section due one week from the lab with the exception of section D, due November 12 at 1:30.
Project ideas:
• Measure flicker-fusion frequency as a function of flash brightness and/or background
light brightness.
Determine the time course of light- and dark adaptation. Give pulses at some interval and get a
baseline in one or the other condition, then change the light level and measure the changes and
their time course.
32
33
NEUSCI 302 Autumn 2020 Lab 3 – Group A: Echo response
Lab 3 – Group A: Recording of Electromotor Behavior and ‘Echo-

Responses’ in Gnathonemus petersii
Warnings for this lab

• For the next two labs with tropical weakly electric fish we will increase the room
temperature to about 27˚ C (80˚ F). This will prevent the water in your experimental
tanks from cooling. Because you’ll have to spend about three hours in this temperature,
dress accordingly.
• The fish species we will be using have very good hearing abilities (for fish), so you must
be careful about abrupt sounds that will startle the fish and interfere with your
experiments. Do not bang the table or fish tank with electrodes, pens, notebooks, etc.
• Please keep your hands out of the fish water, especially if there is any residual soap,
lotion, cosmetics, lunch, or tobacco on your skin. Smokers are relegated to working with
the computers. Smokers do not put your hands in the water or touch any of the equipment
that will be in contact with the fish.
• Please read carefully Appendix 1 to Laboratory 6, at the end of this section.
Aims
• Learn how to record brief, transient signals with the oscilloscope and Labchart.
• Learn how to record electromotor behavior (temporal pattern of EODs) during
swimming, encounters with objects, and social interactions.
• Discover how Gnathonemus petersii responds to sudden changes in its surroundings.
• Learn how to record a simple social communication behavior (“echo-response”) in
Gnathonemus petersii.
Introduction
In this lab, we are going to record the electric signals Gnathonemus is producing, as well as the
temporal pattern of Electric Organ Discharge (EOD) production during different behaviors.
Certain factors can influence this signaling pattern. We will try to evoke those changes by
presenting objects to the fish and by changing the electric properties of objects. In addition, we
will try to evoke a social behavior – the echo response – which the fish use to communicate with
other individuals.
An echo response can be evoked by the presence of a conspecific in the territory of a
Gnathonemus. It consists of an EOD-pulse, which is emitted immediately after (latency = ca. 12
ms) the other fish has produced an EOD, and therefore acts like an "echo" to the other fish’s
signals. Echo responses are usually emitted by dominant fish, which thereby signal their
superiority to the intruder. A weaker (or subjectively weaker) animal will not respond with an
echo, but rather will avoid signaling right after the dominant fish has produced an EOD. This
response is called a preferred latency response. The problem in evoking an echo response is that
the fish has to feel superior to the other fish, and therefore should not be frightened or nervous.
In a lab setting, especially when 6 groups of students try to work in the same room, experimental
34
animals can easily be stressed and fearful. If that’s the case, Gnathonemus might not produce
either an echo response or a preferred latency response and our experiments will not work. So,
try to be calm and avoid loud noises or stressing your experimental animals in any way.
Equipment Setup
You will be provided with a small experimental aquarium, thermometer, fish net, fish tube,
recording electrodes, and stimulating electrodes. Before obtaining a fish and the water from your
TA you will need to connect your cables for recording.
Recording Wiring
You will be using the extracellular amplifier and Labchart to record EODs from Gnathonemus.
The recording electrodes that you will be using should already be connected to the extracellular
amplifier via the 5-pin connector. These should be on channel 1 or 4 of the amplifier.
• For the first part of the experiments, you have to twist the two white wires so that the
ends of the wires are separated by about one centimeter. Then you can use this like a
probe to follow the fish around. For the later experiments, you will have to untwist the
wires and connect them to the tube containing the electric fish in order to pick up its
EODs.
• The signal from the extracellular amplifier should go to channel 1 of the MacLab and
channel 1 or 4 of the oscilloscope. The signal should also go to the audio monitor.
• Make sure the amplifier gain is set to 100.
• Get a ground wire ready to ground the bath.
Stimulus Wiring
Since most of today's lab involves only recording the EOD and presenting objects to the fish, any
necessary stimulus wiring will be described later in the experimental section that requires it.
Computer Setup
You will be using Labchart today to record the fish's EOD. This week you will be using a feature
of Labchart that you have not used previously.
The feature is called a Frequency Vs. Time Plot. This type of recording places time along the x-
axis of your trace (as usual), but the y-axis is converted to display frequency. When you record
in this mode, you are asking the computer to take average frequency measurements as you
collect spikes. So, the Labchart display will scroll as usual, but rather than displaying spikes, a
line will be drawn that corresponds to the firing rate being recorded at any specific time. As the
fish increases its firing rate, the line will trace upward and vice versa. After your data is
collected, you will be able to simply look at the line to see what the fish's frequency was at a
desired time during the recording session (i.e. before, during, and after stimulation with objects).
Animal Preparation
• The fish will be at your station when you arrive to lab. Try not to disturb the fish since a
stressed fish can change your experimental results. It is important that to keep your
35
hands out of the water as much as possible. If you must put your hands into the tank,
please rinse them off with warm tap water only (no soap!) before putting them into the
tank. This will remove any chemical residue.
• Check that the temperature reads between 23° and 27° C. It is important to keep the
temperature in this range over the course of the experiment to not overly stress the fish.
In addition, changes in temperature may affect the frequency of the EOD. Therefore, you
should note the temperature in your lab reports for each experiment. We have increased
the room temperature so that the tank temperature should remain stable. However if you
see the temperature begin to fall, plug in the small heater at your setup and place it in the
recording chamber. Noise from the heater will interfere with your recording, so only keep
it plugged in if absolutely necessary while you are setting up and in between recordings.
• Be very gentle with the fish. Fish do not tolerate handling well, and fish with damaged
scales become susceptible to fatal fungal infections. Make sure the white tube we will use
later for restraining the fish is already in the water. The fish may choose to swim into it
on its own. The white tube is made of a material that is transparent to electrical current.
• Clip your ground electrode to the green wire taped in the corner of the tank.
Data Collection
Record the fish’s EOD

• Insert your recording probe in the water and slowly approach the fish with it. Do not
scare your fish with the probe!
• Obtain the EOD signals on the oscilloscope if they are not there already. Adjust the time
scale and the trigger to visualize the signal. You should also make sure that the voltage
range is set so that you can easily see your signals. Make sure that the probe is set for
"channel 1" and that the trigger is set to run on "auto." If you have problems or lose your
signal, make sure that the trigger level is correct and, remember you have a panic button
labeled "autoset." Also, set the source in the trigger menu to channel 1. Obtain a nice
EOD signal for your records. Because one of the objectives of this lab is to learn to use
an oscilloscope to display short, transient signals you should try it yourself first.
Experiment a little, your TA should be your last resort.
• Turn on the audio monitor and slowly turn up the volume until you can hear the EODs.
Because the fish is moving around, your signal will be changing all the time. Why?
• Try to get a stable recording for some time. Note the waveform of the EOD, its
amplitude, and the variations in inter-EOD intervals.
Record the temporal pattern of the fish's EOD

These recordings can either be made with the fish swimming around the aquarium or with the
fish inside the tube. If you can not get a stable recording of a sequence of EODs because the fish
is moving around too much, it may be better to secure it inside the tube.
• Put some pebbles in the tube – these will weigh down the tube and prevent it from rolling
around. Gently shoo the fish with the net into the tube. When the fish is inside, close the
opening of the tube with the provided cover. Put the tube down and secure it with the two
pebbles to prevent it from rolling around. Make sure the fish is submerged completely!
36
• Untwist the recording electrodes (the two white wires) and connect them to the tube by
placing one wire in each hole at the ends of the tube.
• Now, record a few cycles of the EOD on LabChart. Display the signal on the screen in a
way such that you can nicely see the details of single EOD pulses. Do you notice
anything strange about the way the waveform looks? Is it squared off at the top and
bottom? If so, this is a great example of clipping. Clipping occurs when your signal is
larger than the amplitude limits of your trace. Since the fish produces a large electric
pulse and this pulse is amplified before you see it, the amplitude of the signal may be
much larger than the range of amplitude you are sampling. There are two ways to take
care of the clipping problem. The first way is to increase the amplitude limits in
Labchart. Do this and take another trace. Is the clipping gone? If not, there is another
way that involves reducing the amplitude of the signal. As beginning
electrophysiologists, think about how to reduce the amplitude of the signal you are
recording. Hint (in the form of a question): What happens to signal amplitude as you
move an electrode closer to a neuron that you are recording from?
• To record the fish's temporal pattern of EODs, set up Labchart in such a way that you can
see a sequence of EODs on the screen. The problem with displaying a sequence of EODs
is that the single events are very short. Therefore the sampling frequency has to be quite
high to get the positive and negative peaks of all signals. Vary the sampling rate to find
an optimal setting. Try to do it yourself by starting with a high sampling frequency and
then decrease the sampling rate by half until the waveform shape appears to become
distorted. Now, increase the sampling frequency until the distortion is removed and the
waveform shape looks the same with any increase in sampling frequency. Hint: one
objective measure of “looks the same” might be “has the same amplitude”.
• Record a sequence of EODs (10-20s trace). After you have obtained a trace, you can
change the channel setup in such a way that it displays the instantaneous frequency of the
EOD series versus time. Go to Setup > Channel Settings and then to Display Settings and
choose Cycle Variables for channel 1. Choose Frequency.
• A tricky problem is to set the threshold in such a way that your results are correctly
reflecting the instantaneous EOD frequency. In order for an EOD to be "counted" by the
frequency converted it has to surpass the threshold criterion. Set the noise threshold by
moving the slider. Set the threshold high enough that you do not get multiple triggers per
EOD. Be patient, eventually you will find a good setting.
• Display a frequency versus time plot of your recorded sequence and save it for your
records. What temporal patterns of EOD production can you observe?
Stimulating the fish with objects – electrolocation
Important Note: For the all the following experiments, it is extremely important that the noise
level in the lab is kept at a minimum. As you have noted in your recordings thus far, the fish is
very sensitive to any disturbances, including loud noises and vibrations. The latter are produced
every time you touch the tank or walk around the room. So, please keep all your movements to a
minimum. By doing so you will get results that you can analyze. Try not to walk by other
recording stations while the students are taking recordings, especially the stations leading to the
lab room door.
• While being careful not to bump the fish tank or fish tube with the object, approach the
tube with one of the metal plates provided (keep your hands out of the water). Record the
37
response of the fish before and during the approach noting when the object is put into the
water and when it is removed.
• Produce a frequency versus time plot for your records. When does the fish respond by
changing its EOD frequency and is this change an increase or decrease in frequency?
Why?
Changing object properties – electrolocation

• Take a cable with two banana plugs and hold one end in the water next to one side of the
tube. Do not touch the tube and keep your hands out of the water. Do not make too many
water movements when you do this. Start your recording and insert the other end of the
wire in the water on the opposite side of the tube.
• Do the same with one end of the wire at the head-end of the tube and the other end of the
wire at the tail-end of the tube.
• Record the responses of the fish for both situations. How can you explain these
responses?
Connecting two tanks

Instead of presenting artificial fish pulses to your Gnathonemus, you can also use another electric
fish as a stimulus. To do this you have to work together with the group across the table. When
both groups are done with the previous procedures, work together on this experiment.
• Both groups should set up a recording protocol in Labchart. Be prepared to record for a
short period before and after the two fish tanks are connected. It may be necessary for
only one group at a time to take recordings after the other group has disconnected their
fish tank from their rack.
• In order to use the other group’s fish to stimulate yours, you have to connect the two
tanks. Use a long wire and connect the two tanks.
• Record the EOD behavior of the fish in an amplitude versus time plot shortly before and
after you connect the tanks. How does the fish respond when it can sense the other fish in
the neighboring tank? Can you observe an echo response?
Evoking an echo response electronically - electrocommunication

Now we want to stimulate the fish with an electric signal that mimics an EOD of another fish.
Here is where the stimulus wiring comes in. This first part is the “experimental set up” and the
“dry run.” All stimulus and recording parameters should be worked out before the fish is
included in the experiment. This prevents the fish from being over-stimulated, which would
corrupt the experimental results.
• Do not connect the alligator clips to the wires in the tank yet, but connect the “BNC-to-
two crocodile clips” cable to output 1 of the PowerLab. Also send the output signal to
PowerLab input channel 2 and oscilloscope channel 2. Use a T-adapter and more BNC
cables to do this. This will allow you to see the waveform that you send into the tank in
Labchart. At this time, be sure to keep the clips apart and not connected to the tank
electrodes.
• Set up Labchart to record the fish's signal on one channel and your stimulus on the
second channel. Before you start your experiment try sampling the stimulus waveform.
Again, without the alligator clips connected to the stimulus wires. The directions for
configuring the stimulus are below.
38
• Now, go to the Labchart Setup menu and open the Stimulator. Set the stimulator to
deliver a single, square pulse of a short duration–1ms. Set the amplitude Range to 10 V
and the frequency Range to 20 Hz. The stimulus Amplitude should be set to 5 V and the
Frequency should be set to 4 Hz.
• For data collection and analysis it makes sense to construct a peristimulus time histogram
(PSTH). Set up Labchart in such a way that you obtain a PSTH similar to when you
recorded from the mechanoreceptors of the cockroach leg. Think carefully about the
settings before you start recording. Now, if everything is ready and the stimulus output is
off, we can include the fish and perform a stimulus-response experiment.
• Record a baseline response and obtain a "control PSTH." Without the crocodile clips
connected to the stimulation wires, start Labchart to obtain a PSTH. Present 25–50
control stimuli to the fish for one PSTH.
• Now, use the two stimulation wires attached to the walls on both sides of the tank to put
the signal into the water. Connect the two crocodile clips to the stimulation electrodes
and start Labchart to obtain an “experimental PSTH.” Present 25–50 stimuli to the fish
for one PSTH. Think about how the PSTH should look if the fish responds to the
stimulus with an Echo Response, during which the fish "echoes" each stimulus with an
EOD at a latency of about 12 ms. What about a preferred latency response?
• If you do not see any response from the fish, your stimulation amplitude may be too low.
Increase it and try again. The fish will not be hurt at all even if you increase the
amplitude to 10 volts.
• In the PSTH you should see a distribution of spikes (EODs) following your stimulus.
Compare this distribution to the control PSTH where you did everything the same as
during stimulation except for connecting the crocodile clips to the stimulation electrodes.
Sometimes it is a good practice to obtain another control PSTH after an experimental
manipulation to determine if there is a change from the baseline response of the animal
simply due to the experimental manipulation. How might the fish’s response change
after exposure to another “phantom” fish?
Assignment (due separately from that for second fish lab)
DUE: At start of each lab section in the next week (Nov 16-18) via Canvas.
This lab report is worth 10 points, and consists of answering the questions below (so, not
standard lab report format). Answer all the questions (typed or word processed). Limit the
responses to 2 pages total minus figures. Any figure must be labeled appropriately. Include
figures as appropriate (where you were asked to make a record for yourself). In addition, include
an Abstract describing the question and results succinctly.
• On page 37 you are asked to print a frequency vs time plot.

Present this figure.
What temporal patterns did you observe?
What would change if you moved your electrodes farther from the animal?
• Presentation of an aluminum “plate” in the water surrounding the fish.
39
Present the figure.

Did the animal respond, and if so what was the response? Explain
Did it matter if the plate was moving or stationary? Explain
• Presentation of a transparent “plate” in the water surrounding the fish.

Compare these responses to the aluminum plate.
Explain any differences.
• Presentation of a banana plug in the water surrounding the fish.

Did the orientation of the plugs make a difference to the animal’s response?
Did one side only have any effect?
Was there a difference in a head to tail vs a side to side presentation?
Explain any differences.
• Using a piece of wire, you connected your fish to your neighbor’s fish. Was your fish the
dominant fish? Explain your results presenting data as appropriate.
• You generated a signal from the computer and recorded the fish’s response. Explain your
Results.
40
NEUSCI 302 Autumn 2020 Lab 3 – Group B: Jamming-avoidance
response
Lab 3 – Group B: Jamming avoidance response in the weakly

electric fish Eigenmannia sp. or Apteronotus albifrons
Additional reading
• http://www.alumni.caltech.edu/~rasnow
Aims
• Record the EOD of the weakly electric fish, Eigenmannia sp.
• Discover how Eigenmannia sp. copes with the interference of EOD signals from other
weakly electric wavefish.
• Measure quantitatively which sensory stimuli can evoke a JAR and show the dynamics of
the fish’s response.
New Tools
• Function generator
Introduction
The jamming avoidance response (JAR), which we will be studying today, is a very important
behavior for many weakly electric fishes. In this lab, we will be looking at the JAR of the wave-
type electric fish Eigenmannia sp, which continuously discharge their electric organs and
produce sinusoidal electrical signals. The frequency of the signal varies among individuals
between 200 and 600 Hz. The JAR allows multiple conspecific wavefish to function in the
vicinity of each other without interfering with each other’s pattern of EODs.
The gymnotiform wavefish Eigenmannia constantly produces an electric signal with its electric
organ. During active electrolocation, it can detect nearby objects because they lead to slow
changes in amplitude and phase (timing) of the signal at the epidermal receptor organs. When
swimming near an object, amplitude and phase modulations of the perceived electric stimulus
occur at a modulation frequency of a few Hertz. However, if another Eigenmannia comes close,
very similar changes in sensory input can occur if the intruder’s EOD frequency is similar to the
frequency of the first fish. This similarity leads to jamming of the fish’s electrolocation ability.
Because Eigenmannia continuously discharge their electric organs, an individual fish will
receive its own EOD as well as a nearby neighbor’s. In other words, the EODs of the two fish
overlap and “add together.” The two signals combined cause a beat pattern. That is, a rhythmic
increase and decrease in signal amplitude accompanied by a rhythmic change in signal phase.
The frequency of this amplitude and phase modulation equals the frequency difference between
the two signals. For example, if the resident fish discharges its electric organ at 400 Hz and the
intruder discharges at 398 Hz, the beat pattern has a modulation frequency of 2 Hz. The same
modulation frequency would occur, however, if the intruder fish had an EOD frequency of 402
Hz. Hmm, this could be a dilemma. How would the resident fish determine if the intruder is 2 Hz
higher or 2 Hz lower than its own frequency?
41
response
In order to avoid being jammed by its neighbors, Eigenmannia performs a jamming avoidance
response (JAR). If a fish detects a signal of another wavefish of a frequency similar to its own, it
will shift its EOD frequency away from that of the neighbor. For example, a fish originally
discharging at 400 Hz meeting a neighbor discharging of 398 Hz will increase its EOD
frequency to a higher value around 410 Hz. The intruder will drop its frequency about the same
amount. If the intruder discharges at 402 Hz, the resident fish will lower its EOD frequency and
will end up with an EOD of about 390 Hz—guess what the intruder will do. In either case, the
resident fish increases the difference in frequency between its own EOD and that of the neighbor,
which will ensure that the ability of the fish to electrolocate is not impaired. The JAR results in a
faster beat pattern of the two fish's signals so that the combined signals do not interfere with the
slower amplitude and phase changes the fish are using to sense their environment.
The problem the fish faces when confronted with an electrical stimulus is that both higher and
lower frequencies of a foreign EOD (combined with the resident fish’s own signal) can create the
same beat frequency. Even though the beat patterns in both cases look very similar, the fish
always performs the behavior in the right direction. That is, frequency goes up if the neighbor’s
frequency is lower, and goes down when the neighbor’s frequency is higher.
A JAR can be elicited if the frequency difference (∆F) between the two fishes’ EODs is less than
20 Hz. Delta F is defined as:
∆F = Fneighbor – Ffish
The sign of ∆F determines the direction the fish has to shift its EOD frequency. When ∆F is
positive—the fish’s EOD has a lower frequency than that of the neighbor—the fish has to lower
its EOD frequency. If ∆F is negative, the fish must raise its EOD frequency. The response of the
fish, referred to as ∆R, is defined as the fish’s EOD frequency before encountering another fish
(or an artificial electrical stimulation) minus the EOD frequency the fish ends up with after
performing the JAR. Delta R is defined as:
∆R = Fbefore – Fduring
42
response
Equipment Setup
You will be provided with a tub, thermometer, fish net, fish tube, recording electrodes, and
stimulating electrodes. Before obtaining a fish and the water from your TA you will need to
connect your cables for recoding.
Recording Wiring
Today you will be using the extracellular amplifier and Labchart to record EODs from
Eigenmannia. The recording electrodes that you will be using should already be connected to the
extracellular amplifier via the 5-pin connector. These should be on channel 1 or 4 of the
amplifier.
• The signal from the extracellular amplifier should go to the input of the Humbug.
• The output of the Humbug should split and go to channel 1 of the PowerLab, channel 1 of
the oscilloscope, and to the audio monitor.
• Obtain a wire to connect to the green wire on the recording chamber so that you can
ground the recording tank water.
• In order to pick up EODs, connect the electrodes (the two white wires) to the tube where
later the head and the tail of the electric fish will be.
• Finally, set the gain on the amplifier to 100X.
Stimulus Wiring
After the recording wiring is set, you need to set up the stimulus. A function generator will be
used today to create a signal that mimics the EOD of another weakly electric fish.
• The signal from the function generator needs to split and go to channel 2 of the
PowerLab, to channel 2 of the oscilloscope, and to the tank. The step written below
discusses the connection to the tank in more detail.
• For now, leave the alligator clips unconnected, but connect the “BNC-to-two alligator
clips" cable to the output of the function generator—also make sure the alligator clips do
not touch each other. This is the wire that will carry the signal from the function
generator to your fish through the two white wires taped to your tub. You will not
connect the alligator clips to these wires until you are ready to stimulate the fish.
Computer Setup
You will be using Labchart today to record the fish's EOD. You should know your way around
Labchart fairly well by now, however, you will be using an unfamiliar feature of Labchart—the
Fast Fourier Transform (FFT). We usually look at signals in the time domain. For example, the
typical display in Labchart has time on the x-axis and amplitude on the y-axis. The FFT allows
us to look at signals in the frequency domain by keeping amplitude on the y-axis, but changing
the x-axis to frequency. This concept should become clear once you see Labchart’s FFT function
in action. You will also come to appreciate how much easier the FFT makes your job in contrast
to having to compute frequencies constantly during the experiment.
For now, simply open Labchart. More directions regarding settings will be given in the "Data
Collection" section of today's exercise.
43
response
Animal Preparation
• Nicotine can affect the receptors of the fish. If you smoke do not touch the fish tank or
anything that will be put into the fish tank.
• Rinse your hands with warm water to remove any oils, residual soap etc., which could
damage your fish. DO NOT USE SOAP!
• A fish should be at your station. Important Note: Try to keep your hands out of the water
as much as possible. If you must put your hands into the tank, please rinse them off with
warm tap water only (no soap!) to remove any chemical residue.
• OK, this part will take a little patience; try not to harass the fish. Use the net to gently
shoosh the fish into the tube. When the fish is inside, close the opening of the tube with
the provided cover.
• Put the tube down and secure it with the two pebbles to prevent it from rolling around.
Make sure the fish is submerged completely. Also make sure that the two wires
(recording electrodes) are still attached to the tube’s ends.
• Check that the temperature in your tub is between 23 and 27˚ C. It is important to keep
the temperature in this range over the course of the experiment to prevent stressing the
fish. Also, changes in temperature affect the frequency of the EOD. Therefore, for each
experiment that follows, record the before and after tank temperature in your lab reports.
The room temperature has been increased so the temperature should not vary. However,
if you see the temperature begin to fall, tell your TA and they will increase temperature
with a small heater. Noise from the heater will interfere with your recording, so we will
try to avoid using them. If you do use a heater only keep it plugged in while you are
setting up and in between recordings.
• Finally, clip your ground electrode to the green wire taped in the corner of the tub.
Data Collection
Record the Fish’s EOD

Note: We are still not stimulating the fish, so leave the alligator clips from the function
generator disconnected and not touching.
• Obtain the fish's signal on the oscilloscope if it is not there already.
• Adjust the time scale to visualize your signal how you want it. You should also make
sure that the voltage range is set so that you can easily see your signals. Make sure that
the probe is set for channel 1 and channel 2 (in the channel 1 and channel 2 menus,
respectively) and that the trigger is set to run on "auto." Also, set the source in the trigger
menu to channel 1. If you have problems or lose your signal, make sure that your trigger
level is correct.
• Set up your “Frequency Counters.” The oscilloscope can show a running display of the
frequency of the EOD by setting it to measure the frequency of channel 1. Press the
measure button. Set your source to channel 1. Push the second to last selector button until
it reads "frequency." Now, the oscilloscope will calculate the signal frequency in real
time. You will use this display in this exercise as one of the ways to record and keep
track of your fish’s EOD frequency as well as your stimulus frequency.
• Turn on the audio monitor and slowly turn up the volume until you can hear the EOD—
don’t blast everyone else in the room.
44
response
• Record a few cycles of the EOD in Labchart for your records to use as an example of
your fish’s EOD before stimulation.
• Now set Labchart to show the power spectrum (FFT) by going to Window->Spectrum.
This will convert your x-axis to frequency. The peaks that you see now are the frequency
of your fish and later the peaks will correspond to the frequencies of the fish and of your
stimulus.
• You can use the Spectrum function of Labchart to measure the frequency of the fish’s
EOD. For the analysis to be accurate enough to measure even small frequency variations
of the EOD, you have to find the optimal settings of the program. In order to measure the
JAR exactly, you need a frequency resolution of approximately 1 Hz. A good setting for
number of samples is 2560 and time is 1s. Now you can use the cursors to measure the
frequency of the peaks in the spectrum display.
• Play around with spectrum function to learn what happens to the frequency resolution at
different settings.
• Measure the EOD frequency of your fish in FFT mode for your records prior to
stimulation.
Stimulating the Fish (preparation)

• Now prepare to stimulate your fish, but DO NOT connect the alligator clips yet. The two
white wires submerged in the recording tank on either side of the fish are the stimulating
electrodes. Only after you have determined the fish’s baseline EOD frequency (while no
stimulation is present), and then appropriately adjusted the stimulation frequency to a
value you want to test, will you connect the alligator clips to the white stimulating wires.
• Create a sine wave stimulus. Look at the output from the function generator on the
oscilloscope to do this. You will be giving a continuous sine wave stimulus to the fish to
mimic a conspecific’s EOD. Set the frequency of the sine wave to be about 2–4 Hz above
the frequency of the fish. Use the continuous frequency read-out on the oscilloscope to
help as you adjust the stimulus frequency. For fine-tuning the stimulus frequency, make
an FFT analysis of your stimulus signal with Labchart and use the cursors to read values.
• Important Note: Once you have created the appropriate signal, but before you clip the
stimulating electrodes, make sure that the amplitude of the stimulus is turned all the way
down. Also, change the source in the trigger menu to channel 2, so that you can watch the
fish’s EOD frequency speed up or slow down relative to the constant stimulus frequency.
• Make sure Labchart is set up properly for the experiment. Make sure that data from
channels 1 and 2 are being recorded, on two separate traces. You may want to see both
channels overlaid on the same trace. To do this, double click the line that divides the two
traces. It helps to discriminate between the two signals if channel 1 and 2 are different
colors. Make sure that the range of your y-axis is set so that you can easily visualize both
signals. Once you have done this, open the Spectrum window. This will convert your x-
axis to frequency. The peaks that you see are the frequency of your fish and your
stimulus.
Stimulating the Fish (the real thing)

Address these two questions before you start: Did you turn the stimulus amplitude all the way
down? Did you record the “before” temperature?
45
response
• When the appropriate stimulus is created, note the pre-stimulus EOD and calculate what
the beat frequency will be when you deliver this jamming signal to your fish.
• Make sure you can hear the audio monitor—get ready to listen for “beating.” Now clip
your leads onto the white stimulating electrodes to deliver the stimulus. Turn up the
amplitude of your signal slowly until you see your fish respond with a shift in EOD
frequency. Did you hear the change in beat frequency? Did the beat frequency increase or
decrease?
• Keep the stimulus on. Otherwise, the fish will change its EOD frequency back to the
original frequency. What is the beat frequency a few minutes after the fish has shifted its
EOD frequency? Is it stable? About how long did it take to stabilize if it did change?
• Note the new EOD. Record an example of this EOD in Labchart.
• Turn off your stimulus (by disconnecting one alligator clip) and watch the EOD behavior
of the fish.
• Note the new EOD after the stimulus has been off for a few minutes. Measure the EOD
frequency and record an example of this EOD on Labchart. Did you record the "after"
temperature? If the stimulus amplitude worked well, leave it at that setting for the next
experiment.
Recording the Time-Course of the JAR

• Make sure the fish has had about five minutes to rest. Set the frequency of your stimulus
to a value about 2–4 Hz below the EOD frequency of the fish. If possible, use the same
stimulus amplitude as before. If necessary, the stimulus amplitude can be set as high as
15 V peak-to-peak. As a rule of thumb, the heights of the lines you obtain when doing a
spectral analysis can be about equal when overlaid. The stimulus line, however, should
not be larger than the line for the fish’s EOD. Now, you will be simply repeating the
stimulation procedure from above to make the fish shift its EOD frequency, but you will
be recording the time-course of the response and recovery (see #2 below). How’s that
bath temperature?
• Record the fish’s EOD before stimulation (to get a new baseline), during stimulation,
and after stimulation (to get recovery data). Note the EOD frequency as often as possible
during these stages of the experiment. Later, you will make a graph that illustrates the
time-course of the JAR. On this graph, you will plot EOD frequency versus time for the
increase in EOD frequency during the stimulation and the return of the EOD frequency
back toward baseline. Use “reverse time” for the recovery phase—your TA will show
you how. On this graph, also mark the time point of the beginning and end of the
stimulus. Think of how to obtain the necessary data for this graph. For example, you
could record the fish’s EOD in Labchart every 2 seconds, perform an FFT analysis of
these recordings, and later measure the EOD frequency of each recording. Because the
fish’s response is quite rapid at the beginning of stimulation, you should be prepared to
record more frequently early in the experiment. Later, when the frequency plateaus, it
might be sufficient to make fewer recordings with a longer pause in-between. You should
only have to stimulate for a few minutes and record the recovery for a few minutes. Did
you record the bath temperature?
• After you plot the data, look at the graph and note if the recovery phase follows the same
time-course as the stimulation phase? Look up “hysteresis” in the dictionary.
46
response
The JAR's Dependence On Stimulus Frequency
• Only stimulus frequencies close to the frequency of the fish are effective in evoking a
JAR. Why? In this experiment you will evoke a JAR at different values of ∆F and note
the magnitude of the fish's response, ∆R, for each value of ∆F.
• Use the following ∆F values: -20, -15, -10, -5, -2, 0, 2, 5, 10, 15, 20 Hz. Test them
pseudorandomly by alternating between positive ∆F values and negative ∆F values. If
you don't have enough time to obtain data for all these values, think about which are the
least important and exclude them from your testing.
• Let the fish rest for a few minutes between each test. Be careful to keep the stimulus
amplitude constant. Why is constant stimulus amplitude important? For each single trial,
note the magnitude of the ∆R of the JAR. Remember that ∆R is the difference between
the fish's EOD before stimulation and the fish's EOD after the EOD frequency has
leveled off at a final value. Note that a ∆R could have either a positive or negative value.
• Make a graph where you plot ∆R versus ∆F.
47
response
Assignment
DUE: On November 25, 11:59 PM to avoid conflict with Exam 3 on November 23; via Canvas.
This write up is worth 15 points. It will include an Abstract, Introduction, Materials and
Methods (either a brief summary of lab protocol or reference the lab manual), Results
(including 5 properly labeled figures), and a discussion section. The format is 12 font, 1 inch
margins all around, double spaced with a 10 page maximum to the text.
• Figure 1. Record a few cycles of EOD on Labchart (see pg. 67, #5).
• Figure 2. EOD of fish in spectrum mode (see pg. 67, #9).
• Figure 3. EOD during fish stimulation in scope mode (see pg. 68, Stimulating the
Fish, #4)
• Figure 4. EOD – before, during and after stimulation (see pg. 68 & 69, Time course
of the J.A.R., # 2 & 3)
• Figure 5. Plot of Δ R versus Δ F (see pg. 69 #4)
48
response
Lab 3B: Possible alternative lab, depending on fish availability

NEUSCI 302 Lab Exercise – Electric Organ Discharge (EOD) and Waveform characterization
Weakly electric fishes possess electric organs that generate three-dimensional electric
dipole fields around their bodies, which can be used to detect and actively locate objects in their
environment. The weakly bioelectric fields produced by the electric organ discharges (EODs)
have amplitudes in the range of millivolts to a few volts and are used for electrolocation and
social communication.
There are two types of freshwater weakly electric fish: Mormyriformes and
Gymnotiformes. The mormyrids are a pulse species and the gymnotoids are a wave species (Fig.
1). Mormyrid fish such as Gnathonemus petersii produce pulse EODs that are single-cycle clicks
repeated at rates from below 1 Hz to about 65 Hz at rest. The EOD pulses are often separated by
pauses that are long (and often variable) compared to the duration of the EOD. The amplitude
spectrum of a single pulse contains energy that has a broad and continuous frequency range with
a flat peak region (thus, the signal is ‘broadband’). Frequencies of peak amplitude are usually
below 10 kHz (but can be as high as 25 kHz).
Figure taken from: Kramer (2009) Electric organ discharge. In: Binder, Marc D. und Hirokawa, Nobutaka und Windhorst, Uwe, (eds.)
Encyclopedia of Neuroscience. Springer, Berlin.
49
response
Gymnotoid fish such as Apteronotus albifrons produce a continuous wave type EOD.
The EODs are produced in a quasi-sinusoidal manner with the duration of pulses being
comparable to the duration of the interpulse interval. Compared to pulse EODs, wave EODs are
of higher repetition rate and weaker in amplitude. Wave type EODs compensate for being weak
by strongly contrasting
from background noise via the harmonic structure of the wave EOD (see Fig. 1). There is little or
no D.C. component to the EOD of wave fishes (only a few studied) which makes fish with these
EODs less prone to detection by certain predators (Bennett 1971, Bass 1986, Kramer 1996).
References
Bennett MVL (1971) Electric organs. In: Hoar WS, Randall DJ
(eds) Fish physiology, vol 5. Academic, London,p 347–491.
Bass AH (1986) Electric organs revisited: evolution of a

vertebrate communication and orientation organ. In: Bullock
TH, Heiligenberg W (eds) Electroreception. Wiley, New
York, pp 13–70
Kramer B (1996) Electroreception and communication in

fishes. Gustav Fischer Verlag, Stuttgart
Figure 2. EODs from several species of electric fish (from Hoar and
Randall, 1971).
In this exercise, you are asked to characterized the waveform of a gymnotoid (Apteronotus
albifrons) and if possible a mormyrid species (Gnathonemus petersii) of weakly electric fish.
The waveform of these fish can convey information about the individual’s species, sex, social
rank and reproductive status (i.e., adult vs juvenile).
1) Gymnotoid species - measure the frequency (discharges per second) of the continuous wave
type EODs produced by the blackghost knivefish (Apteronotus albifrons). Make 6-10
measurements of the EOD frequency and then compute an average EOD rate.
Next, measure the amplitude and duration of the EOD waveform. Is the amplitude and
duration of the EOD stable? Make 6-10 measurements of the amplitude and duration of the
EOD’s monophase peak and compute the EOD’s average amplitude and duration.
50
response
Do any of the EOD waveform measurements vary with animal size?
Try and estimate the total length of the fish (head to tip of tail). You can do this by measuring
the length of one square of the mesh tubing (which contains the animal) and then extrapolate
the length of the animal based on the number of squares that corresponds to the animal’s total
length. Compare your data with that of the measurements from other groups.
Plot the following: EOD frequency vs. total length and EOD waveform duration vs total
length. Do any of these measures correlate with total length of the animal?
2) Mormyrid species - measure the frequency (discharges per second) of the pulse type EODs
produced by Gnathonemus petersii. Note that the pulse discharges can be quite variable. Try
to get a resting measure of EOD pulse rate when the animal is at rest (i.e, you should
minimize arousal of the animal). Make 6-10 measurements of the pulse EOD frequency and
then compute an average EOD rate.
Next, measure the amplitude and duration of the EOD waveform (see figure below). Is the
amplitude and duration of the EOD stable? Make 6-10 measurements of each of the EOD’s 4
pulse phases (P 1-4). Compute the average duration for each phase. Also, make 6-10
amplitude measurements of the positive and negative peaks. Compute the average amplitude
for each peak. Which peak is greater? How do the amplitudes of these peaks compare to the
peak amplitude of the wave type fish?
Do any of the EOD waveform measurements vary with animal size?

Again, try and estimate the total length of the fish (head to tip of tail). You can do this by
measuring the length of one square of the mesh tubing (which contains the animal) and then
extrapolate the length of the animal based on the number of squares that corresponds to the
animal’s total length. Compare your data with that of the measurements from other groups.
Plot the following: each phase duration (P1-4) vs total length and total EOD duration (P1 +
P2 + P3 +P4) vs total length.
Other EOD characteristics such the durations of the major positive and major negative
phases have been used to describe sex differences. Males are known to have a phase 2
duration > 24 msec and a phase 3 duration > 15 msec whereas females are known to a phase
2 duration < 23 msec and a phase 4 duration <15 msec (Landsman 1993). How does your fish
compare?
51
NEUSCI 302 Autumn 2020 Lab 4: Cockroach flight
Lab 4: Electromyographs (EMGs) of the Cockroach Flight

Muscles: Behavioral Evidence for the Existence of Central Pattern
Generators (CPGs)
**This laboratory exercise was adapted from one developed by Dr. David Yager and Dr. Sarah
Durand (Univ. of Maryland) with the help and advice of Dennis Dever.
Aims
• Learn to record chronically in behaving whole animals.
Introduction
Now that you have worked with various parts of the cockroach, it is time to experience the
triumphs and failures of “whole animal” in vivo electrophysiology. Although recordings from a
whole animal as it performs a specific behavior are sometimes difficult to achieve, the recordings
often represent the closest approximation to understanding function in the natural condition. The
cockroach is exceptionally well adapted to its niche. The cockroach family has been around for
about 350 million years, far longer than humans have been dropping food scraps on the kitchen
floor. If you put today into a temporal perspective, consider that you are going to be accessing
evidence for a neural mechanism that has changed little since this animal evolved. In essence you
will be recording neural networks that existed 350 million years ago!
Figure 1. Cockroach anatomy. From J.G. Houseman, Animal Form and Function (2003).
Insects, including the cockroach have four wings, an anterior (mesothoracic) and a posterior
(metathoracic) pair. The goal today is to record the wing beat frequencies and phase relationships
of the elevator and depressor muscles between the wings of each pair and the phase relationship
between the anterior and posterior pairs. Also, we want YOU to come up with some ideas for
manipulations that could alter these relationships and then test these ideas. In other words, while
much of the exercise is straightforward and laid out for you, today you get to be the
52
experimentalist and do some hypothesis testing. You now have almost two quarters worth of
neurophysiological experience; try to pull together all you know for this lab.
Figure 2. Muscle/Wing relationship. From Yager and Durand (1998).
CAUTION: Before you start, please realize that cockroaches can successfully colonize almost
any niche, even the Health Sciences Complex. You are responsible for your subject cockroach.
Do not let it run up your sleeve! Do not let it fall into your backpack! Do Not Let It Escape!
Equipment Setup
Come on! You know how to do this by now! You will need to use Labchart to record all of the
responses to your manipulations today. You will be running four channels, so you need to set up
the amplifier with the appropriate number of cables and set up Labchart accordingly.
Unfortunately, you only have only one Humbug, so only one channel can be filtered. Since
filtering only one channel would make comparison between channels difficult, do not include the
Humbug in your circuit today.
Animal Preparation
Cockroaches (Periplaneta americana) have already been “posted” in preparation for this lab.
Posting is a procedure of attaching a holder to an animal so that it can be restrained in some
manner. In vertebrate recording, typically a post is attached to the skull. For the cockroach, a
post is attached to the prothorax (front thorax), anterior to the wing-bearing mesothorax (middle
thorax) and metathorax (distant or posterior thoracic segment). Does this remind you of the
prosencephalon, mesencephalon, and metancephalon divisions of the vertebrate brain? The head
with eyes and antennae remain unencumbered. The cockroach post also has a screw in the
coelom of the prothorax that can serve as the reference electrode; this will be the “common”
reference electrode for all four channels of active electrodes (see below).
53
CAUTION: you must take care not to damage or glue the wings or wing hinge joints during this
exercise. Treat the cockroach with care so that its performance is not compromised.
• Obtain a chilled out cockroach and place it into a chilled Sylgard Petri dish. At your
station find the four-inch length of stainless steel tubing with a (reference) wire attached.
This is the “tether rod.”
• Hold the post with forceps and slip one end of the tether over the post on the cockroach.
You want to restrain the cockroach in the Petri dish using the tether rod with the tether
clamped in the micromanipulator on the isolation table at your station. You will need to
fiddle with the roach, Petri plate, and manipulator until the cockroach is pegged securely
down on the Sylgard and cannot move. At this point in the procedure, face the cockroach
away from you so that you will be able to view its back. You may want to use the
dissecting scope for the following procedures.
• Once the roach is pinned, place a drop of nickel print (essentially this is a paint with a
large amount of nickel dust in it) on the connection between the tether rod and post. This
helps bind the tether to the post and ensures electrical continuity. After the nickel print
dries, again ensure that the roach is tightly pressed against the Sylgard.
• Using insect pins, spread all four wings out so that you can visualize the dorsal (top)
plates of the meso- and metathoracic segments.
• Now that all four wings are spread out and held in place with insect pins, use a small
piece of Kimwipe or a Q-tip (pull the cotton and twist to a point) dampened with alcohol
and held in forceps to wipe the oil off the cuticle of the cockroach between the wings.
• While the alcohol is drying, make a quick sketch of the thoracic segments and wings and
note which are bigger or smaller.
• After the alcohol has dried, use an insect pin or syringe needle (CAREFUL—don’t poke
yourself) to drill a hole in the center of each segment’s dorsal plate.
• Now, using forceps, push one of the “bullet” electrodes into each hole until the entire
bullet passes through the cuticle. Insert all four bullets.
54
Figure 3. Electrode placement. From Yager and Durand (1998)
• If there is significant leakage of coelomic fluid you may need to “plug” the hole with
VetBond (veterinarian superglue). If there is no significant coelomic fluid leakage you
should not apply VetBond. Wipe any excess coelomic fluid from around the penetrations
and seal the holes with the tiniest subdrop of Vetbond: ****the best way to do this is to
use a toothpick to wick a small amount (not a big drop) of glue onto the cuticle
holes.**** Be careful not to glue the wings or wing hinge joints. It is better to let the
holes leak than to get glue all over the cockroach. Let the Vetbond dry for about five
minutes. (Please keep moisture away from the Vetbond. Hydroxyl groups in water cause
it to start hardening and the whole bottle can go bad if it wicks in moisture.) [Note: In
previous years, we have not always found this to be necessary.]
WARNING: after the cockroach is implanted, be sure to touch ground
before touching the tether rod, cockroach, or recording wires and cables.
A common mistake in chronic recordings is to forget to touch ground
immediately before or while touching the animal. The static discharge
can kill the neurons being recorded.
• This is an important point to take notes: which color electrode corresponds to which
wing. Label the picture you drew—you will need it to interpret the later recordings.
• Route the electrode wires between the wings close to the thorax. Position the wires so
that they do not interfere with the wings during “flight.” Attach the wires to the tether rod
with “tacky wax” so that they don’t interfere with the wings. Make sure the wires are as
short as possible between the cockroach and the wax so that no movement artifact is
recorded in the EMG signal. One electrode wire will go to each channel of the
extracelluar amplifier. Connect each electrode wire to the active side (black) of the
amplifier cable and the reference wire (on the tether) to the white wire of all four
55
amplifier cables. N.B.: Keep track of which wing segment/electrode cable color
corresponds to which amp channel. Secure the amp cables so that they are stable enough
that they do not put any strain on the recording wires. Connect a ground wire to the top
of the tether rod.
• Now that the cockroach is implanted with electrodes, you will want to position the
cockroach so that you can blow on it from different directions. Fill the plastic cup with
warm water and put the Ping-Pong ball into the cup. The warm water will help the roach
recover from the cold anesthesia and the ping pong ball will be a substrate for the roach
to walk on between “flights.” ****This is a tricky part**** The manipulator should be
near the bottom of its travel during non-flight intervals (when the roach is sitting on the
ping pong ball) but when the manipulator is retracted/elevated, it should be possible to
displace the ping pong ball from the roach’s feet. You may need to push the ball from the
roach’s feet with the forceps. When the ball is displaced from the roach’s feet, the
cockroach will begin to fly—this is when you want to start collecting data.
Figure 4. Preparation for flight recording. Adapted from Yager and Durand (1998).
Data Collection
56
By now you should be able to wire up the rig yourselves (if you can hook up a VCR, DVD,
computer, etc., it’s not that different). You will want to record four channels of data
simultaneously and have the audio monitor linked to one. You may use Labchart to record four
channels of data simultaneously on one graph. We suggest that you have the roach perform a
“test flight” before collecting critical data with the computer. This will ensure that all the
equipment is working and that the cockroach will actually fly when prompted. Also, this baseline
recording will help you determine what the amplifier filter frequencies should be (consider the
possibility that the CPG has a rhythm of about 60 Hz). (300-5000)
Retract the manipulator so that the cockroach lifts the ping-pong ball above the water level. If the
animal does not drop the ball, ground yourself and then push the ball off with forceps or a
wooden applicator stick. If the roach does not start flying itself, gently blow on its head or use
the handheld fan positioned a couple feet away from the head (you don’t want to blow the roach
off of the tether!) and move it closer until flight begins. To stop flight, lower the roach with the
manipulator until it can again grasp the ball. Keep the flight bouts short so that the cockroach
does not get too tired. Hmmm, will the phase relationships be different at the beginning and end
of a very long flight?
When everything is working well, acquire about five seconds of good flight data. The beginning
and end of a bout may have complex signals because takeoff and landing on a floating ping pong
ball can be tricky. Make sure the cockroach is flying straight and not tilted. If it is needed, keep
the handheld fan at a constant distance and directed straight toward the roach’s head.
Look at the data for one wing. You will see compound EMGs from both the elevator and
depressor muscles. One will be shorter in duration and “faster,” the other will be broader with a
slower rate of rise. Can you figure out which is the depressor potential and which is the elevator
potential? Think about the work the wing is doing. What is the period for a wing beat cycle?
Congratulations, you are recording from a behaving animal.
If you have kept track of which channel corresponds to which wing, it should now be possible to
compare the relative phase of the left versus right wings and the anterior versus posterior wings.
How do the wing beat cycles compare? Is there any phase difference between left and right
wings? How about fore and aft wings? Calculate the degrees of any differences. Do any wings
lead or lag the others? You may have to measure several cycles across each paired comparison
and calculate a mean value for phase.
OK, so now you have some baseline flight data. However, insects do not normally fly straight
and level because they need to navigate around obstacles, they are buffeted by wind gusts, and
their wings sometimes get damaged by predators. So how might the EMGs (i.e., the CPGs)
change in these circumstances? Do some testing to see if phase relationships can change.
Here are some ideas, but you can come up with your own and run them by the TA’s. Do the wing
beat cycles slow as the roach is coming in for a landing? Does it need to see the “perch”
approaching? What if it encounters a burst of wind? What if it is tilted (roll or pitch)? What if
one wing is loaded more than the others? What if a wing gets “damaged?” Can you perform a
visual illusion on the roach and see a change? What if the body temperature changes? What if it
57
is given sugar water to drink? Or an octopamine injection? Can you think of other
manipulations?
Acquire data that address four different manipulations, comparing wing beat frequencies and
phase differences. You will need to summarize your findings in the lab write up.
Lastly, to euthanize the cockroaches, we will have to remove the head so that any potential
escapees cannot find mates and reproduce. This is required by the Health Department. So to
maximize your learning experience per animal, if you wish to do so, you may formulate a test to
see if the flight CPGs are located in the head. How do you test this? Does the animal still fly? If
so, do the frequencies and/or phase relationships change? Do they walk more or less now? What
does this say about descending control?
Figure 5. Cockroach anatomy with CNS included. From Yager and Durand (1998).
***When you have completed your testing, please ask Michael or the TAs for assistance in
disposing of your subject cockroach. Carefully remove the bullet electrodes from the
animals, making sure you do not break the solder connection between the bullets and the
wire. We need the electrodes back and we don’t want the cockroaches to escape.
Assignment
Normal lab report writeup.

Due: Due on Friday, December 4, at 11:59 PM to avoid conflict with lab presentations the week
of December 7; via Canvas.
58
NEUSCI 302 Autumn 2020 Additional Reading Material
Additional Reading Material

Weeks 6-7: Electrosensory and electromotor systems
Reading: These notes; Delcomyn chapter 14: 354-357, and chapter 20: 472-485
Introduction:
Even though it is quite common among fishes, the electric sense is one of the most recently
discovered animal senses. Because it needs a conductive medium, electroreception is always
associated with aquatic organisms. Electroreceptive structures can be found in many fishes and
in aquatic amphibians, in the Platypus and the Echidna, but not in invertebrates.
To use this sense, fishes have evolved special receptor organs, each of which contain several
electroreceptor cells. These receptor cells respond to environmental electric fields, which can be
of abiotic (originating from geochemical or seismographic processes) or biotic (coming from
other animals or from the receiving animal itself) origin. The animals can detect these electric
signals for orientation and navigation or for electrocommunication.
Besides “listening” to signals generated from outside sources, some electrosensitive fish species
have in addition evolved the ability to produce their own electric signals. These “electric fishes”
possess a specialized organ, called an “electric organ,” which is ontogenetically and
phylogenetically derived either from skeletal muscle or from motorneurons which no longer
innervate skeletal muscle. With these organs, some species can produce strong electric signals of
high voltage or current, which are perceptible for humans. These “strong electric fishes” use their
discharges for defense against predators and for stunning or killing their prey. In contrast,
“weakly electric fishes” produce “electric organ discharges” (EODs) of millivolts to only a few
volts. They use their self-produced electric signals in combination with their electric sense for
“active electrolocation” and for electro-communication.
This lecture will mainly be concerned with weakly electric fish.
Topics covered in this section:

1. Diversity of electroreceptive fish and EODs
2. Electric organs and the neurobiology of generating electric organ discharges
3. Electroreceptororgans, electroreceptor cells, and the neural basis of electroreception
5. The biology and neural basis of active and passive electrolocation
6. The 'jamming avoidance response': from sensation to action
Production of Electric Organ Discharges

The electric signals of weakly electric fish are of low amplitude (< 5 V), so low that neither a
human (even if we hold a fish in the hand) nor any non-electric fish can perceive them. Strongly
electric fish, like the electric eel (Electrophorus), who lives in South American rivers and lakes,
or the salt water electric ray (Torpedo), produce discharges of up to 600 V (freshwater species)
or up to 100 Amps (salt water species). Both types of signals are produced by electric organs,
which are built similarly in strong and weak electric fishes.
59
An electric organ consists of “electrocytes”, which are ‘former’ muscle cells that became
specialized for generation of EODs. That is, over time some myocytes lost their contractile
proteins and became cells that still fire action potentials, but no longer contract. These
electrocytes often are flat cells that are arranged in a very regular fashion within the electric
organ, like coins in a roll. Each electrocyte is innervated by a motoneuron from the spinal cord,
which receives input from the brain. An action potential in the motoneuron activates a
cholinergic synapse at the electrocyte and causes one electrocyte action potential. Because all
electrocytes in the electric organ are innervated and fire synchronously, the whole electric organ
discharges as one unit. Realize that, even though the fish does not move as a result of firing
these former muscle cells, discharging the electric organ is a still a motor behavior.
Usually, only one face of the electrocyte is innervated by the motorneuron. Depending on the
species, the two faces of the electrocytes can either both produce action potentials, or one or both
produce EPSPs only. The temporal and spatial relation of the potential changes of the faces of
the electrocytes determine the shape of the EOD waveform.
Strongly electric fish often produce short monopolar electric pulses. In contrast, various species
of weakly electric fish produce a variety of EOD waveforms, which play an important role in
electrolocation and electrocommunication.
Fish with weak electric organ discharges belong to two groups: the Gymnotiforms which occur
in freshwater throughout tropical South America, and the Mormyriforms which occur in Africa,
again in tropical freshwater. Within each group, different species have either a wave-type (i.e.,
continuous) or a pulse-type (i.e., discrete or intermittent) EOD. About half of all gymnotiform
species (South America) and only one African species (Gymnotus) produce wave-type EODs. In
the lab, we will have one South American wave-type fish (Eigenmannia) as an example of this
group. Wave-type EODs are continuous signals, which more or less resemble a sine-wave. The
frequency of the wave can be between 50 and more than 2000 Hz, and can be extremely constant
over short time periods. However, during certain kinds of behavior, e.g. during the jamming
avoidance response (see below) or during electrocommunication, the EOD frequency can be
varied by the fish. In extreme and rare cases, wave-fish can even shut down their electric organs
completely and can be silent for short periods of time.
Weakly electric fish that emit pulse-type EODs live in Africa and South America. These EODs
have a constant amplitude (all-or-none events) and are often very brief (down to less then 200
µs). They are followed by a silent period, which always is significantly longer than the EOD
duration itself. African pulse fish produce electric pulses at a very irregular temporal pattern,
which is varied constantly by the fish. This pattern contains information, which is used by other
fish during electrocommunication. In the lab, we will work with the elephant nose fish
(Gnathonemus petersii) as an example for this group. South American pulse fish are more
regular in their discharges. One example species (Brachyhypopomus pinnicaudatus) often
produces a regular rhythm of EODs of about 30 Hz. Like in wave-fish, this rhythm can be
changed in certain behavioral contexts.
Active and passive electrolocation

When a fish discharges its electric organ and an EOD is emitted, an electric field builds up in the
water around the fish. If the field is perceived by the same fish that has produced it, the field can
be used for “active electrolocation”. In this mode, a fish actively produces a signal, “sends” it
out into the environment and perceives the consequences of this motor act. Objects in the vicinity
of the fish cause distortions of the self-produced field, which lead to changes of the perceived
60
electrical stimulus at certain parts of the fish’s skin. By analyzing these field distortions, the fish
can detect, localize and analyze objects. The electric signals are perceived by the electroreceptor
organs, which are embedded in the fish’s skin and are distributed over almost the entire body
surface of the animal (see below). (Note that if the field of an EOD emitted by one fish is
perceived by another electric fish, we also can speak of a case of electrocommunication.)
In contrast to active electrolocation, during passive electrolocation, in contrast, the fish “listen”
to electric signals coming out of the environment, either from animate or inanimate sources (see
above). In this mode they do not actively produce electric signals but instead attend to outside
electrical sources. Passive electrolocation is used by these fish for detecting prey or enemies,
and/or for orientation and navigation in their habitat. Weakly electric fish can perform both
active and passive electrolocation (simultaneously!), while fish that have no electric organs but
are electroreceptive (e.g. sharks and rays) can only perform passive electrolocation.
Electroreceptors
All electrosensitive fish have ampullary electroreceptororgans, with which they can detect weak,
low-frequency electrical signals. The ampullary receptororgans of elasmobranchs (sharks and
rays) are called the Ampullae of Lorenzini, and are the most sensitive electroreceptor organs
known in nature, being able to detect electrical gradients of only 0.005 to 0.02 µV/cm. [This is
equivalent to being able to detect the electrical current generated by a 1.5 volt battery if one lead
were placed in the ocean 1800 miles from the other !] Ampullary receptors are used for passive
electrolocation, i.e. for the detection of signals not produced by the receiving fish itself.
Tuberous electroreceptororgans can only be found in fish with an electric organ, which produces
the EOD electric signals. They are used to detect high-frequency changes in voltage caused by
the fish’s own EOD or the EOD of conspecific fish, and hence are used in active electrolocation
and in electrocommunication (detecting the EODs of other electric fishes). African Mormyrids
have 2 types of tuberous receptor organs, one used for active electrolocation (the Mormyromasts)
and one used exclusively for electrocommunication (the Knollenorgans). Mormyromasts are
amplitude coders, i.e. they are specialized to detect small changes in the amplitude of the self-
produced EOD. In addition, they also respond very sensitively to changes in EOD waveform,
which occur during the electrolocation of certain types of objects (capacitive objects).
Knollenorgans are a second type of electroreceptororgans found in Mormyrid fish. They are
specialized for detection of temporal information, and are used for communication.
Knollenorgans are time-coders, which fire with high sensitivity a single action potential to the
occurrence of one temporal aspect of another EOD.
Gymnotiforms also have two types of tuberous electroreceptor organs, one of which is used for
coding of temporal aspects of an electric stimulus, while the other one codes with high precision
even minute amplitude changes. In contrast to mormyriforms, gymnotiforms use both types of
receptor organs for active electrolocation and for electrocommunication.
All electroreceptororgans contain several receptor cells, usually up to 30 but some ampullary
organs can contain several hundred of receptor cells. Electroreceptor cells are secondary receptor
cells, i.e. they don’t possess their own axon but instead are innervated by an afferent nerve fiber
through a chemical (sometimes mixed chemical/electrical) synapse. In contrast to light
transduction in rods and cones, or to sound transduction in cochlear hair cells, because the
adequate stimulus for electroreception is already electrical, no transduction process in the
receptor cells is required. (Transduction usually transforms the modality of the stimulus (e.g.,
light, sound, odor, touch, etc.) into electrical energy, which is used by nerve cells.) The receptor
61
cells are directly de- or hyperpolarized by the stimulus, a process which only has to be amplified
by the receptor cells, e.g. through voltage gated ion channels. The stimulus-caused gradual
change in voltage (receptor potential) is ‘translated’ into a gradual change in transmitter release
at the synapse between the receptor cell and its afferent nerve fiber. The resulting EPSP in the
afferent nerve fiber is then translated into a series of action potentials at the dendritic spike
initiation zone (the fiber innervating the receptor cell is a dendrite!), and the neural signal is
conducted towards the brain.
The afferent fibers coming from the electroreceptor cells project through the lateral line nerve to
the brain into a nucleus called the electrosensory lateral line lobe (ELL) in weakly electric fish.
This nucleus, and also several other nuclei of the electrosensory pathway in the brain, is
organized topographically according to the location of the electrosensory organs on the surface
of the body. Therefore, these nuclei contain somatotopical maps of the fish’s electroreceptive
body surface. In some brain regions, such as in the ELL, there are several parallel somatotopic
maps, each of which processes one aspect of the electrosensory input, e.g. amplitude or phase of
the stimulus. In even higher stages of the brain, the information which was first processed in
parallel converges to allow the extraction of complex stimulus properties, which require the
combinational processing of several stimulus parameters.
The jamming avoidance response of Eigenmannia

The gymnotiform wavefish Eigenmannia constantly produces an electric signal with its electric
organ. During active electrolocation, it can detect nearby objects because they lead to changes in
amplitude and phase (timing) of the signal at the epidermal receptor organs. When swimming
along an object, amplitude and phase modulations of the perceived electric stimulus occur at a
modulation frequency of a few Hertz. However, if another individual Eigenmannia comes close,
very similar changes in sensory input occur, if the intruder’s EOD frequency is similar to the
frequency of the first fish. This similarity leads to jamming of the fish’s electrolocation ability.
Because two Eigenmannia individuals that meet constantly discharge their electric organs, the
EODs of the two fish overlap. When combined, the two signals cause a beat pattern, i.e. a
rhythmic up and down in signal amplitude accompanied by a rhythmic change in signal phase.
The frequency of this amplitude and phase modulation equals the frequency difference between
the two EOD signals. For example, if one fish discharges its electric organ at 400 Hz and the
other one at 398 Hz, the beat pattern has a modulation frequency of 2 Hz. The same modulation
frequency would occur, however, if the intruder fish had an EOD frequency of 402 Hz. (We will
focus on one “resident” fish, but realize that both the resident and intruder will experience a
combined waveform that will beat at a frequency equal to the difference in the two fish’s EOD
frequency.)
In order to avoid being jammed by its neighbors, Eigenmannia performs a so called jamming
avoidance response (JAR). If a fish detects the signal of another wavefish of a frequency similar
to its own, it will shift its EOD frequency away from that of the neighbor’s. For example, a
resident fish originally discharging at 400 Hz meeting a neighbor discharging of 398 Hz will
increase its EOD frequency to higher values, i.e. to about 410 Hz. If the intruder discharges at
402 Hz, the resident fish will lower its EOD frequency and will end up with an EOD of about
390 Hz. (What will the intruder do?)
The problem the fish faces when confronted with an electrical stimulus from a conspecific is that
both higher and lower frequencies of a foreign EOD cause the same frequency in the resulting
beat pattern (e.g., 402 Hz - 400 Hz= 2 beats per second, and 400 Hz – 398 Hz = 2 beats per
62
second). Even though the beat frequencies in both cases are very similar, the resident fish
performs the behavior in the “correct” direction, i.e. its EOD frequency goes up if the neighbor’s
EOD frequency is lower than its own, and its EOD frequency goes down when the neighbor’s
frequency is higher than its own. How is this possible? (If YOU are humming one frequency,
how would you determine if your neighbor is humming at a higher or lower frequency? Well,
it’s far more complicated for electric fish.)
In the lecture we will follow the processing of the sensory stimuli from the electroreceptor
organs of the fish through the brain to the electromotor centers, which finally command the
electric organ response (the motor behavior that increases or decreases the EOD frequency).
Thanks to the pioneering work of the late Walter Heiligenberg, we now understand very well
how the fish perceive foreign EODs, determine the frequency difference relative to their own
EOD, and perform the appropriate motor behavior to avoid being jammed by the neighbor’s
EOD.
Fig. Schematic representation of the JAR-pathway in the brain of Eigenmannia
brain
TS
NEdown ELL
NEup SPPn spinal cord
PPnG
Rn
Pn
....
towards electromotor neurons
from P- and T-
electroreceptororgans
electrocytes of
ELL: electrosensory lateral line lobe
NE: nucleus electrosensorius, down-region
electric organ
....
down
NE: nucleus electrosensorius, up-region

up
Rn: relay nucleus

SPPn: sublemniscal prepacemaker nucleus
TS: torus semicircularis
Pn: pacemaker nucleus
PPnG: prepacemaker nucleus, gradual rise region
In order to perform a jamming avoidance response, fish have to first determine the sign of the
frequency difference between their own and their neighbor’s signal. They achieve this by
measuring the phase (with their T-unit tuberous electroreceptororgans) and the amplitude (with
their P-unit tuberous electroreceptororgans) of the combined signal they perceive on one part of
their body and compare that information with the signal received with receptors on a different
part of their body. The somatotopically organized information from the T- and P-units is
processed in parallel by the hindbrain ELL, the first station in the brain. Several types of neurons
are found in the ELL, which respond to changes in amplitude or phase in different ways. In the
next brain station, layer 6 of the midbrain nucleus torus semicircularis (TS) (which pair of
mammalian collicli is the TS homologous with?), amplitude and phase is recombined and
63
specialized neurons exist, which respond to certain combinations of amplitude and phase of the
signal. From the torus, efferent neurons project to the nucleus electrosensorius (NE), which has
two subdivision named NEup and NEdown. In NEup, neurons only respond when the fish has to
increase its EOD frequency, (i.e., when the neighbor’s frequency is lower than its own). The
opposite is true for NEdown neurons. The NE serves as a senso-motor interface providing input to
several electric premotor nuclei. In particular, NEup projects to the PPnG, which in turn excites
the pacemaker nucleus (Pn) and thereby increases the EOD frequency produced by the electric
organ. NEdown projects to the SPPn, a tonically active nucleus in the midbrain which usually
excites the relay nucleus (Rn) and thereby keeps the EOD frequency and at a slightly elevated
level. NEdown actually inhibits the SPPn, which in turn releases the Rn from the excitation
provided by this nucleus. Because of the resulting reduced activity of Rn, the EOD frequency of
the fish decreases.
The JAR is a remarkable constant and precise behavior that can be triggered by sensory
stimulation even in restrained fish. Because it is still performed when the fish is fixed in an
electrophysiological set-up, its neural basis has been studied extensively and in great detail. The
unraveling of the neural mechanisms of the JAR has been very successful and has turned this
behavior in an excellent neuroethological model system. The JAR is one of the few behaviors of
animals that is understood now in detail, from the sensory periphery through the brain with its
sensory, sensory-motor interfaces, and motor nuclei, to the motor (electric) organs.
Week 8: Motor control
Although much of our course so far has been concerned with the ways in which sensory
information is integrated and analyzed by the brain, sensory processing is only half the story
about how nervous systems provide the basis for behavior. Some motor behaviors, especially
apparently ‘simple’ motor acts such as startle or escape responses, consist of single episodic
motor actions. Other behavioral output of animals, for example during locomotion, is driven by
rhythmic patterns of motor activity. Voluntary activities like some ‘fixed action patterns’,
walking, flying (even singing in some animals!) require the precise coordination of sets of
muscles that are contracted or relaxed. Many involuntary actions, such as the beating of a heart
or the coordinated movement of food through the gut require similar coordination of muscle
activity.
In the next few lectures, we will explore the ways in which neural circuits can produce either
episodic (non-rhythmic) motor programs or motor programs that consist of rhythmically repeated
motor acts.
64
Week 8: Motor systems 1
Readings
1. Motor unit, stretch reflex, central pattern generators (CPGs): Delcomyn chapter 15 and
16; Kandel, Schwartz & Jessell: Chapters 34-36 (there is more detail here than we need,
but it is a very comprehensive account of the principles of organization of movement in
vertebrates)
2. Locust flight CPG: Carew, Ch 6, pp155-172
3. Crayfish escape response: Delcomyn chapter 18, pg. 417-424; Carew Ch 17, pg. 187-206
Central pattern generators:

Cellular properties of neurons can cause a single neuron (or muscle cell as in the example of the
pacemaker cells of the heart) to be rhythmically active. As in the example of the heart
pacemaker, the endogenous activity of such a neuronal oscillator (or pacemaker neuron) is
caused by a cyclic interplay of different ion-currents and -channels. There is not a single
mechanism valid for all neural oscillators, because different oscillators often have a different set
of ion channels. In addition, other properties of a neuron (such as tonic activity, accommodation,
postinhibitory activity, or bistable membrane potentials) can influence its rhythmic activity.
Neurons that produce rhythmic activity never work in isolation. In contrast, they are embedded
in a network of neurons which are interconnected. Single neurons are never 'dictators' of a
rhythm of such a network, but rather cooperate in order to produce a coherent rhythm of the
whole network. Often it is the specific connections between the neurons in a network that cause a
specific rhythm. A network that produces oscillatory activity is called a 'network oscillator'. In
the lecture we will talk about different types of such network oscillators, that employ different
mechanisms to produce a neural output rhythm.
A well-studied example of a central pattern generator controlling vertebrate locomotion is in the
lamprey. The lamprey has a brainstem and spinal cord with all basic vertebrate features, but with
orders of magnitude fewer nerve cells of each type than higher vertebrates. Moreover, the
lamprey’s spinal cord and brainstem can be maintained in vitro over a period of several days,
with spontaneous or induced activity in the locomotor CPGs. These conditions are very favorable
from an experimental point of view and have enabled Sten Grillner and his colleagues from
Stockholm/Sweden to detail the networks that underlie the locomotor behavior in cellular terms.
The lamprey swims by producing an alternating activation of motorneurons on the left and the
right side of each segment along the body. In addition, the 100 different segments are activated
successively with a phase delay. The phase delay is about 1% of the cycle duration. This results
in the propagation of an undulatory muscular wave from rostral to caudal. This caudally directed
wave pushes the animal forward through the water. While swimming, the animal generally
maintains the dorsal side up, and if this position is disturbed, it will be corrected rapidly, mainly
as a result of signals from the vestibular apparatus. These signals act on the reticulospinal
neurons, which project down the spinal cord. These neurons become activated maximally at
different angles of tilt. Brainstem neurons are thus responsible for the initiation of locomotion as
well as for providing some sensory input, while the spinal cord provides the motor pattern.
The spinal CPGs also receive input from excitatory and inhibitory sensory stretch-receptor
neurons that sense the lateral bending movements occurring during locomotion. The spinal
network contains excitatory glutamatergic interneurons that project to all types of ipsilateral
interneurons and to motorneurons, and inhibitory glycinergic interneurons with a crossed axonal
65
process that inhibits all types of cells in the locomotor network on the contralateral side. The
cycle-to-cycle pattern generation of this network is produce by the interacting glutamatergic and
glycinergic neurons, with NMDA receptor channels playing an important role at lower rates of
locomotion. The fine tuning of the network, produced by serotonin, dopamine, and GABA-
systems, involves a modulation of Ca2+-depenedent K+ channels, high- and low-threshold
voltage-activated Ca2+ channels, and presynaptic inhibitory mechanisms. Mathematical modeling
has been used to explore the capacity of these biological networks.
The control system of the lamprey, phylogenetically old among vertebrates, has most likely
evolved and been modified to encompass the control of fins and limbs in more recently evolved
species. Although the locomotor behavior in lamprey is much simpler than that occurring in
terrestrial vertebrates, the basic mode of operation of this control system is similar in all
vertebrates.
The principle of multiple and coupled oscillators for locomotion in lampreys (each segment has
its own CPG) can also be applied to locomotion of tetrapods. In these animals, each leg is
controlled by its own separate oscillator network (CPG). Coordinating connections are used to
adjust the activity between the contra- and ipsilateral extremities and between the front- and
hindlegs. The different gaits of animals (walk, trot, pace, gallop etc.), as well as a change from
forward to backward locomotion, are achieved by changing the connection pattern between the
CPGs of each leg.
Command neurons and the crayfish escape response

An example of motor output controlled by command neurons is the escape response of crayfish.
Flexor muscles in the tail are innervated (among other connections) by two pairs of giant
interneurons (GI) of the ventral nerve cord. GIs have large-diameter axons, specialized for rapid
conduction of action potentials, which make specific synaptic contacts with other interneurons
and with motor neurons that control the abdominal musculature. The two medial GIs receive
sensory input from receptor cells at the anterior part of the animal (mainly from the head). They
evoke a tail-flip, which causes the animal to jump backwards, away from the stimulus. The two
lateral giants receive input from sensory receptor at the tail of the animal. They cause a tailflip
which causes the crayfish to jump upwards and forward. These different orientations of the two
escape responses, which make sense because in both cases the animal is projected away from the
threatening stimulus, are caused by the specific pattern of connections to postsynaptic neurons in
the animal's abdomen. The medial giants make electrical synapses with large flexor motor
neurons in all segments of the abdomen. When they fire, all flexor motor neurons are excited and
all abdominal segments are flexed. The abdomen suddenly bends inward and a tailflip is created
that propels the animal backwards. The lateral giants, on the other hand, have synapses with
large motor flexor neurons only of the first three segments of the animal's abdomen. If they fire,
the posterior segments are not flexed and remain flat, while the first 3 segments are bend
inwards. As a consequence the main flexor component is directed downwards and the animals is
tilted forward. The pattern of activation (in this case represented as a ‘space code’ rather than the
temporal code we see in some other central pattern generators) leads to a distinct behavioral
response.
The GIs can be considered to be 'command neurons' because each organizes the complete escape
sequence for which it is responsible. Besides activating the appropriate motor neurons, they
inhibit some other muscles to make sure there are no competing instructions from other neurons.
The concept of the 'command neuron' implies that a single neuron is both necessary and
66
sufficient to evoke a behavior. However, researchers now recognize that no behavior is

exclusively initiated by single command neurons. In contrast, most if not all behaviors involve
more than one type of neuron for its control and regulation, and can be initiated through more
than one pathway. In crayfish, escape swimming can be triggered in the absence of the giant
interneurons. However, it then takes longer to elicit and a stronger sensory stimulus is required.
In the intact animal this would make no adaptive sense in terms of survival, because for an
escape reaction to be useful it has to be initiated extremely fast and efficiently to take the animal
away from a sudden danger.
The Mauthner-Neuron-System in fishes:

A sudden and unexpected vibratory stimulus can cause an escape response in many fishes, which
is, as in the crayfish, directed away from the stimulus. Most fishes have in their brainstem two
bilaterally symmetrical Mauthner neurons whose somas (somata) and dendrites are extraordinary
large. Each Mauthner cell receives sensory input from the auditory, visual, vestibular,
somatosensory, and mechanosensory lateral line (hydrodynamic stimuli) afferents. The axon of
each Mauthner cell crosses the midline (decussates) and projects down the spinal cord and
synapses on the trunk (body) musculature that is contralateral to the soma. If the Mauthner cell is
depolarized above threshold by a sensory input, the muscles of the contralateral side contract
causing a sudden tailflip which turns the animal away from the stimulus.
Besides activating certain muscle groups, the Mauthner system has inhibitory connections with a
similar function as in the crayfish escape circuits. In addition, the two Mauthner neurons inhibit
each other reciprocally through interneurons with chemical and very unusual electrical inhibitory
synapses. This inhibition ensures that at a given time only one Mauthner neuron is active. This is
important because vibratory stimuli in the water tend to stimulate both sides of the animal after a
small time delay, which could depolarize both Mauthner neurons. This would lead to a
contraction of the whole trunk musculature of the fish and thus to its paralysis.
67
Appendix A: A short primer for the Tektronix TDS 210

Oscilloscope
Three data recording tools used to be common in every electrophysiology lab. A cathode ray
oscilloscope (CRO or ‘scope’) used to be one of the most important and was (still is) found in
virtually every lab. It allowed the researcher to view the output (membrane potential) of a neuron
as a function of time. An oscilloscope provides very high temporal resolution, allows
visualization of events on millisecond or even microsecond time scales. Hard copies were
produced by photographing the image on the screen, a process aided by ‘storage’ modes on some
scopes. Two other major tools were used for this task (often at the same time). A chart recorder
produced a direct ‘hard copy’ of data, and kept continuous record of very long segments of data,
but had relatively poor ability to discriminate events on a short time scale (poor resolution)
because of the low-pass filtering induced by inertia of the pen. The third tool in many labs was a
tape recorder, which didn’t allow ready visualization of the response, but kept a high fidelity
record of it that could be played back on a chart recorder or oscilloscope at any future time.
To a large extent, we have replaced these tools with computerized data acquisition systems (like
the Powerlab). It is no coincidence that the software that we have provided for you, Labchart, are
named thus. The Maclab system includes many of the functions of oscilloscope, chart and tape
recorder, as well as including some capabilities of a function generator. It is designed to be a
flexible ‘virtual instrument’. Many labs use other software packages that are more specifically
dedicated to particular tasks (e.g. Axon Instruments ‘PClamp’ software for voltage and patch-
clamp recordings).
Despite the growing role of computers in data acquisition, many of us still use an oscilloscope as
an additional aid to recording responses, for the following reasons:
• A physical scope provides an additional set of input channels and amplifiers for viewing
data in different ways, so that we can reserve the computer for other tasks. For example,
when set up for triggered data acquisition in short bursts (e.g. as we did in recording from
cockroach mechanosensory spines) you can no longer view the data source for the rest of
the time on the computer screen. A scope can be set up to display the same signal in a
similar way, but with continuous triggering, precluding the need to scroll through menus
to change trigger settings just to view the current activity of a neuron.
• Changing the way in which you view the data on the scope doesn’t affect the process of
data collection. To ‘zoom in’ on very brief events using the computer software requires
you to use a higher sample rate, and this may then limit you to sampling a shorter
segment of data. The settings on the oscilloscope are, however, independent of whatever
you do with the same data on a computer.
• Once learned, the interface for manipulating the scope is much faster and easier than
setting up a computer to record a response, because it has hardware (buttons and knobs)
that are available continuously for manipulating the signal, rather than being buried
within menus.
The Tektronix TDS 210/220 oscilloscope is a digital oscilloscope. It digitizes data, either
continuously or in triggered chunks and plots the data as lines on the display. Think of it is as a
miniature computer data acquisition system similar to the Labchart software program, but with a
dedicated hardware interface for the user (as well as some software menus that are accessed by
pressing certain buttons). These oscilloscopes are much cheaper (around $800) than the older
analog scopes that had some similar capabilities. They are also cheaper than a computer with
data acquisition hardware and software. Unfortunately, they suffer from some of the same
68
problems that effect computer packages: The software running on the TDS210 makes it a little
more flexible than an analog scope, but also a little more complicated. It is easy to get lost within
submenus and sometimes difficult to figure out why something isn’t working.
The following primer is aimed at describing the main features of the scope.
First, note that the controls on the scope are in several groups. These are related to the key
functions of the scope, so if you think about the job that it needs to do, you can work out what
each control is for. It has two input amplifiers, each of which is interfaced to the outside world
by means of a BNC connector, and a single Time Base amplifier that effects the timing of both
channels.
TIME BASE: The amplifier on the right hand side is the Time Base amplifier: This controls the
number of seconds displayed for each sweep of the beam. On a digital oscilloscope, this also
determines the effective sample rate. You need a fast sweep speed to sample brief events
faithfully. The only control that we need to use is the ‘Sec/Div’ knob. Turning this changes the
sweep speed, and thus zooms in or out on a signal that we are recording.
CH1/CH2: These are inputs for your signal(s). The screen has a separate ‘beam’ for each, which
you can move up or down in using the ‘position’ knob. A little cursor on the left hand side of the
display tells you where the zero volt level is for your signal. It is normal sensible to position the
zero point on one of the grid lines. Each also has a GAIN control, labeled ‘VOLTS/DIV’ for
changing the vertical magnification of the signal. The gain setting is displayed at the bottom of
the screen for each channel in units of ‘volts per division’. A ‘division’ means one of the squares
that make up the grid on the display.
MENU buttons: To the left of the amplifiers are a set of buttons with lines connecting to points
on the right hand side of the display: These are buttons for changing settings on a menu.
Selecting one of these buttons is a bit like using the mouse to make a menu selection in
Labchart. The menu that you choose from changes according to which of the variety of menu
buttons you press on the control panel and the function of each button changes at the same time.
For example, pressing the “CH1 Menu button’ brings up a set of options for how to view the
signal on CH1 (NOTE: once a channel menu is displayed, pushing the Ch1 or Ch2 menu button
again will display or hide the input from that channel).
There are a set of 6 special menus brought up by the buttons at the top of the unit: Most useful
among these are the ‘Acquire’ and ‘Measure’ menus: You will use these later in the quarter when
working with electric fish.
Trigger options: Perhaps the most important menu is the trigger menu, brought up from the
appropriate button on the trigger panel to the right of the unit. In addition to the options brought
up on the screen, additional buttons and the ‘level’ knob provide additional control over the way
you trigger the scope to display a trace of data. You can choose to set the trigger MODE (second
button from the bottom on the screen menu to ‘AUTO’, so that a new trace triggers each time the
beam reaches the right hand side of the screen. At low sweep speeds, the data will draw to the
screen as it is acquired (just as in Labchart on the MacLab system). At higher sweep speeds, the
scope will automatically switch over to a mode where data is presented at the end of each sweep.
Alternatively, setting the trigger mode to ‘NORMAL’ will trigger a sweep only when a source
69
generates a pulse to the trigger source specified by the ‘Source’ option above (the middle button
in the five on the right hand side of the screen). The trigger source could be the signal on CH1 or
CH2 (input via the BNC) or an external trigger pulse, supplied to the Time base amplifier input
(‘Ext Trig’).
Take time to play with the oscilloscope and see how the various options effect the display. If you
like the way it is set up in a particular experiment, you can save your settings using the
‘save/recall’ button at top left: press this and then save your settings by pressing the ‘save’ menu
option button. You can change which setting number it saves your options to by using the ‘setup’
button. You can recall any of 5 saved option sets at any time by pressing ‘save/recall’ followed
by ‘recall’ (having chosen the number again using ‘setup’.
HINT!! If you get into real trouble, there is a button at the top right of the unit labeled ‘autoset’:
Pressing this, scans the signals on both channels, and adjusts time base, gain and trigger settings
in a way that the TDS 210 thinks most appropriate for these signals. It is pretty clever at getting
the signals to display well, but tends to set the time-base into too high a magnification. But try
pressing this button and then turning the ‘secs/div’ knob back to a point where your timing
displays better.
70

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NEUSCI 302, University of Washington

Autumn Quarter, 2020

Instructor: Dr. Marti Bosma

Jim Canfield • Charlie Hass • Jordanna Henry • Michael Kennedy

Additional modifications by Dennis Dever and David Perkel

This manual was produced by:

Marti Bosma • David J. Perkel • Michael Kennedy

Not for duplication without authors’ permission.

Syllabus for NEUSCI 302, Autumn 2020

Updates and extra information will be published there.

Teaching Interns (TIs) (extra office hours and contact information)

Madison Bravo (Maddie)(she/her) Monday 9:00-11:00 mabravo3@uw.edu

Lab write-ups 100 Points

We reserve the right to adjust this point distribution.

Lecture and Exam Schedule

Weeks 1-2 Introduction/Auditory/Sound localization (September 30 – October 9)

Friday, October 9 In-class Exam 1

Week 3 Visual system (October 12 – October 16)

Week 4 Somatosensory system (October 19 - October 23)

Monday, October 26 In-class Exam 2

Weeks 5-7 Electrosensory and electromotor systems (October 26 – ~November 13)

Wednesday, November 11 No class/lab - Veteran’s Day

Week 8 Neuromodulation. (~November 16 – November 20)

Monday, November 23 In-class Exam 3

Thursday, Friday, November 26,27 No classes - Thanksgiving Holiday

Week 11 Development of the visual system (December 7 – December 11)

Friday, December 11 In-class Exam 4

Schedule of laboratory activities

Days Exercise Points

Oct 28, Nov 2, 3

Lab reports and grading

As in NEUSCI 301, however, your written work should be exclusively yours.

Lab 1: A quantitative analysis of neural coding using the cockroach

Simple Tuning Curves

Spiking Responses Are Noisy

Noise Influences Experimental Design

Measuring the Frequency Response of a Spine

Figure 2. Raster display and PSTH of neural response to periodic stimulation.

• Use the stimulator panel to increase the stimulus frequency to 50Hz.

Designing Your Own Experiments

The Effect of Varying Stimulus Frequency on Firing Rate

The Effect of Varying Stimulus Amplitude on Firing Rate

Other Possible Experiments

The following points should be addressed somewhere in your write-up:

Lab 2: Photoreceptor Physiology: Recording the ERG from fly

Figure 1. Illustration of the latency and half-width of a response to stimulation.

Figure 2. Trace showing the lamina cell step response to stimulation.

Figure 3. Illustration of fly eye anatomy with pipette trajectory shown.

Computer Setup (and stimulus testing)

>Setup >Stimulator >Setup >Sampling!

>Setup menu >Stimulator

Figure 5. Illustration of recording and stimulation geometry.

Testing the Setup

• Determine the input-output relationship of the response by giving a series of flashes of 2

>Setup >Stimulator >Setup >Sampling!

• Reduce the duration of the flash to 1 ms:

>Setup >Stimulator >Setup >Sampling!

>Setup >Stimulator >Setup >Sampling!

Temporal properties and dark adaptation of photoreceptors

Figure 6. Example traces showing flicker fusion.

Temporal properties and light adaptation of photoreceptors

Normal lab write-up.

Lab 3 – Group A: Recording of Electromotor Behavior and ‘Echo-