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FraidyRat

2016 Fall: Mechanisms of Cued Fear Conditioning & Extinction

Creative(Commons(Copyright(

(
Dr.(Franklin(B.(Krasne,(2012,(2014(
krasne@psych.ucla.edu(
TABLE OF CONTENTS

GENERAL INTRODUCTION p. 03
Overview
Fear Conditioning in Real Rats
General Framework for Thinking

WEEK 1: INTRODUCTORY LAB p. 07


Learning to Use FraidyRat
Specifying an Experiment the Short Way
Running an Experiment
Re-Plotting and Animating an Experiment
Wiping FraidyRats Brain Clean
Recording Single Unit Activity
Drug Infustion
Electrical Stimulation
Scheduling Experiments Using the Long Method
Miscellaneous

WEEK 1: BACKGROUND EXPERIMENTS p. 16


Background Experiment I (to be done as group)
Background Experiment II (to be done as a group)

WEEK 2 & WEEK 3 GOALS p. 17

WEEK 2 (approx.):
ANALYZING THE LOCUS AND PROPERTIES
OF CUED FEAR CONDITIONING p. 18
Brief Review of LTPs Properties
Week 2 Lab Research Questions
Week 2 Lab Experimental Approaches to Answering Research Questions
Week 2 Lab Preparation Questions (to be turned in before lab)
Week 2 Lab Agenda
Week 2 Lab Final Report Questions

WEEK 3: DISCOVERING MECHANISMS OF EXTINCTION p. 26


Some Specific Hypotheses to Test
How Hypotheses I-IV Relate to Renewal
Week 3 Lab Extinction Questions
Week 3 Lab Experimental Approaches to Answering Research Questions
Week 3 Lab Preparation Questions (to be turned in before lab)
Week 3 Lab Agenda
Week 3 Lab Final Report Questions

BACKGROUND STUDY p. 37

NOTES ON FraidyRats PHARMOCOPEIA p. 38

SCHEDULING EXPERIMENTS USING THE LONG METHOD p. 39

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FraidyRat Tutorial

GENERAL INTRODUCTION

Overview

Learning plays a large and essential role in the behavior of all vertebrates and often is important even in lower
animals such as insects and molluscs. Learning therefore is a central focus in the study of psychology. When
animals learn, properties of certain neurons presumably change, and psychologists and behavioral
neuroscientists want to know where these changing neurons are, what the nature of the changes are, and how
they are produced. Despite many attempts, very few answers emerged from research prior to the 1960s. During
the 60s, the locus of the cellular changes ("engrams") underlying a few kinds of learning, as well as some
information about the nature of the (neuronal) changes during learning began to be discovered. Since then, there
has been considerable progress. The kind of learning most amenable to analysis has been Pavlovian
("classical") conditioning. The prototypes for this kind of learning are the dogs that Russian physiologist Pavlov
taught to expect the arrival of food [the so-called unconditioned or unconditional stimulus (US)] when a
previously neutral stimulus [the so-called conditioned or conditional stimulus (CS)] was presented. Trained
dogs salivated copiously whenever CSs occurred that had been previously paired with a US (food), as though
they were thinking about the expected food. Pavlov referred to the effects of this conditioning as the US's
"reinforcing" effect, and it has become common to label any event that causes learning as a reinforcement.
Modern physiological researchers, using training procedures very similar to Pavlov's, have usually studied either
eye-blink conditioning, in which a neutral stimulus warns of a puff of air to the eye, or fear conditioning, in
which a stimulus warns of a painful or frightening stimulus such as a moderate electrical shock. These
experiments have revealed a great deal about the brain loci and nature of the changes responsible for both these
kinds of classical conditioning.

In this lab you will try to discover aspects of the anatomical and physiological bases of fear learning in a
"virtual" (i.e. computer program) animal that we have named FraidyRat. The behavioral aspects of fear learning
in FraidyRat are similar to those of real rats, and the way that Fraidy works conforms to some current theories
about how real rats work. But Fraidy is an automaton, not a real rat. What you discover about classical fear
conditioning in FraidyRat may be similar to what has been found for real rats, but it won't be exactly the same.
Think of FraidyRat as a mutant or alien rat that you have been presented for study. You will learn a lot about
the nature of learning and its neural basis from this exercise; but its real purpose is to give you a taste of doing
this kind of science. With the click of a button, you will be able to do experiments that would be technically
difficult and very time consuming in real animals. Nonetheless, the mental processes involved in deciding what
experiments to do and interpreting what you find will be identical to what real scientists have done and continue
to do. Moreover, because the effort factor is so low, you will be able to do experiments on FraidyRat that have
never been tried on real animals, and you might even discover things that give novel insights into real animals.

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Fear Conditioning in Real Rats

When a discrete stimulus such as a tone or light (a CS) warns of shock onset, animals come to fear the stimulus
[fear is the conditioned response (CR)]. They also become afraid of the situation or "context" in which the
shock occurred (a contextual conditioned response). If subsequently the CS or context occur repeatedly
without shock, fear to the stimuli diminishes (extinction). Fear learning is very fast and extinction is slow.

If behavior in response to stimuli alters, there must be alterations within the nervous system. Perhaps synapses
between certain nerve cells become stronger or weaker. Perhaps critical firing levels of certain neurons alter.
Perhaps certain neurons that were formerly were inactive become tonically active and provide input to other
neurons thereby changing their responsiveness to stimuli. Or perhaps tonic inhibitory activity suppresses
potentially spontaneous responses, and conditioning causes this inhibition to go away, thus allowing the
responses to be expressed. In order to discover what is changing during learning, one must find the location of
those neurons involved and then see how their operation alters. The changes responsible for learning must result
from particular kinds of input to specific neurons, and it is key to know what these kinds of input are. When
extinction occurs, the changes that caused the original learning might be erased, or perhaps new, suppressive or
inhibitory learning occurs that prevents fear from being expressed while leaving the original fear learning intact.
If we could understand the extinction process, perhaps we could find ways to undo or suppress unwanted
learning such as that responsible for anxiety and traumatic stress disorders. These are a small sample of the
questions that one would like to answer for learning in general and fear learning in particular. We can address
any of the above questions and more in FraidyRat.

As you may know from previous courses, there are some kinds of learning that are highly dependent on the
hippocampus and others that are not. The rapid learning that allows us to recognize people we have seen before
and allows us to remember particular events in our lives are among those that depend heavily on the
hippocampus and related medial temporal lobe structures. An especially intriguing aspect of hippocampus-
dependent learning is that it appears that whereas the hippocampus is initially crucial for laying down and
storing the memory in question, over time the information seems to move elsewhere (probably neo-cortex).
Moreover there is a widespread belief that transfer from hippocampus elsewhere depends on the hippocampus
sending information to other parts of the brain while we are asleep!!! As you will see in our first lab session,
learning to be afraid of situations (contexts) where bad things happen (but not of specific transient cues that
warn of USs"cued" fear learning) seems especially hippocampus-dependent. During these three weeks, we
will focus on context fear learning and on comparisons between it and cued fear learning. We will try to figure
out where learning in FraidyRat's brain occurs, where it is consolidated over time, etc.

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FraidyRat Tutorial

General Framework for Thinking

In addressing the mechanisms of learning it is helpful to have a working hypothesis in mind. So we will begin
by thinking a little about classical conditioning in general.

Many current investigators think about conditioning using a


model something like that in Figure 1. They suppose that
when a CS is presented, there are cells in the nervous system
that become active to represent the presence of this stimulus XX ?
(cells Y1 and Y2 in Figure 1) and tell other cells that it is US
there. We call these representation neurons for the
stimulus. The neural wiring that allows particular cells to
respond selectively to the particular stimulus they represent U
might be pre-wired as the result of the developmental
processes, or their establishment might itself require CS1 Y1
learning. Such learning might simply involve exposure to XA ZA CRA
(or experience with) the stimulus or it might require that the CS2 Y2
stimulus in question actually be used as a CS to condition Neurons
that
something, though in our case, not necessarily to condition a produce
fear response. You could suppose that animals in a lab have other CRs
existing representation cells for things like particular sound
frequencies (pitches of sound), and colors of light if the
animals are not color-blind. But you might imagine that Figure 1
perhaps each of us has neurons that become active only
when we see our mother or our dog or something else as meaningful. If so, you would expect that we were not
born with such neurons, but that learning played some major role in their development. Representation cells for
particular stimuli, whether innate or learned, can be thought of as useful "hooks" to which associations to those
stimuli can be attached.

When an animal is successfully conditioned to make a conditioned response (e.g. a fear response, represented as
CRA in the Figure 1) to a stimulus (CS1), neurons responsible for the production of CRA (represented by XA)
become strongly activated by CS1 (whereas before conditioning they were not). As a result of the training,
these cells increase the extent to which they respond to activity of representation cells for CS1. For the purposes
of this discussion we will call such cells conditioning cells. One possibility is that this conditioning could
involve the growth of brand new connections between the representation neurons whose activity indicates the
presence of CS1 and the XA cells. But another, more likely possibility is that very weak connectionsthat are
already therebecome greatly strengthened as a result of some change in the physiology or microanatomy of
the relevant synapses. Many investigators believe that such strengthening involves LTP-like mechanisms (in a
later section there is a review of LTP properties). In some cases there is evidence that supports this hypothesis;
but this is still under investigation. It may turn out that existing synapses strengthen due to mechanisms
different from LTP or that new connections really do form, at least in some cases.

It is also at least theoretically possible that CS-representing neurons (Y neurons in Figure 1) become able to
cause firing of conditioning cells (X neurons), not because synapses between Y and X got stronger, but because,
as the result of training, neurons somewhere else (XX cells) become active and depolarize X cells enough so
that when summed with input from CS-representing cells, the X cells fire (though neither Y or XX input alone
causes X to fire). There is some evidence that this latter sort of thing may sometimes contribute to some kinds
of learning. For example, it is widely thought (though recent experiments suggest this might be wrong) that
extinction of fear conditioning is due to inhibition of amygdala fear conditioning cells (or cells downstream
from them) by neurons that originate in the pre-frontal cortex, and that the actual learning is an NMDA-
dependent process that occurs pre-frontally. In that case the pre-frontal cells would be like the XX cells of
Figure 1, though in this case they would be involved in suppressing a response rather than causing one. XX-
like cells might fire continually ("tonically") or just briefly ("phasically") in response to particular CSs. Of

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course if discovering that an XX-like mechanism is involved, one must also try to find out what causes the XX
neurons themselves to alter their activity.

The X neurons are what sets a given kind of conditioned response going, but they do not usually directly specify
what muscles or glands should do something in the particular temporal pattern characteristic of the response.
Rather, the X neurons command the animal to do something, but the details of that something are worked out by
downstream circuitry that is sometimes called "motor pattern generating" circuitry. In our diagram neurons that
are part of this circuitry for fear responses are indicate by ZA in Figure 1. Actually, this way of picturing it is
over-simplified in that the motor pattern generating circuits may be very complicated circuits and the X neurons
may not in fact innervate all the neurons of a given circuit. Also, note that the X neurons for a conditioned
response are not single neurons. They are populations of cells, some of which might also be part of the set of
conditioning cells for other kinds of conditioned responses.

Obviously, successful conditioning depends on the occurrence of the US along with the CS during training, so
the US must enter the picture in some way. A common belief is that the US causes strong input to XA,
depolarizing XA, and allowing hippocampus-like LTP to develop at those synapses on XA whose presynaptic
side is active. [Note that evidence validating the idea that X-like neurons get strongly depolarized by the US
during fear conditioning is not strong. Nonetheless, it is a working hypothesis of many real investigators.]
Figure 1 portrays the presumptive neurons via which the US activates XA as U neurons.

Note in Figure 1 that learning to specifically make response CRA to CS1 requires increasing the ability Y1 to
fire XA, either by altering the intrinsic properties of the synapse itself or by some sort of extrinsic modulation
from XX-like cells. Increasing effective transmission at the synapses shown on Y1 as opposed to those on XA
would promote responses to CS1 generally but not specifically CRA; however, increasing transmission on Y-like
("representation") cells would be just the kind of mechanism the nervous system would use to form
representation cells for stimuli that don't have representation cells formed automatically during development
(like cells that respond to one's grandmother).

Above we use the term "representation cell" to refer to a neuron that is active only when a particular stimulus is
present and that innervates what we have called possible "conditioning" neurons. It would also be appropriate to
call any cell that signals the presence of a particular stimulus a "representation cell" for that particular stimulus,
whether or not it is involved in learning. Since we will be concerned only with ones that do innervate
conditioning cells, however, we won't bother to make a distinction.

In the above discussion we have made a clear distinction between circuitry on the sensory or input side and the
motor or output side of our hypothetical circuitry. Moreover, for purposes of clarity we have imagined that
conditioning occurs on neurons that are innervated by sensory-side cells and that innervate motor-pattern
generating cells. This might be an over-simplification. In real animals or in FraidyRat, there could be networks
of cells that lie in between strictly sensory and motor circuitry that are hybrids between what we have defined as
Y-like and X-like cells. Nevertheless, we will make the working assumption that things are organized as
Figure 1 diagrams unless the facts of what we find in experiments on FraidyRat force us to abandon our
simplification. It is key to make some "working assumptions" like this to avoid feeling paralyzed by the
possible complexity of things when trying to do scientific research.

The neural changes responsible for learning, whether they are changes of the synapses on XA or alterations in
circuitry of neurons analogous to those we have called XX neurons in Figure 1 are often referred to as the
engram of the learning. Changes underlying the formation of representation cells that feed conditioning cells
should probably also be considered part of the engram. "Engram" is a term coined many years ago by Karl
Lashley, the first person who devoted a scientific career to trying to locate the places in the brain where changes
responsible for learning occur. He was not very successful in finding any engrams, but since the 1960s
scientists have been somewhat more successful, and hopefully you too will be in your experiments on
FraidyRat.

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FraidyRat Tutorial

WEEK 1 LAB: INTRODUCTORY LAB

Learning to Use FraidyRat

In this section you will learn to use the FraidyRat program. We will go over this together in class.

There are two versions of the FraidyRat program: Windows and Mac. The procedures to initiate the program
depend on the operating system. If you are working in the student lab, FraidyRat is already installed on the lab
computers, and your instructors will show you how to launch the program. You will certainly have to use the
program outside the lab, on either a public/ campus computer or your own computer. If using a public/ campus
computer, where you cannot download and permanently save the program, you can only use the Windows
version of FraidyRat. FraidyRat for Windows is the only version that can be run from an external drive; it does
not require installation. If, however, you are using you own computer, you can install and run either version,
Windows or Mac. Both versions are available to download for free from the FraidyRat webpage:
https://mdcune.psych.ucla.edu/modules/frat. You should carefully follow the instructions in the FraidyRat
readme file, also available on the webpage (and described below).

FraidyRat for Windows does not require installation. If you decide to put the program on an external drive (e.g.
a flash drive), make sure there is at least 3 GB of free space to accommodate it. Also make sure that the
computer you use to run the program has Microsoft Visual C++ 2005 SP1 Redistributable Package (x86).
Current versions of Windows should already have this software, but if not, you can get it here:
http://www.microsoft.com/en-us/download/details.aspx?id=5638. Download the FraidyRat for Windows
program file, fratWM4Windows_2016a.zip. Right-click this file, then select Extract All... to extract it to a
folder named fratWM4Window_2016. You may move the program folder to any location except for your
Documents folder, because it occasionally will not work there.

FraidyRat for Mac requires installation. Download the FraidyRat for Mac program zip file,
fratWM4MacDsktp.zip. Double-click this file to extract it to a folder named fratWM4MacDsktp; do NOT
change the name of this folder or it will not run. Move the folder directly on the Mac Desktop; any other
location will prevent the program from running.

Note that there will be various circumstances (described below) when you will need to put things into or take
things out of a folder called Exps. That folder can usually be found directly in the FraidyRat for Windows
program folder (fratWM4Windows) and the FraidyRat for Mac program folder (fratWM4MacDsktp). If you
dont see it, check the sub-folders to make sure that it exists.

To launch the program, open the program folder and double-click either the startfratWMa.bat file (Windows)
or the run_fratWMa.command file (Mac) JUST ONCE. Be patient, this process may take a few minutes. The
program is ready when the fratWMa window, the programs user interface (control panel), and an Animation
window (viewport) appear. If, instead, a rectangular window opens and rapidly goes away, or you receive some
sort of error message when you try to start the program, you are probably missing a support package (referred to
above), or a file did not unzip properly, and you may want to start from scratch or seek help.

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A fully launched FraidyRat programs windows are shown below (Figure 2).

Figure 2

The normal procedure used to classically condition an animal (or person) is called "delay conditioning". First,
the CS is turned on from time to time, typically for some seconds. Next, the US is turned on for a short time.
Finally the CS and US are terminated together. In some instances, though, the US is turned on just as the CS
terminates. Either procedure produces good conditioning. Alternatively, if there is a space of time between the
offset of the CS and the onset of the US, conditioning is considerably slower and has somewhat different
properties (so-called trace conditioning). FraidyRat does not learn with trace procedures.

The program allows you to present either of two CSs: CS1 or CS2. FraidyRat can be placed in any of three very
different environments (contexts A, B, or C), or in its home cage (context 0), where you can't mess with him.
You can give him a US (shock) whenever you want so long as he is not in his home cage.

You may set up a conditioning experiment in either of two ways in the FraidyRat program: the "short" way or
the "long" way (see the upper left of the control panel). The short way is simpler but has less flexibility; the
long way (which will be explained later) is a little more complicated but allows you to do some things that can't
be done with the short method.

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FraidyRat Tutorial

Specifying an Experiment the Short Way

Hit the Short button in the Make or Get Schedule section at the upper-left of the control panel. You will see a
new window with 5 similar green panels separated by white rows and 1 turquoise panel at the bottom, as in
Figure 3. The green rows allow you to schedule 5 periods of the experiment separated by rests. Some
parameters that are common to all the periods are entered in the turquoise region.

Suppose you wanted to schedule an experiment in which you gave 5 conditioning


trials using CS1 in context A followed by 20 extinction trials in context B with no
rest in between. You would replace the zeros with the correct values, detailed below
(and also shown in Figure 3).

In the first green block:

For N Per1 (the number of trials in period 1), enter 5.

For Cntxt Per1 (the context for period 1), enter A.

For CS/US Per1 (the CS to be given, followed by a plus sign '+' if a shock is to
be given during the CS and by nothing if a shock is not to be given), enter 1+
with NO SPACE between the CS designation and the '+' sign). [Note that if you
just wanted to give a US (shock) and didn't want to present a CS (which should
always be represented as a 0), as might be the case if you just wanted to condition
fear to the context, you would enter 0+].

Rests are the number of days in the home cage between one part of an experiment
and the next. In this case the Rest is 0, so the second period of the experiment
directly follows the first.

In the next green block we have programmed the 20 trials of extinction, which
occurs in Context B:

For N Per2, enter 20.

For Cntxt Per2, enter B.

For CS/US Per2, enter 1. [Note that since there is no US during extinction, there
Figure 3 is NO + following the CS designation.]

At the bottom in the turquoise region:

For ITI (inter-trial interval), enter 90 (to represent 90 seconds). [Note that the ITI refers to the time between
the simultaneous offset of the CS and US and the start of the next CS. All CS periods, even the first, are
preceded by a duration of one ITI.]

For CSdurAcq (the CS duration during acquisition), enter 15 (to represent 15 seconds).

For USdur (the US duration), enter 5 (to represent 5 seconds).

For CSdurExt (the duration of the CS on extinction trials), enter 15 (to represent 15 seconds).

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The last two rows allow you to automatically insert an extra rest of a specified number of seconds at the start
of each period (aclimitization interval, ACLdur), or at the end of each period (ENDdur); you will probably
have little use for these and will usually leave them at 0. We will explain the long form of programming
later.

Please note that every box must have an entry. If a box is not relevant for a given experiment, its entry should
be 0. When you are done entering the necessary information, hit the Done button.

Any time after hitting the Done button, you can save the schedule you are using. In the Make or Get Schedule
section of the control panel, in the fname w/o ext box, enter a name for your schedule without an extension (i.e.
no dot) then hit the Keep button. Any time you want to make it operative (where it will replace any current
schedule), enter its name in the box and hit the Get button. You can always see any short schedule that has been
entered by hitting the Short button (and you can modify it if you wish) and then hitting the Done button again.
If instead you want to save a modified schedule and not over-write a previously saved one, be sure to give it a
different name before hitting the Keep button. The schedules you keep in this way get stored in the folder
called Exps within the folder than contains your program.

Note that you can adjust the strength of the US by altering the value in the box called Shock Strength (a
maximum of 100 is the default value, but you may change it). We will not utilize this feature now, but for some
experiments you may want to get weak conditioned fear, which can be done by lowering the strength of shock.
Fear is easily conditioned in a single trial when using a very strong shockit often asymptotes in one trial. So
using fewer training trials with strong shock wont produce weaker conditioning.

Running an Experiment

To run the experiment that you have just prepared, hit the Run/Continue button. If nothing happens, be patient.
Sometimes the program needs time to warm up before it is ready for use. Eventually, charts will appear
showing the extent of freezing per second (0 means uninterrupted movement, and 1 means total freezing) as well
as a graph showing events such as the CS, US, context changes, as a function of time (time axis in seconds).
The graphs for this experiment should look like Figure 4, below:

0.8

0.6

0.4

0.2

0
0 500 1000 1500 2000 2500
us
cs2
cs1
cntxt
0 500 1000 1500 2000 2500
Seconds

Figure 4

In this case notice that freezing was zero until the first CS/US offset, at which point FraidyRat froze almost (but
not quite) maximally. This freezing was due to fear of the context. No freezing was seen during the US because
FraidyRat's innate response to foot shock is to dance wildly about. After the first CS/US pairing and prior to the
next onset of the CS, freezing gradually abated. But when the CS came on again, there was an increase in fear
due to FraidyRat having learned to be somewhat afraid of the CS on the first pairing. Freezing drops back to
zero again when the second US is presented. After the second pairing, the CS evoked maximal freezing
(maximal fear).

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FraidyRat Tutorial

After five pairings, extinction training commenced in context B. Notice that as soon as the context switched to
B, the baseline freezing that had been seen between CS presentations in context A vanished; that is because
FraidyRat was not afraid of context B, only context A. When the first CS of extinction occurred (the CS was
not followed by shock), FraidyRat froze, and this happened every time there was a CS, but the freezing to the
CS gradually extinguished (went down). Also notice that after a few CSs had evoked fear in context B, some
freezing developed transiently during the inter-trial interval. This is probably because FraidyRat became afraid
of context B since frightening things (the CS1) happened there (this is an example of higher order conditioning);
this fear of context B rapidly extinguished (went away) as the fear of the CS itself also did.

Investigators who are primarily interested in responses to the CS, as opposed to fear of the context, usually test
responses to the CS in a different context from that in which conditioning occurred, as we did here. This
strategy allows for a more pure measure of fear of the CS that is not mixed together with fear of the context.
The advantage of this strategy is shown by examining the response to the CS on the last conditioning trial: when
the CS1 turns on, the freezing score goes from about 0.2 to 1.0 (i.e. total freezing). You cannot tell from this
whether CS1 alone without context would have caused total freezing, but it becomes apparent when FraidyRat is
tested with CS1 in a neutral context that freezing is maximal on the first trial of extinction.

Re-Plotting and Animating an Experiment

The program plots the entire experiment on one graph, and things can get pretty compressed in time. To spread
them out use the Replot/Animate feature on the control panel. Simply enter the first and last x-axis value you
want to plot in the From and To boxes, respectively, and hit the Replot button. Try re-plotting from 1 to 500.

You can also see an animation of what FraidyRat is actually doing. To see this, check the Animate box then hit
the Replot button. In the animation, the context or situation in which FraidyRat finds itself is indicated by the
color of the sides of the display (red, green, or blue). The CSs are tones, one pitch for CS1 and another for CS2,
which you can hear if your speaker system is turned on. We have also provided two circular lights that turn on,
one during CS1 and another during CS2, so that you can tell when CSs are occurring if you don't have a speaker
system. Note that FraidyRat can't see these; they are just for you. The animation plays substantially faster than
real time. Nevertheless, you should not specify more than about 300 sec of animation, because it will take too
long to play. You will see that before any conditioning, FraidyRat continually explores his testing chamber.
But when a cue occurs of which he has become afraid, he stops moving.

Wiping FraidyRats Brain Clean

You can wipe FraidyRat's brain clean by hitting the NewFraidy button in the upper right corner of the control
panel. When you wipe Fraidy's brain clean, you always get rid of any changes in the brain that you have
produced by training. In real life when you start with a new animal, you of course must re-implant any probes
you want placed in the brain, etc. For simplicity, there is a check-box on the control panel called Reimplant
Probes, that sits to the left of the NewFraidy button. When this box is checked, they any probes you previously
placed will automatically be re-implanted for you when you hit the NewFraidy button. If, however, you were
recording from a nerve cell (see below), previous probes will be lost, and you will have to go fishing for new
cells again.

FraidyRat's program allows you do a variety of kinds of experiments to study the mechanisms of fear learning,
using much the same methods that have been used by scientists to investigate such learning in real animals. You
can make lesions in the brain, infuse pharmacological agents (such as NMDA receptor inhibitiors, GABA
receptor inhibitors, and other agents), record extracellular single unit activity, stimulate electrically and see the
resulting effect, etc. We will now explain how to do these things and learn a little more about FraidyRat in the
process.

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Recording Single Unit Activity

To record single unit activity you must implant an electrode, specify that it is a recording electrode, and then
advance it until it finds a single unit. By advancing it further, you will lose the unit (neuron) from which you
were recording, but you will eventually find another.

Before moving further make sure that you have hit NewFraidy. If in doubt, do it again.

We will start by having you record extracellularly from units (neurons) in a region of the brain called Grisham's
Nucleus. Grisham's Nucleus has not yet been discovered in real rats. Grisham's Nucleus is filled with
spontaneously active neurons that drive FraidyRat's exploratory behavior. If these neurons stop firing,
FraidyRat stops exploring. When FraidyRat is afraid, the neurons of Grisham's Nucleus get inhibited.

You have available three probes that can be implanted in FraidyRat's brain. These are multipurpose probes
that can be used to do either single unit recording, infusion of chemical agents, or electrical stimulation, but a
given probe cannot be used for more than one thing at a time.

Let us begin by implanting a probe (Probe 1) into Grisham's Nucleus. If you hit the Show Atlas button on the
control panel, you will see a map of FraidyRat's brain with coordinates for stereotaxic implantation of probes.
Unlike real brains, FraidyRat's brain is two-dimensionalso you are seeing the whole brain in this one picture.
The various named regions of the brain are labeled, frCerebellum, frSensCtx, etc. The prefix fr is there to
remind you that although the anatomy of FraidyRat has some parallels to real rat brains, it is definitely not the
same (so don't use what you learn here as the basis for a neuroanatomy exam!!!!!). If you want to see things
better, you can drag a corner to expand the atlas picture, then print out a copy for reference (which you can do
AT HOME from the File menu of the Atlas window). Grisham's Nucleus is labeled frGrishams'sNuc in the
atlas and is centered at about -13.5 on the x-axis and 29.5 (note that down is positive) on the y-axis. To implant
a probe in Grishams nucleus, enter these x- and y- values into the X and Y boxes for Probe 1 in the Implant
Probes section of the control panel, and then hit the Implant button, which places your probe into the specified
region (assuming your stereotaxic coordinates are right).

Having implanted Probe 1 in Grisham's Nucleus, specify that the electrode is designated for recording. In the
Recording section at the top right corner of the control panel, select Record at Probe 1. To advance your
electrode and begin to look for units (neurons), hit the Move button. Each time you do this you will advance the
microelectrode a little bit and hopefully pick up a unit. After doing this, you can see what your electrode is
recording using the Manual Stim section toward the middle of the control panel. Select a context (A, B, or C)
in the Cntxt box in which to do your recording (note that you cannot record when FraidyRat is in his home
cage), then hit the Go button. You will get a new window that displays the responses picked up by your
recording electrode as a function of time in milliseconds. When you advanced your microelectrode you might
or might not have picked up a neuron. If you did not, your trace will appear as a noisy flat line. If you did, your
trace will appear as a steady train action potentials (in blue), because all the neurons in Grisham's Nucleus are
continually active unless they are suppressed by fear. Keep moving your electrode and testing until you have
seen what things look like both when your electrode has and has not found a unit.

You will also see a red dashed line on the Activity window (graph screen). This line represents the average
firing rate obtained if you sampled from this electrode at this placement multiple times. This average is given as
a proportion of the maximum possible firing rate. Thus, if the firing rate is 1.0, the unit is firing as fast as is
possible. If the firing rate is zero, the unit is not firing at all (or at least no more than a very low baseline rate
that all of FraidyRat's units showsee below). We have built the calculation of the red dashed line into the
program as a convenience to you; in real life you would have to make many measurements and average them to
get the information provided by the red line. You will often want to measure the exact y-axis value of this line.
To measure anything on any graph produced by this program, hit the Data Cursor icon (the yellow box with a
bottom left corner plus) in the toolbar at the top of the graph screen, then click with the resulting + cursor that

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FraidyRat Tutorial
appears (possibly with a little delay) on the part of the trace you want to measure. You will get a numerical
readout, which is extremely useful for making accurate measurements.

Units in Grisham's Nucleus are always very active unless inhibited. Units in most parts of FraidyRat's brain,
however, fire at a very low, random rate if not excited by some input. In order for you to discern if you are
actually recording from a neuron, it is important for you to observe random firing. The units in dorsal PAG fire
randomly at a low rate. Implant Probe 2 into this region and record units there (leave Probe 1 in place but
remember to select Probe 2 as your recording electrode). If your electrode finds a unit, you will almost
certainly see at least one spike within 10 hits of the Go button. Play a bit and see what it looks like when you do
and when you don't have a unit.

Now lets see how the activity in Grisham's Nucleus changes when we teach FraidyRat to be afraid. Select
Probe 1 for recording again and find an active unit in Grisham's Nucleus. Hit the Short button to bring up the
experimental schedule that you set up at the start of the lab. Modify the schedule so that instead of running one
extinction session after the conditioning session, you have two additional sessions of 2 trials each, the first in B
and the second in C, and neither with any CS presentations. Finally, put a minus sign before the 0 in the NPer2
and NPer3 boxes (with no space between the minus sign and the number); these minus signs will pause the
running of the schedule at the start of sessions 2 and 3, which will be useful below when we talk about drug
infusion. You can pause the schedule in this way either at the start of an experimental period or at the start of a
rest. And you may do this at as many places as you like in a schedule. These pauses allow you to make
measurements or alter some conditions at specified points during the experiment. We have constructed this
program so that stimulation you do to make measurements will not itself cause any learning. Thus, you can go
back to your experiment without having altered FraidyRat's brain. You can imagine that you have infused a
cocktail of drugs that block all learning during this time-out period. [Note that an exception to this is when you
do tetanic stimulation to produce LTP or LTD, but we will discuss that at a later time.] When an experiment
pauses due to your having inserted a minus sign, the Run/Continue button will stay red. You should be sure to
complete the experiment so that the button goes white before trying to schedule a new experiment.

Now (after hitting the Done button on the Short window), proceed to condition FraidyRat by hitting the
Run/Continue button on the control panel. This conditioning will establish considerable contextual fear to
context A, and Fraidy will also be very afraid of CS1. To see the effect on activity in Grisham's Nucleus, look
at the effect of a brief presentation of CS1 with FraidyRat in context B, of which he should not be afraid. Do
this by choosing Cntxt B and Stim CS1 in the Manual Stim section of the control panel, then hitting the Go
button. [Do this while still recording from the same unit that you found before; but if you lost it, look for
another.] You will see that for the first 12 or so milliseconds of the trace, the Grisham's Nucleus unit will be
firing at maximum rate (providing maximum drive to the neural circuits that implement exploration), but once
CS1 comes on, the neuron will pretty much stop firing. If you now select Cntxt A and do the same thing, you
will see that even the context alone suppresses firing in Grisham's Nucleus.

If you set up recording from a unit before running an experimental training schedule, the program will make a
graph of the activity of that unit as a function of time over training. In other words, the graph plots the average
firing rate per second. To see this for the entire experiment, look for the "Mean Spike Rate of Recorded Cell"
and the "Graph of Freezing (Stillness)" windows (graphs). Notice that for the most part, whenever the firing
rate of the Grisham's Nucleus cell (which is driving exploration) goes down, freezing score goes up. But during
USs, FraidyRat jerks about during the shock, so the freezing score also goes down.

Leave things as they are for what we will do next. Your experimental schedule is still paused; we will deal with
that later.

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Drug Infusion

One of the most powerful tools that neuroscientists have is the use of pharmacological agents to modify nervous
system activity. In FraidyRat you can infuse a variety of agents listed in the Infusion section of the control
panel. You can do this either via probes that you have implanted in specific brain nuclei or systemically. Over
time drugs will spread out from where they are continuously infused and will also tend to get destroyed by
endogenous brain chemicals. In FraidyRat we have chosen the concentration of each available drug so that the
drug's radius of effective action is 0.2 mm for every unit of its infusion rate. In the Infusion section of the
control panel, enter the infusion rate you want (0-100) into the Rate box. A useful rule of thumb is that the
radius of effective action (in mm) will be 5 times the infusion rate. Therefore, if you want to affect all the cells
in a nucleus maximally and to have no effect farther away, set the rate to 5 times the nucleus radius. It is
sometimes the case, however, that just a few cells not being affected by the drug will have a very large effect on
neural activity. So we recommend that if you want your drug to affect an entire nucleus, set the infusion rate to
5-10% higher than what should be just right.

Let's play with this. FraidyRat is still conditioned and you should still be recording from a Grisham's Nucleus
cell (if you are not, find another). In the Manual Stim section of the control panel, select Cntxt C and NoStim.
When you hit the Go button, you should see a steady train of spikes. Now we will set up to infuse a drug into
Grisham's Nucleus. Introduce Probe 3 into Grisham's Nucleus; go for dead center of the nucleus (this is
important if you want to get all the cells of the circular nucleus and no others). In the Infusion section of the
control panel, in the Where? box select Probe 3 for infusion, then select (check) GABA-R agonist. The radius
of Grisham's Nucleus is 2 mm, so we should get a maximal effect of the drug at a rate of 10. Lets set the rate to
5 and toggle the On/Off button to on (red). Hit the Go button, and you will see your cell firing at about half its
maximum rate. Play around with various flow rates and see their effects.

We have already discussed the fact that Grisham's cell firing controls exploration. Lets look at this directly.
Turn off your drug and hit the Run/Continue button to run the scheduled period in context B; this will provide a
control period with no drug. Now turn the GABA agonist on at a flow rate of 10, which should totally suppress
firing of Grisham's cells. That competes the scheduled experiment. If you now look at the graph of the
experiment, you will see no freezing in context C when Grisham's cells were being allowed to fire unfettered
and you will see in the bottom spike rate box that the firing rate of you recorded cell is maximal in context B
and zero in context C. To convince yourself that FraidyRat's behavior really does do what Grisham's cells tell it
to, run the animation from about 650 to 850 seconds where you can watch what happens as the Grisham's
cellswhich are firing maximally in context Bstop firing in context C.

Finally, note that you can also infuse drugs systemically (i.e. to the entire rat). In that case the value entered in
the rate box will be approximately the percentage of the maximum possible effect of the drug.

Electrical Stimulation

Another method used in neuroscience is electrical stimulation of the brain. In FraidyRat, you can stimulate
brain regions via probes you have implanted. We will illustrate stimulation by using a probe in dorsal PAG and
passing a brief pulse of current to make some neurons there fire while recording the effect in Grisham's Nucleus,
which receives its sole input from dorsal PAG (we will work out this anatomy later in this lab session). You
already have Probe 1 in Grisham's Nucleus and Probe 2 in dorsal PAG. Hit the NewFraidy button in the control
panel, and make sure you are still recording a unit in Grisham's Nucleus. In the Infusion section, select None
for the locus of infusion (Where? box), in the Manual Stim section, select Stim Probe 2, and set Stim Volts to
100 (the maximum allowed). Put FraidyRat in any context (A, B, or C). Now hit the Go button, and you will
see that stimulating dorsal PAG causes a small slowing of the firing rate of neurons in Grisham's Nucleus. This
decrease in rate means that neurons in dorsal PAG inhibit the spontaneous firing of units in Grisham's Nucleus
(you could probably block this inhibitory effect by applying a GABA receptor inhibitor, but we won't take time
to try that now). You saw a much greater reduction in Grishams Nucleus firing when you presented a feared

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stimulus; the difference may be that electrical stimulation can only recruit a relatively small proportion of the
neurons in dorsal PAG whereas natural input may be able to recruit many more.

Antidromic Stimulation: Electrical stimulation can also sometime be useful as a way of determining whether
one is recording from a cell that sends its axon to a particular place. If the axon is large enough and branches
suitably in the target region, then one may be able to evoke a backward propagating ("antidromic") spike by a
shock to the target region. Antidromic propagation proves that the recorded neuron's axon projected to the
stimulated region. Antidromic propagation works for the neurons that run from dorsal PAG to Grisham's
Nucleus from dorsal PAG to Grisham's Nucleus. In the Manual Stim section, select Probe 1 (in Grisham's
Nucleus), and set Stim Volts to 95. In the Recording section, select Record at Probe 2 (in dorsal PAG), and
advance (Move) your electrode until you have a unit. Now hit the Go button. Unless your recorded unit is
refractory because there happened to be a spike just before you stimulated, you will see a single ("antidromic")
spike arrive at your recording site about 2.5 msec after the stimulus pulse.

Scheduling Experiments Using the Long Method

This is a method that you will probably not need for what we are doing this quarter, so I have appended it to the
end of this manual, and unless you need it, you can forget about it.

Miscellaneous

You now know how to work almost everything. The exceptions are the Tet function (which allows you to do
LTP experiments and will be explained later) and an anatomical tool (which will be explain in the next section).

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WEEK 1: BACKGROUND EXPERIMENTS

We will get some essential background information that we will need for almost everything we do. The
following background experiments (I and II) will be conducted as a group.

Background Experiment I: Using Backfills to Find Interconnections Between Brain Areas

We need to know the interconnections of the various nuclei of FraidyRat's brain. We will employ a modern
technique used by real anatomists, in which a dye is injected into a selected brain region that will be picked up
by nerve terminals and transported back to the neuron's cell body. So if region A receives input from regions B
and C, and dye is injected into region A, the dye will show up in regions B and C. Backfills need to be done for
all the named nuclei in the atlas. Implant a probe in the target region, specify that that probe is to be used for
infusion, and hit the Backfill button at the bottom of the Infusion section of the control panel. The program will
do the rest. After you inject dye in one place, delete the atlas frame with the dye fills on it if you plan to do
more, because otherwise all your fills will all get superimposed. Your instructor may have the class do this as a
group and share the information.

Background Experiment II: Discovering the Expression Pathways for Conditioned Fear

A final preliminary experiment will determine brain regions crucial for the performance of a conditioned
response. Give FraidyRat three conditioning trials with a 10-sec CS, a 3-sec US, and a 90-sec ITI, all in context
A. Then inactivate or lesion an area and test responses to the CS and the context.

One way to do this is to schedule an experiment in which you stop the program to lesion or inactivate a region
by infusing a GABA agonist, and then Continue the experiment with suitable test trials. In this case you will
be using the actual behavioral end-point of freezing to determine whether the lesion/inactivation prevented the
expression of the previously learned fear. If you do the experiment this way, however, you will have to do the
entire experiment over again, including training FraidyRat for each region you want to test. A more efficient
way would be to train FraidyRat just once and then assess conditioning by recording Grishams Nucleus cell
firing in response to the CS and context using manual stimulation. You can then make temporary functional
lesions of each area in turn by injecting GABA receptor agonist into the region of interest (as is done with
muscimola GABA agonistin real life. Whichever way you do the experiment, be sure to check both
contextual fear and fear of the CS-proper for each region inactivated. You will be able to see a pure expression
of contextual fear by looking at times just before the onset of the CS.

When you are done with this section, you should be able to make a chart or figure that tells how each region is
connected with each other. You should also indicate on your figure which regions are needed for expression of
conditioning that was done prior to your inactivation manipulations (we will call this the pathways defined by
these experiments the cued fear and context fear expression pathways). You should get this information into
some form that is convenient for easy reference, since you will need to consult it frequently.

Make a drawing of what is connected to what specifically in the expression pathway. Include in the diagram
only the regions that are part of the cued fear or context fear expression pathways. Moreoverand this is
importantmake your diagram without reference to the actual location of each nucleus in the brain (i.e. a
schematiclike a subway maprather than a real map). When you do this, you will find that a quite neat and
simple picture emerges.

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FraidyRat Tutorial

WEEK 2 AND 3 GOALS

We are going to use FraidyRat to address two sets of questions:

(1) Where in FraidyRat's brain do the changes responsible for fear conditioning reside, and what is the nature
of those changes? [Week 2timing approximate]

(2) What is the basis for extinction in FraidyRat (e.g. erasure vs. inhibition, etc)?

Notably, you will not find specific instructions below for the experiments you will do. Rather, we will explain
the questions to answer by experimentation and will discuss possible ways of answering those questions. It will
be your job (with some help) to design and carry out experiments to get data that will allow you to answer the
research questions posed. This approach is intended to give you experience not only with some of the methods
of behavioral neuroscience, but also to give you a taste of how the scientific process actually works.

Each week's section of this document will have the following organization:

(1) General orientation for the week. Determine which questions will be the focus of the week's
experiments, and decide experimental approaches for answering them.

(2) A question set will be turned in at the start of the lab. Its intention is to make you think about what you
are going to do and why you are going to do it. The first 45 minutes of lab will be devoted to discussing your
answers.

(3) Experimental agenda.

(4) Questions for this week for the final report (these might get slightly revised as we go along).

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WEEK 2 LAB (approx):
ANALYZING THE LOCUS AND PROPERTIES OF CUED FEAR CONDITIONING

In real animals cued fear (which is our focus) is likely due to LTP of synapses on amygdala neurons that
promote freezing responses and other behavioral aspects of fear when they fire. The locus and changes
responsible for fear conditioning may be different in FraidyRat than in real rats. For example, it could
conceivably be that fear learning in FraidyRat is not due to LTP at synapses between cue and context
representing neurons and fear-producing ones (i.e. X-like cells) but rather to input from the XX-like cells
discussed in the section titled "General Framework for Thinking" near the start of this manual (which you may
find it helpful to consult often).

Brief Review of LTP's Properties

You have all been introduced to long-term potentiation (LTP) and perhaps also to long-term depression (LTD)
in earlier courses. Because this is so important in thinking about learning mechanisms in both real animals and
FraidyRat, here is a brief rundown on some things it will be helpful for you to know:

LTP was first discovered in the hippocampus. As now understood, hippocampal LTP is a long-lasting (probably
at least weeks and maybe much, much longer) increase in the efficacy of susceptible synapses when presynaptic
endings release glutamate while the postsynaptic cell is fairly strongly depolarized. [Note that if the synapses
are excitatory, "increased efficacy" means that excitation is strengthened, resulting in more depolarization per
presynaptic spike, and if the synapses are inhibitory, it means more effective inhibition per presynaptic spike.]
At hippocampal synapses subject to LTP, the postsynaptic membrane has, in addition to ordinary glutamate
receptors, a special kind of receptor called an NMDA receptor (NMDA stands for a long chemical name almost
nobody uses). When the glutamate released by excitatory neurons binds to NMDA receptors, they open up to
calcium ions, which enters the cell. These calcium ions in the dendritres are the immediate inducer of LTP.

Pavlovian conditioning is widely believed to be due to the kind of LTP discovered in the hippocampus. The
relevant synapses, however, are usually elsewhere. In the case of fear conditioning, they are in the amygdala. It
is thought that the unconditioned stimulus depolarizes conditioning neurons via pre-existing circuitry, and the
neurons fired by the CS release glutamate to them. Therefore, LTP occurs, and thereafter the excitation
produced in the conditioning cell by the CS cell is augmented and more likely to cause the neuron to fire.

While all this was being discovered, drugs that block NMDA receptors were also discovered. These drugs
prevent development of LTP and are therefore very useful in determining whether LTP is responsible for any
given kind of learning.

Further, if there is a lesser calcium ion elevation, due for example to a weaker postsynaptic depolarization
during induction of the synaptic change, a long-lasting form of depression, rather than a potentiation of
excitation, often occurs; this is "long-term depression" (LTD).

As synapses all over the nervous system were studied, it was discovered that there are forms of LTP and LTD
with properties somewhat different from those above. Some of these synapses elevate postsynaptic calcium ion
concentration via postsynaptic receptors other than NMDA receptors, and they have different pharmacological
properties. One common motif is for so-called metabotropic glutamate receptors to play a role somewhat
similar to what NMDA receptors do at the hippocampal synapses that have been so well studied. At synapses
subject to some kinds of LTP, the production of LTP is contingent not only on the postsynaptic cell being
depolarized when the presynaptic side of the LTP-susceptible synapse releases glutamate, but also on the
presence of various neuromodulatory substances like dopamine or serotonin. To make things even more
complicated, there are some kinds of synapses that produce LTD when postsynaptic depolarization is large and
LTP when it is small, just the opposite to the situation in the hippocampus. One well-studied example of this

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situation occurs on cerebellar Purkinje cells, which are inhibitory neurons (these neurons are intimately involved
in cerebellar eye-blink conditioning, which you might have heard about in other neuroscience classes). Some
forms of learning are thought to be due to these more recently discovered forms of synaptic plasticity. Our
working assumption, however, will be that any forms of synaptic plasticity occurring in FraidyRat are of the
classical hippocampal type. We will occasionally propose experiments to test that working hypothesis, and we
should have an open mind about the possibility that some synapses in FraidyRat are subject to these more exotic
types of synaptic plasticity. But unless experiments force us to conclude that some kind of FraidyRat synapse
exhibits these more exotic properties, you should assume that any plasticity found in FraidyRat is of the
hippocampal kind.

Week 2 Lab Research Questions

What we want to try to find out from our first week's experiments is:

Research Question 1: Where are the "conditioning neurons" (X-like cells) for fear conditioning in FraidyRat's
brain?

Research Question 2: Do fear conditioning cells alter their responses to CSs because synapses on them
undergo Hebbian LTP or because changes in tonic input of some kind alters their responses to the CS? Or for
neither of these reasons?

Week 2 Lab Experimental Approaches to Answering Research Questions

There are a number of kinds of experiments that should be informative when figuring out Fraidys CNS. Here is
a short list of some salient possibilities (there are often many different ways of answering a certain question, and
all of the following approaches won't necessarily be needed):
cn
Approach 1: Use inactivation of regions in already conditioned FraidyRat to see what
regions need to be active in order for already established fear conditioning to be
expressed. th

By the time you read this, we will have already done this as a class. We refer to the regions
cx
found to be necessary as the "fear expression pathway". Figure 5 to the right summarizes
hc
these experiments. The expression pathway for cued fear is shown in blue, and the
expression pathway for context fear is shown in red. Knowing these pathways is very am
useful. It would seem that the engram must reside within the expression circuit: This
follows because removal or inactivation of the parts of the brain where learned information
that controls conditioned responding is stored (i.e. the engram) would prevent performance pagd
of conditioned responses. And this makes it, by definition, part of the expression circuit.
But of course the engram would presumably be limited to particular portions of the
expression circuit, and one of our tasks will to be to figure out which one(s) these are. bsxp

Approach 2: Silence neurons in portions of the expression pathway during Figure 5


conditioning and see whether this prevents conditioning.

If learning is due to changes that develop in neurons in some region during training, then preventing neurons in
that region from firing might well prevent the changes of those neurons from occurring and so might prevent
learning. For example, as you know, development of LTP requires strong depolarization of the post-synaptic
neuron. So if strong depolarization were prevented by using a GABA agonist, no LTP should develop.
Therefore the ideal experiment would be to somehow suppress firing of the neurons with a GABA agonist
during conditioning and then test to see if anything was learned with everything working normally again.
Experiments of this kind can be very informative, but interpreting them can be a little trickier than one might

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think; this is partly because when we suppress activity in a region, we not only prevent its neurons from
developing whatever changes are responsible for learning, but we are also likely to prevent information flow to
downstream regions where changes responsible for learning might be occurring.

Approach 3: Use NMDA receptor antagonists to prevent classical LTP.

NMDA receptor antagonists are drugs that inactivate the normal ability of NMDA receptors to let calcium ions
and thereby cause development of LTP in the cells. However the cells operate normally in other regards. Thus,
unlike GABA agonists, which act like inhibitory input and prevent cells from becoming depolarized and
therefore prevent firing and also prevent development of LTP (because the cells cannot depolarize), NMDA
antagonists allow the cell to work normally except that NMDA-dependent plastic changes (LTP and LTD)
cannot develop in them.

The use of these drugs can both tell you where blockage of LTP can prevent learning whether LTP is the
mechanism responsible for the learning. We will make the working assumption that any LTP that is the basis
for learning in FraidyRat is the type that is triggered via NMDA receptors. We should be prepared, however, for
the possibility that experimental findings might force us to abandon that assumption.

Approach 4: Record from neurons to see where training causes changes in how neurons of the expression
pathway respond.

If, for example, the neurons you were recording were "conditioning" or "X"-like cells as defined in the section
titled "General Framework for Thinking", then you would perhaps expect them to increase their rate of firing in
response to the CS after they were trained. And if there are neurons whose level of tonic activity changes as the
result of training, you might hope to find them by recording in suitable locations. There are two ways to do
recording experiments: (i) the "cross-sectional" approach in which one surveys the activity of cells in a region
both before training and after training, not necessarily even in the same animals, and (ii) the "longitudinal"
approach in which one follows the activity of single cells through the training process and see whether and how
it changes. It is usually a good idea to start with a cross-sectional approach, especially if there are many types of
cells in the region of the brain being studied, because it will be much less time-consuming to study the behavior
of many cells in the trained vs. untrained situation than it would be to have to train FraidyRat for every cell you
sample (then kill him and use a fresh Fraidy for the next cell). Imagine trying to proceed with only the
longitudinal approach if there were a dozen or more different cell types, as there in fact are in FraidyRat's
posterior cortex. When doing this sort of study, it is just luck as to which of possibly many types of cells your
electrodes record on any given occasion. Therefore, it is never certain if all the different existing cell types have
been identified. Indeed, it can occur that the kind of cell you are hoping to find is but one of many, and maybe
even in minority. At the end of such an experiment you would want to be in a position to specify how many
different kinds of cells there are and what are the properties of each type. After employing this cross-sectional
approach, it is a good idea to follow up with some longitudinal experiments.

In real animals, such experiments are quite time-consuming and laborious. It is therefore a good idea to begin
with approaches other than recording to determine where recording studies are likely to be most informative
given the questions you hope to answer.

Approach 5: See where and whether responses of cells to electrical activation of inputs to them are
altered as the result of conditioning.

For example, if recording from a "conditioning cell" in some region of the brain, you might expect the cell to
respond to electrical stimulation from presynaptic neurons differently after training than before, and seeing
where this sort of thing happens would be one way of trying to locate conditioning neurons. It is important to
note that sometimes there are multiple routes to a given structure (e.g. in FraidyRat, the amygdala gets input
from the cortex, both directly and via the hippocampus). In such cases you would want to conduct experiments
to find out over which route relevant information is flowing. One way to do this is with experiments where one
of the parallel routes is silenced.

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FraidyRat Tutorial

Week 2 Lab Preparation Questions


[Note: These questions are due at the VERY START of Week 2 lab.]

The following questions (Prep Questions 1-13) are intended to prime you for designing and thinking about the
experiments that will be needed to answer the research questions we have posed.

Your answers to the multiple choice questions should be marked on the scantron you will be given for handing
in.

You should also mark your answers on a copy of this question sheet as well as providing written answers to the
underlined "Explain...." portions of the questions. This too must be handed in at the start of the lab session.
Please, however, be sure to keep copies of all your answers that you can refer to in class. You will need them
during our experiment-planning session at the start of the lab.

We already have some information that will help in trying to identify the conditioning cells we seek, because in
class we together worked out the fear expression pathway. As we argued above, we expect this pathway to be
where the action is. To move us even a little farther along before we start doing experiments, we have provided
a writeup at the end of this manual (using data from a previous class) of an experiment using what we above
called Experimental Approach 2. Please read the document near the end of this manual called
BACKGROUND STUDY: EFFECTS ON CONDITIONING OF INACTIVATING REGIONS DURING
CONDITIONING. You will need to study it before trying to answer the following questions.

Also keep in mind the overall questions we are addressing this week:

(i) Where do conditioning neurons (X-like cells) reside in FraidyRat?

(ii) Do fear conditioning cells alter their responses to CSs because synapses on them undergo Hebbian LTP
or because changes in tonic input of some kind alters their responses to the CS? Or is it for neither of these
reasons?
Before conditioning After conditioning
Suppose we were recording cells in a region that we thought might
2 2

either CS1 or CS2 both CS1 and CS2


IA
1.5 1.5

contain conditioning cells, and we found three kinds of cells (A, B, and 1 1
Activity

Activity
C) that respond to CS 1 and CS2 as shown in Figure 6 to the right:
0.5 0.5

CS1
2 2

0 0

1.5 1.5

0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Milliseconds Milliseconds
1 1
Activity

Activity

Prep Question 1: Which type are best considered pre-wired


0.5 0.5

CS2 0 0

representation cells for CS1?


2 2

0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
1.5 Milliseconds 1.5 Milliseconds

(a) A B
II 1 1
Activity

Activity

0.5 0.5

(b) B CS1 2 2

0 0

1.5 1.5

(c) C
0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Milliseconds Milliseconds
1 1
Activity

Activity

0.5 0.5

CS2 0
2 0
2

Prep Question 2: Which type are best considered experience- 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50

C
1.5
Milliseconds 1.5 Milliseconds

dependent representation cells for CS1? III 1


1
Activity

Activity

0.5
0.5

CS1 2

(a) A
2

0
0
1.5
1.5

0 5 10 15 20 25 30 35 40 45 50
0 5 10 15 20 25 30 35 40 45 50

(b) B
Milliseconds
1 Milliseconds
1
Activity

Activity

0.5
0.5

(c) C CS2 0
0

0 5 10 15 20 25 30 35 40 45 50
Milliseconds 0 5 10 15 20 25 30 35 40 45 50
Milliseconds

Prep Questions 3: Which type are best considered conditioning cells?


Figure 6
(a) A
(b) B
(c) C

Explain why you answered Prep Questions 1, 2, and 3 as you did.

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Prep Question 4: In the study (mentioned above and appended), various regions of FraidyRat's brain were
silenced with a GABA agonist during conditioning and then Fraidy tested under normal conditions. From this
data, what is your best guess as to the location of conditioning cells?
(a) cochlear nucleus or thalalmus (lumped here to allow responses on a scantron)
(b) sensory cortex
(c) amygdala
(d) dorsal PAG
(e) Grisham's nucleus (AKA brain-stem exploration nucleus)

Prep Question 5: Is it possible, based on the above data, that there is also a second region where conditioning
can occur?
(a) yes
(b) no

Explain your reasoning in answering Prep Questions 4 and 5.

Prep Question 6: One of the questions we wish to address is whether cells we have tentatively identified as
cued fear conditioning cells increase their responses to the CS because the synapses between CS-representing
neurons and the conditioning cells are subject to LTP. If this is in fact the case, would you expect these cells to
respond to a US prior to conditioning?
(a) yes
(b) no

Explain your reasons for your answer to Prep Question 6.

Actually DO the above experiment in an untrained FraidyRat in a region where you think there are conditioning
cells that might increase their responses due to LTP of the synapses on them.

There may, of course, be multiple kinds of cells in a given region, so it might be that some cells will and some
won't respond to the US before conditioning. For the current experiment we suggest that you assume that seeing
what 10 cells do (not including cases where your electrode does not encounter a cell) will give you an adequate
idea of what sorts of cells there are in the region(s) regions where you will be conducting your experiment.
[Note, however, that this rule of thumb would NOT be sufficient for some parts of FraidyRat's brain.]

Prep Question 7: In what region did you do your experiment?


(a) Grisham's nucleus
(b) dorsal PAG
(c) amygdala
(d) posterior cortex
(e) thalamus

Prep Question 8: Did the cells you recorded respond to the US as you would have expected if LTP of their
synapses is responsible for fear conditioning?
(a) yes
(b) no

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FraidyRat Tutorial

Let's take stock: Below is a list of things we want to know that we might or might not yet know based on
information so far available. We should choose further experiments to fill in the gaps (as well as to try to get
more than one sort of evidence regarding important questions)

Prep Question 9: Do we know at least one place where it is very likely that there are conditioning cells?
(a) yes
(b) no

Prep Question 10: Do we know whether there are conditioning cells in multiple places?
(a) yes
(b) no

Prep Question 11: Do we know whether LTP occurs at synapses on conditioning cells during conditioning?
(a) yes
(b) no

Prep Question 12: Refer to Figure 1 (reposted right). Do


we know whether conditioning cells alter their responses to
CSs because transmission between CS-representing cells and
XX ?
conditioning cells gets modulated by activity of some other
cells (i.e. by what we have called "XX-like" cells)? US
(a) yes
(b) no
U
CS1 Y1
XA ZA CRA
CS2 Y2
Neurons
that
produce
other CRs

Figure 1 (repost)

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Prep Question 13: Refer to Figure 7 to the right. Suppose we found that
there were four types of cells in a region where we expected to find cued fear
I
conditioning cells. In the figure the CS occurs at the red triangle, and prior to
conditioning none of the four types responded to the CS. Suppose,
moreover, that we have decided that the cells making the kind of response we
have labeled type I were conditioning cells because they are the only ones
that send their axons to downstream regions involved in fear responses. We
are interested in the question of whether any of the other three types might be II
XX-like cells. Which of the remaining types respond in a way that seems
consistent with their possibly playing an XX-like role?
(a) II
(b) III
(c) IV III
(d) II and III
(e) II, III, and IV

Prep Question 14: Of the five Experimental Approaches for addressing the
above issues, we have likely discovered as much as we can utilizing the first
two approaches, but there are three remaining approaches that we might be
able to use to our advantage:
IV
Approach 3: NMDA Receptor Antagonists
Approach 4: Recording
Approach 5: Electrical Stimulation
Unfortunately, we may only have time to do two. If so, which ones should
they be? Figure 7
(a) Approaches 3 and 4
(b) Approaches 4 and 5
(c) Approaches 3 and 5

Explain your choice for Prep Question 14.

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FraidyRat Tutorial

Week 2 Lab Agenda

After discussing the above questions, we will do experiments utilizing at least the two approaches most favored
in Prep Question 13 above.

Experiment I: Use NMDA receptor antagonists to prevent classical LTP to get at unanswered questions
(Approach 3).

Experiment II: Use electrical recording to get at unanswered questions (Approach 4).

Experiment III: See where and whether responses of cells to electrical activation of inputs to them are
altered as the result of conditioning as a way of getting at unanswered questions (Approach 5).

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Week 2 Lab Final Report Questions

Answer the following questions (Final Questions 1-4) for your lab writeup. Some of these questions closely
parallel ones asked during preparation for this lab. When asked for explanations, use only text. If we want
charts and data, we will explicitly ask for it. When we do ask for data, make sure that all your graphs are well
labeled and fully explained. [As Prof. Grisham is fond of saying, "Don't make your readers think!"]

Final Question 1:

(A) Where is at least one place that we are pretty sure there are conditioning cells?

(B) Explain in words all your evidence for this choice.

(C) Present and explain data from two kinds of experiments (that you have done yourself) that support this
conclusion.

Final Question 2:

(A) Are there conditioning cells in places other than the above (and if so, where)?

(B) Explain your reasons.

Final Question 3:

(A) Is LTP at synapses on conditioning cells responsible for conditioning (i.e. cued fear conditioning)?

(B) Explain fully why this is your answer.

Final Question 4:

(A) Do we know whether XX-like cells (in the sense of our "Frameworks" discussion) are responsible for
the increased responses of conditioning cells to CSs after conditioning?

Please specify one:


(i) They probably are not.
(ii) They probably are.
(iii) We have no relevant information.

(B) Explain fully why you answered as you did and present any relevant data.

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FraidyRat Tutorial

WEEK 3 LAB: DISCOVERING MECHANISMS OF EXTINCTION

Our focus during the third week of this module will be to learn something about how FraidyRat extinguishes.
Broadly speaking, there are two, not necessarily exclusive possible mechanisms for extinction: (1) erasure of the
changes that were responsible for conditioning in the first place, and (2) inhibition of fear whose underlying
basis remains intact.

In real animals the behavioral phenomenon of "renewal" has convinced most everyone that extinction is at least
in part due to inhibition, and many people subsequently assume that inhibition is necessarily the whole story
(however, this may not be truethere is in fact also some evidence of erasure). There has also been
considerable work on the details of how the inhibition is produced and its underlying anatomy. One line of
experimentation has convinced many researchers that the inhibition is driven from pre-frontal cortex (PFC) and
results from LTP occurring within PFC during extinction. These experiments also suggest that that the
conditioning cells themselves dont get inhibited, but neurons downstream from them do. Thus, it is often
thought that the conditioning neurons themselves keep responding to the CS even after extinction, but their
ability to drive cells farther along the pathway is blocked.

Another line of experimentation suggests that extinction results from inhibition originating within the amygdala
itself. Throughout the entire nervous system, it is very common for neurons that provide excitatory input to
some target neuron to also excite inhibitory neurons that innervate the same targets (so-called "feed-forward"
inhibition), which may have a variety of purposes. There is some evidence that when fear extinguishes, it may
extinguish in part because of augmented feed-forward inhibition of conditioning neurons.

Our broad goal this week is to figure out what is responsible for extinction in FraidyRaterasure and inhibition
are the candidates on the table. In order to focus our experiments and our discussion, we are going to put
forward some explicit (but not necessarily mutually exclusive) hypotheses based on general erasure and
inhibition hypotheses and motivated by some of the ideas described above for real animals. Our goal is to try to
get evidence for and/or against these specific hypotheses in FraidyRat. You might think that we should start
with experiments and let the results guide us in formulating such specific hypotheses. But often in science, one
moves farther faster by formulating and testing specific hypotheses (based on logic, conjecture, or perhaps very
sparse information) than by just doing experiments and then trying to figure out what they might mean. Indeed,
a very useful thing to do when planning experiments is to ask, "If a given hypothesis is true, what result would I
expect from my planned experiment?" A good experiment should have some possible outcomes that are
consistent with the hypothesis and other outcomes that are not. When an experimental result forces us to reject
an hypothesis, we know that we have to revise it and try again. That is how we eventually create a valid picture
of things.

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Some Specific Hypotheses to Test

Refer to Figure 8 to the right. We


assume that when FraidyRat is I II III IV
conditioned to be afraid of a cue it Erasure Inhib via PFC Augmented Inhib of
feed-forward inhib downtream
becomes afraid of both the cue and by CS. (A)Via neurons by
the context in which the conditioning pot.input to CS. (A) Via
inhibitor and (B) pot.of input to
occurred. In all four hypotheses, we PFC
pot. of inhib inhibitor and
postulate that this conditioning is due synapse itself. (B) pot of inhib
synapse itself.
to LTP at synapses (drawn green)
Cntxt CS Cntxt CS Cntxt CS Cntxt CS
between the cue and context-
representing neurons that were active
A? A?
when the US was given. Please note 1
that when a single neuron is drawn, it
is meant to indicate that all the
neurons involved are of only one B?
"Conditioning"
type, though there might be many cells 2
more than one neuron of this type.
B?
When a stack of neurons is shown, it
means there are multiple types of
...

...

...
...
neurons of this general kind; for
example, the CS is represented by a Figure 8
stack of neurons because there are
neurons representing each of the many different possible CSs.

Hypothesis I (Erasure): When the US is omitted after having conditioned FraidyRat, potentiation of synapses
that are active during the extinction gradually loose their potentiated state. Since the CS that was conditioned
will always occur during extinction, its synapse with the conditioning cell will always weaken. Furthermore, if
extinction is done in same context where conditioning occurred, the cells representing that contextwhich
would have become potentiated during conditioningalso lose their potentiated state.

Hypothesis II: This parallels the common belief for real animals that the feared CS-representing and context-
representing neurons are active during extinction and excite neurons in PFC. These PFC neurons, in turn,
inhibit neurons downstream of the conditioning neurons, and thus preventing the expression of fear.
Development of the CS-representing and context-representing neurons ability to drive the PFC neurons is
known to be an LTP-dependent process (in real animals), but the exact details are not specified (and are not
known in real animals).

Hypothesis III: This postulates that extinction is due to inhibition driven by feed-forward inhibitors located in
the same region as the conditioning cells. It supposes that either: (A) the synapses on inhibitory neurons from
context-representing and CS-representing cells that are active during extinction become strengthened, or (B) that
the inhibitors always fire, but that their synapses with the conditioning cells become strengthened in extinction.
If evidence were to favor Hypothesis III, one should also try to distinguish whether option A or B (or both) were
correct.

Hypothesis IV: This is similar to Hypothesis III, except that the inhibitors are postulated to have their effect
downstream of the region where conditioning occurs (there is some evidence that this happens in real animals).

The above hypotheses are not mutually exclusive. In principal, all could be correct to some extent. Our goal
will be to try to determine which are consistent with reality (FraidyRat reality) and which are not.

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FraidyRat Tutorial

How Hypotheses I-IV Relate to Renewal

Refer to Figure 9 to the right. Since


the phenomenon of renewal plays In Cntxt
In B B In Cntxt
In C C
such a prominent role in discussions
of extinction, it is worth considering Cntrl A1 A2
Not
what predictions each hypothesis extinguished Crit. firing level
makes about renewal. For now, we
Resting level
will just consider experiments in CS
which FraidyRat is conditioned in Extinguished B1 B2
context A, extinguished in context Hypothesis I
B, then tested for renewal in
Context C (so-called "ABC" C1 C2
renewal). Figure 9 cartoons the Extinguished
membrane potential of a Hypothesis IIIA
conditioning neuron just prior to the
occurrence of a CS with its onset at
the red triangle and continuing for a
D1 D2
Extinguished
little while after. The resting level Hypothesis IIIB
and critical firing levels are
indicated in blue. The "In Cntxt B"
column shows EPSPs and firing of a Figure 9
hypothetical conditioning cell in
Context B at the end of an extinction session. The "In Cntxt C" column shows the EPSPs and firing during a
brief subsequent test in Context C.

The top row ("Cntrl Not extinguished") shows a hypothetical control experiment in which no extinction occurs.
Notice that since these traces are in contexts B and C, where FraidyRat has had no experience with shock, there
is no depolarization above the resting level due to the context alone, but there is a big response when the CS
comes on. If FraidyRat were in context A, there would be some depolarization and firing of the conditioning
neuron even before onset of the discrete conditioned stimulus.

The second row ("Extinguished Hypothesis I") shows the expected outcome under Hypothesis I following
extinction in context B. Extinction causes the potentiated synapses to conditioning cells from the CS
representing neurons to weaken. We assume throughout that we extinguish in B just enough so that firing
entirely stops. So there is no firing in either context B or C, meaning there is no sign of renewal.

The third row ("Extinguished Hypothesis IIIA") shows the expected outcome under Hypothesis III-A. During
extinction in context B, active synapses on the inhibitory neuron become potentiated. So synapses on inhibitors
from both context B and CS-representing cells become potentiated. As a result, conditioning cells are inhibited
both by context B itself and by the CS. The effect of this inhibition is indicated both by the hyperpolarization
prior to the onset of the CS and by the reduced amount of depolarization relative to the control once the CS
comes on. When subsequently testing in context C, the excitation of the inhibitory neurons produced by context
B-representing cells does not occur, so the firing of the inhibitory neurons diminishes and less inhibition is
produced. Therefore some renewal would be predicted.

The situation is different under Hypothesis III-B (bottom row; "Extinguised Hypothesis IIIB"). In this case (III
B), the synapses from context and CS-representing cells onto the inhibitor are of constant strength, and so they
would always fire the same amount. Extinction occurs because the inhibitory synapses themselves potentate.
But the amount of inhibition is therefore the same no matter where FraidyRat is tested. So no renewal occurs.

From that discussion you should be able to see what would be predicted for Hypotheses II, IV-A, and IV-B.

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Week 3 Lab Extinction Questions

The general goals of the Week 3 lab are to determine the extent to which extinction is due to erasure of the
changes that developed when FraidyRat was conditioned, and to what extent conditioning changes persist but
are suppressed by inhibition. Nonetheless, there are various specific ways that these two broad sorts of
mechanisms might be implemented, and we have put forward four specific hypotheses that are similar to
proposals that have been made for real animals. These hypotheses differ from one another not only with respect
to whether erasure or inhibition is involved, but also with respect to various details of how these mechanisms are
implemented. For example, the hypotheses utilizing inhibition differ from one another in where the inhibition is
applied, where the synaptic changes controlling extent of inhibition reside, etc. We will be trying to find out not
only which general mechanism FraidyRat uses, but also which of the more specific mechanisms it uses.

Week 3 Lab Experimental Approaches to Answering Extinction Questions

Approach 1: Renewal experiments. These provide perhaps the most widely cited evidence from real animals
that extinction is due to inhibition. There are three ways of doing renewal experiments:

(1) Train in context A, extinguish in the same context, and test extinction in a different context B (a so-called
"AAB" renewal test).

(2) Train in A, extinguish in B, then test in A ("ABA" renewal).

(3) Train in A, extinguish in B, then test in C ("ABC" renewal).

In real animals, ABA renewal is easiest to demonstrate, but arguments can be made that it does not really
provide very convincing evidence for inhibition. AAB is very difficult to get in real animals, though it does
occur. ABC is probably the best kind of experiment to do to get convincing evidence for inhibition. We may
discuss in lecture why ABC renewal provides better evidence of inhibition than does ABA.

Approach 2: Use GABA blocking drugs to locate the targets of inhibitory action. Obviously, in so far as
extinction is due to inhibition, one should be able to reverse extinction by applying GABA blocking drugs in the
right places. Localized applications should also allow one to figure out where the inhibition is located
anatomically. Nonetheless, some care must be used since its possible that Hypotheses III and IV are both
correct. It should be pointed out that there are some potential subtleties to the use of this method if inhibition
operates at more than one locus, as would be the case if both Hypotheses III and IV were true. If this were the
case, for instance, and one had extinguished thoroughly that inhibition was very strong at both inhibitory loci,
applying a blocker to either region alone would not reverse extinction, though applying it to both would.
Instead, if one extinguished only modestly, then blocking inhibition at either site would probably cause some
degree of reversal of extinction.

Approach 3: Silence neurons to locate the source of inhibitory drive. Given the set of hypotheses that we
have on the table, we could test the validity of Hypothesis II by examining the effect of silencing PFC with
GABA agonist. We can't, however, use this method to evaluate Hypotheses III and IV by injecting into regions
1 or 2, because we would silence not only the possible inhibitory neurons but also the conditioning neurons
themselves.

Approach 4: Recording. By recording in the right places we can hope to find neurons that behave the way we
expect inhibitors to behave as well as getting other evidence for or against our hypotheses. For example, if
Hypothesis II was correct, we might expect to be able to find PFC neurons that increase their responses to the
CS as the result of extinction training. Recording experiments are always somewhat risky though, because the
kind of cell we are looking for may be one among many, and we may never find instances of it even though it is
there. Thus it is always good to first try other methods to constrain hypotheses if one can. If Hypothesis III-A

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FraidyRat Tutorial
or IV-A were correct we might find neurons in region 1 that increase their responses to the CS during extinction.
And if we were able to find such cells, thus providing evidence for Hypothesis III-A or IV-A, we might be able
to distinguish these two hypotheses from each other by seeing whether the neurons we found could be
antidromically activated from region 2.

Approach 5: Use of methods 2 and 4 in conjunction. This might be especially informative. For example,
one might be able to determine whether erasure as well as inhibition contributed to extinction by trying to
abolish inhibition with a GABA blocker and then looking for decreased responses of fear-causing cells in
regions 2 or 3 as the result of extinction.

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Week 3 Lab Preparation Questions
[Note: These questions are due at the VERY START of Week 3 lab.]

The following questions (Prep Questions 1-9) are intended to prime you for designing and thinking about the
experiments that will be needed to answer the research questions we have posed.

Your answers to the multiple choice questions should be marked on the scantron you will be given for handing
in.

You should also mark your answers on a copy of this question sheet as well as providing written answers to the
underlined "Explain...." portions of the questions. This too must be handed in at the start of the lab session.
Please, however, be sure to keep copies of all your answers that you can refer to in class. You will need them
during our experiment-planning session at the start of the lab.

In real animals, renewal experiments have best convinced researchers that extinction involves inhibition of fear
whose underlying basis seems to persist to some degree despite extinction training. The most convincing
experiments used the so-called ABC design (see above). You should carry out such an experiment on FraidyRat
and see if you get renewal.

Prep Question 1: Did you get renewal?


(a) yes
(b) no

Prep Question 2: Refer to Figure 8 (reposted below). What can you infer about Hypotheses II, III, and IV
from your renewal experiment?
(a) Hypotheses II, III-A, or IV-A could explain my results whereas Hypotheses III-B and IV-B could not.
(b) Extinction seems to involve inhibition.
(c) Hypothesis II can be ruled out, but Hypotheses III and IV remain possible.
(d) My results are consistent with Hypotheses II and IV-B, but Hypothesis III can be ruled out.

Prep Question 3: Refer to Figure 8


I II III IV
(reposted right). From your results, Augmented Inhib of
Erasure Inhib via PFC
what can you infer about Hypothesis feed-forward inhib downtream
I? by CS. (A)Via neurons by
pot.input to CS. (A) Via
(a) Erasure does not occur. PFC inhibitor and (B) pot.of input to
(b) Erasure does occur. pot. of inhib inhibitor and
synapse itself. (B) pot of inhib
(c) No evidence of erasure, but synapse itself.
some might be occurring. Cntxt CS Cntxt CS Cntxt Cntxt
CS CS

Explain your reasoning in A? A?


answering Prep Questions 2 and 3. 1

B?
"Conditioning"
cells 2
B?
...

...

...
...

Figure 8 (repost)

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FraidyRat Tutorial

Although renewal experiments provide evidence that inhibition contributes to extinction in real animals, it
seems risky trying to make firm conclusions about a neurophysiological mechanism based entirely on behavioral
data. It would be nice to look into the matter more directly by studying the effects of GABA blockers on
expression of extinction. If extinction is really due to inhibition, and if a GABA blocker is applied to the right
parts of the brain, then one should be able to reverse the extinction. In FraidyRat you can apply drugs either to
single regions or to the whole brain via a systemic injection (but not to more than one specific region at a time).

Prep Question 4: Suppose that only Hypothesis II were correct, and after extinguishing fear of a CS very
thoroughly, you examined the effect of infusing a GABA blocker into either region 1 or 2 by themselves or
systemically. Which of the following results would be most likely?
Inject 1 Inject 2 Systemic
(a) Little or no effect Little or no effect Little or no effect
(b) Little or no effect Reverse extinction substantially Reverse extinction substantially
(c) Reverse extinction substantially Little or no effect Reverse extinction substantially
(d) Little or no effect Little or no effect Reverse extinction
(e) Reverse extinction substantially Reverse extinction substantially Reverse extinction substantially

Prep Question 5: Suppose that both Hypothesis II and III were correct, and after extinguishing fear of a CS
very thoroughly, you examined the effect of infusing a GABA blocker into either region 1 or 2 alone or
systemically. Which results would be most likely?
Inject 1 Inject 2 Systemic
(a) Little or no effect Little or no effect Little or no effect
(b) Little or no effect Reverse extinction substantially Reverse extinction substantially
(c) Reverse extinction substantially Little or no effect Reverse extinction substantially
(d) Little or no effect Little or no effect Reverse extinction
(e) Reverse extinction substantially Reverse extinction substantially Reverse extinction substantially

Prep Question 6: Prep Question 5 asks you to suppose that extinction, which is assumed to be due to both
mechanisms described in Hypotheses II and III, is very thorough. Thus inhibition would probably be operating
very strongly at the relevant sites in both regions 1 and 2. One might expect somewhat different results, and in
some ways get more information, if extinguishing only partially. Suppose you did the experiment in Question 5,
but instead had only extinguished partially. Which result would be most likely?
Inject 1 Inject 2 Systemic
(a) Little or no effect Little or no effect Little or no effect
(b) Little or no effect Reverse extinction substantially Reverse extinction substantially
(c) Reverse extinction substantially Little or no effect Reverse extinction substantially
(d) Little or no effect Little or no effect Reverse extinction
(e) Reverse extinction substantially Reverse extinction substantially Reverse extinction substantially

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Prep Question 7: Actually, in FraidyRat there is a complication that must be taken into account to see whether
systemic infusion of a GABA blocker will reverse extinction. If you inject FraidyRat systemically with GABA
blocker, what will happen (based on what you know from Week 1 lab)?
(a) Fraidy will always freeze even if it has not been conditioned.
(b) Fraidy will never freeze even if it has been conditioned.
(c) Extinction will be reversed because of the action of GABA blocker on Grisham's nucleus.
(d) Extinction will be reversed if it is partly due to inhibition.

Prep Question 8: What could you measure, instead of freezing, to circumvent the problem in Prep Question 7?
(a) The firing of cells in Grisham's nucleus.
(b) The firing of cells in PAGd.
(c) The firing in cells of PFC.

Although inhibition is thought to play a major role in extinction in real animals, this does not mean that erasure
does not also occur, at least to some extent. The same is true for FraidyRat. Obviously one way to look for
erasure while knowing that inhibition is (also) operating, is to see if there is any sign of extinction in animals
tested with relevant GABA-receptors blocked. In such an experiment, you should: (1) condition, (2) measure
fear (Test 1), (3) extinguish, and (4) measure fear again (Test 2). When, during such an experiment, would you
want to block GABA receptors?

Here are some possibilities:

Systemic GABA block status during various parts of experiment

Arrangement
i ii iii iv
Condition Norm Norm Norm Norm
Test 1 Blkd Norm Norm Norm
Extinguish Norm Norm Blkd Norm
Test 2 Blkd Blkd Blkd Norm

Prep Question 9: Which arrangement would be most important to run (think about what would happen if there
were a small amount of inhibition unrelated to extinction operating)?
(a) i
(b) ii
(c) iii
(d) iv

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FraidyRat Tutorial

Week 3 Lab Agenda


[Note: We have continued the numbering from the Week 2 Lab Agenda section.]

Experiment IV: Use GABA blocker


to look for relatively direct evidence I II III IV
of inhibition and to try to Erasure Inhib via PFC Augmented Inhib of
feed-forward inhib downtream
discriminate Hypotheses II, III, and by CS. (A)Via neurons by
IV. pot.input to CS. (A) Via
inhibitor and (B) pot.of input to
PFC
pot. of inhib inhibitor and
Refer to Figure 8 (reposted right). As synapse itself. (B) pot of inhib
synapse itself.
said above, although renewal
Cntxt CS Cntxt CS Cntxt CS Cntxt CS
experiments provide a valuable avenue
for trying to evaluate whether inhibition A?
A?
plays a role in extinction, one would 1
really like to investigate the matter with
more physiological approaches, and the
obvious one is to see what GABA B?
"Conditioning"
blockers do to established extinction. cells 2
So one question you will want to ask B?
here is whether such experiments
provide evidence of inhibition operating.
...

...

...
...
If inhibition does contribute to Figure 8 (repost)
extinction, you would like to know
where the inhibitory neurons are, what is controlling whether they fire, and where they exert their effects.
Information about these questions can be obtained by studying the effect of infusing GABA blockers into
specific regions. But, as explained in the Week 3 Lab Preparation Questions, there are some complexities that
must be kept in mind when doing these experiments.

These experiments will be most useful for discovering where inhibition operates. You should note that the
target of inhibition is the same in Hypotheses II and IV, but different in Hypothesis III.

Experiment V: Try to find and record from inhibitory neurons.

One obvious way to try to locate inhibitory neurons and see how they behave (e.g. they just turn on and stay on,
or they respond only when an extinguished CS occurs, or something else happens) is to record from them. You
should try to find cells that you think might be inhibitors in regions where you think they might occur. Where
does it make sense to look?

When are done, you should be able to determine what (if anything) your data tells about locations and behavior
of these cells.

Experiment VI: Investigate the effect of GABA blockers on cellular responses of an extinguished CS.

If you find cells that correspond to those illustrated in one of the hypotheses, you should be able to make and
test predictions about what effect GABA blockers would have on each type of cell.

Experiment VII: Use GABA blockers to try to determine whether erasure occurs.

The obvious way to test this idea is to see whether there is any sign that extinction training decreases responses
to the CS when the testing is done with GABA blockers operating. In thinking about how to do your
experiments, you should keep in mind Prep Question 9 above.

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Week 3 Lab Final Report Questions
[Note: We have continued the numbering from the Week 2 Final Questions section.]

Final Question 5:

After having completed Experiments IV, V, VI, and VII, which of our four hypotheses seem valid and which
seem to have been ruled out (the following are not mutually exclusive choices)?

(a) Hypothesis I
(b) Hypothesis II
(c) Hypothesis III
[Note that if you choose this option, also indicate whether A alone vs. B alone would seem best.]
(d) Hypothesis IV
[Note that if you choose this option, also indicate whether A alone vs. B alone would seem best.]
(e) My experiments force me to reject all four hypotheses.

Final Question 6:

Explain why you ruled out any hypotheses you did, and show (and fully explain) the data that convinced you
to do so.

Final Question 7:

Show and explain your evidence that erasure either can or cannot occur (depending on what you decided).

Final Question 8:

Conduct the following experiment, then consider whether the results are consistent with our still viable
hypotheses:

(i) Condition CS1 and CS2 in context A. 60 sec ITI, 5 sec CS, 2 sec US.
(ii) Extinguish CS1 in context B for 30 trials and extinguish CS2 in context C for 30 trials.
(iii) Give 5 presentations of CS1 in context C to look for renewal.

Does renewal occur? Is this consistent with whatever hypotheses have remained viable after all our other
experiments? Explain.

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FraidyRat Tutorial

Week 3 Lab Extra Credit Final Questions

Extra credit will add to your score only within the FraidyRat module; you can't get more than 100% on the
module as a whole. The extra credit questions are intended to be difficult; instructors will not help you with
them, but you are free to discuss them with one another if you should care to.

EC Final Question 11:

Design, do, and present the results of an experiment to determine whether the inhibitory neurons figured in
Hypotheses II, III, and IV become more effective as the result of extinction training (i.e. whether the
inhibitory synapses they make on their target cells become able to inhibit more effectively).

EC Final Question 12:

As previously explained, when a neuron excites some target cell, it often also excites inhibitory neurons that
partially inhibit the same target ("feed-forward inhibition"). When using GABA blockers to try to decide
whether extinction involved inhibition, one asks whether a GABA blocker reverses extinction. If it does, this
indicates that extinction had somehow increased either the firing of inhibitors or their effect (or both). If,
however, one gets a positive result in such an experiment, it might be simply due to removing feed-forward
inhibition that has nothing to do with extinction. Try to find evidence that GABA blocker-induced release
from extinction in FraidyRat is not simply the result of reducing feed-forward inhibition that is always there.

Helpful hint: One way to reduce the amount of conditioning that occurs during training (which is something
you might want to be able to do in this experiment) is to reduce the size of the shock you give. There is an
adjustment for this on the left side of the FraidyRat control panel.

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BACKGROUND STUDY:
EFFECTS ON CONDITIONING OF INACTIVATING REGIONS DURING CONDITIONING

Introduction

Earlier work on Fraidy Rat has established a set of regions that must be operational in order for previously
established fear of a CS to be expressed. We have referred to this set of regions (and their presumed
interconnections) as the "cued fear expression circuit". The neural changes responsible for CS-fear learning are
expected to be within some of the regions of the expression circuit, otherwise it is hard to imagine how CS fear
could be expressed if the regions containing the engram were not operational during testing. Thus the regions
containing the engram would be likely to be amongst the regions needed for fear expression (though it should be
recognized that determination of the fear expression circuit by inactivating only one brain region at a time could
result in some of the relevant regions to be missed).

Inactivation of a region should prevent changes from developing within it. Therefore, one way to get
information on which regions are changed by learning would be to inactivate regions during training (and then
test with everything operational). This was done in the present study.

Methods

Conditioning consisted of 5 conditioning trials with a 60 sec ITI, 30 sec CS, FraidyRat was conditioned in
and 5 sec US that co-terminated with the CS. After completion of one context and tested in a
different one. The regionss
conditioning 5 extinction trials were given in a context different from that of named were infused with
training to assess the amount of fear produced by the conditioned stimulus. GABA agonist during
conditioning , but not during
testing.
GABA receptor agonist was infused into various regions during conditioning
us
cs2
but not during testing. There were three conditions; a condition in which no
cs1
cntxt
0 100 200 300 400 500 600 700 800 900

drugs were applied during conditioning was run as a control.


1
Seconds
0.8

0.6
Control
0.4

0.2

Results
0 100 200 300 400 500 600 700 800 900

Coch nuc suppressed


1

0.8

0.6

The adjacent figure shows freezing responses over the course of the
0.4

0.2

experiment in each of the several conditions studied. [Note that to avoid


0
1
0 100 200 300 400 500 600 700 800 900

confusion, be sure to understand that these records are behavioral freezing


0.8

0.6
Thal suppressed
0.4

responses of Fraidy, not something measured within a nucleus of the brain.]


0.2

Above each graph is the name of the region that was suppressed during
0 100 200 300 400 500 600 700 800 900
1

0.8
Ctx suppressed
training. In the control condition good CS and context conditioning occurred.
0.6

0.4

0.2

There is no context fear during testing because the testing context was
0
1
0 100 200 300 400 500 600 700 800 900

different from that during conditioning. There was about 70% reduction of
0.8

0.6
Amyg suppressed
the freezing response to the CS over the five test trials.
0.4

0.2

0
0 100 200 300 400 500 600 700 800 900
1

0.8
PAGd suppressed
Conditioned responses to the CS developed when PAGd was suppressed
0.6

0.4

during conditioning, but not when any regions upstream of it were suppressed.
0.2

0
0 100 200 300 400 500 600 700 800 900

Figure 10

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FraidyRat Tutorial

NOTES ON FraidyRats PHARMACOPEIA


[Note: This is NOT comprehensive.]

NMDA receptors:

Let calcium ions into cells when glutamate binds while postsynaptic side depolarized. At some hippocampal
synapses low calcium ion entry causes LTD and high entry LTP. Similar effects at many kinds of synapses,
including some in defense behavior circuitry of the invertebrate Aplysia, on which many basic studies of
learning mechanisms have been done. The extent and longevity of changes can be influenced by various
neuromodulators.

mGluR (metabotropic glutamate receptors):

Glutamate binding to these receptors activates various intracellular chemical signaling systems that can in turn
have effects that cause or promote LTP or LTD, depending on the particular nerve cells involved. In cerebellar
Purkinje cells, which are inhibitory neurons, stimulation of these receptors contributes to calcium ion entry, and,
in contrast to hippocampal cells at which "classical" LTP and LTD have been studied, high levels of calcium
cause LTD while low levels cause LTP.

Serotonin:

Neuromodulator that can affect LTP or LTD. Especially well studied in Aplysia gill withdrawal circuit where it
greatly enhances the ability of NMDA receptors to cause LTP.

Dopamine:

Neuromodulator that can affect LTP or LTP in various regions. In striatum it promotes LTP mediated by
NMDA receptors and LTD mediated by mGluR.

NE (Norepinephrine):

Neuromodulator that can affect LTP or LTD cortically and elsewhere.

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SCHEDULING EXPERIMENTS USING THE LONG METHOD
[Note: Refer to this only if needed.]

So far we have been using the easier Short method of scheduling experiments, but there are some things you
can't do with this. Suppose you wanted to see what FraidyRat learns when you present a CS during, rather than
before, a US. A real rat would not learn to fear the CS under these circumstances, because the CS does not
actually warn of the US, which is already occurring when the CS comes on. As a way of illustrating the Long
method of scheduling, lets see what FraidyRat does under such circumstances. You probably wont need this
method very often, but you can refer back to the next few paragraphs when and if you do.

Long schedules are prepared in Excel and imported into FraidyRat. The rules are these:

An experiment is specified by a number of rows, each having 7 entries.


Each row tells the program to do something for a specified period of time.
Each row has 7 items (each in its own column), which are diagramed as follows:

Col A The row number (you must explicitly enter this; our program does not see the inbuilt Excel row
numbers).
Col B The context: 0=Home Cage 1=Context A 2=Context B 3=Context C.
Col C CS1: 0=No 1=Yes
Col D CS2: 0=No 1=Yes
Col E US: 0=No 1=Yes
Col F Execute a Pause in the experiment (a time-out for making measurements, changing drug infusion
status, making a cytotoxic lesion or whatever): 0=No pause 1=Pause (set all other columns
to zero if you are ordering a pause)
Col G Duration: Duration is given in seconds unless you want FraidyRat in his home cage (Context
=0). If you are instructing the program to put FraidyRat in his home cage, duration is in days.

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FraidyRat Tutorial

The following Long schedule has some text to explain what is being scheduled, but real Long schedules must
not have anything except the numbers on them or FraidyRat will crash. This schedule has FraidyRat sitting in
context A for 60 seconds, then the US comes on, then CS1 comes on, then the US is turned off, then after 2
more seconds CS1 shuts off. In other words, the US comes on for a short time in the middle of the CS, as we
had wanted. This is done three times.

Figure 11

The file is then saved as a text file (tab delimited), which is a format option Excel provides. Again, the
spreadsheet may have nothing but the block of numerical information. Any other writing on the sheet will cause
FraidyRat to crash.

You should now open an Excel file and copy and paste the above numerical information into it. You should
also add three lines of your own to specify a test of CS1 in context B. That way, when you run the experiment
you will see if any response to CS1 has been learned without any background contextual fear to possibly
confuse things. Specifically: (1) switch to context B for 60 sec, (2) add in CS1 for 3 sec, and (3) have context B
alone for 60 more sec.

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Save your file as a tab delimited text file (one of the Save As options in Excel). You can name it whatever you
want. Place it in the same folder where the FraidyRat program lives (ask you instructor if you don't know where
that is). Then go back to FraidyRat write the name of your file in the fname box of Make or Get Schedule
(without the .txt extension), and hit Import Excel Sched. Your schedule should now be operative. But do NOT
try to see it by pressing Short, because this is not a Short schedule and the program will probably get confused.
Now hit Run/Continue. What you get should look like this:

Figure 12

Initially in context A, FraidyRat is unafraid and exploring, so the freezing score is zero. When you give the US,
he hops about, so the freezing score stays zero. But when the shock stops, he is now afraid of context A and
freezes. Over the next 60 sec this freezing extinguishes a bit. But when the shock comes on again, he again
hops about, etc. When the context switches to B, all the contextual fear goes away, so the freezing score goes to
zero. When the CS1 comes on a little after 250 sec, there is no sign at all of freezing. Thus, like a real rat,
Fraidy did not become afraid of a CS that began after the US was already on.

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