Document 6580407

Left-overs from Exp II and other things:
1. The "Questions you should be able to answer" section of Exp II read as follows:
Questions you should be able to answer after doing the above experiment. Did you find some cells that you think
might be "conditioning" cells for cued fear conditioning? Where are they? Do you feel that you can rule out the
possibility that they are representation cells that got established as such during the conditioning process rather than what
we have called "conditioning" cells (in the sense of our discussion in Section 1 of this document).
My intention when I wrote the above was that if you felt the experiments you had done did not allow you to conclude that
the possible conditioning cells you had found really were such, you would not just say "no", but try to figure out
experiments that would allow you to decide whether they were and go back and do them. However, I did not make this
sufficiently clear. I think almost nobody did the relevant experiment(s), but at this point we still have too much else to do
for me to want you to go back and do more work on Exp II. So I am sending you this note to discuss the matter, and we
will resolve the matter together during lecture on Monday.
2. I have revised the lab manual somewhat for the remaining experiments on extinction and am sending you a fresh version
of the relevant parts of the manual. It is attached. Please use it, rather than the original version, when doing your
experiments. There is also now a section on "Going Further" added, that you can ignore but that I added for future use.
3. I made some revisions in the Final Report questions, as I warned you might happen. It is attached. This might get
slightly altered even further, but this new version will be pretty close to the final one.
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Exp IV. Where in the cued fear expression pathway do cellular responses to the CS cease as the result of extinction?
By now you have a fairly good idea where cellular responses to a CS develop as the result of conditioning, and you
should by now think you know where "conditioning" neurons (in the sense of Chapter 1) reside. When one extinguishes
FraidyRat, behavioral fear responses to the CS go away, and that must mean that neurons somewhere along the expression
pathway that have learned responses to the CS stop responding. It might be that presumptive "conditioning" neurons
themselves stop responding or it might be that they keep responding but responses stop somewhere farther along the
pathway. The purpose of this experiment is to try to find out where this happens.
We recommend utilizing a longitudinal approach in this experiment. Find a unit in whatever region you are concerned
with that you think is involved in mediating conditioned fear and then extinguish and see whether the unit you are recording
stops responding. Of course if neurons in a given region do stop responding, presumably those downstream of it will as
well. So what you are really looking for is the most upstream region where responses stop.
Questions you should be able to answer after doing the above experiment. Where along the fear expression pathway
do cellular responses that are thought to mediate conditioned fear cease as the result of extinction? It could of course be
that failure of extinguished FraidyRat to make behavioral fear responses is due to failures of response at more than one
locus. Neurons down steam of the most upstream region where failure occurs will also stop or reduce their responding
simply because the neurons exciting them reduce their responses. But perhaps there is some sort of erasure or inhibition
operating downstream of the spot you identified in this experiment. We are not asking you to do experiments on this, but
can you think of kinds of experiments you would do to try to get at this question? This matter is pursued further in Exp XI
in the "Going Further" section below.
Exp V. Does FraidyRat show renewal?
As explained above, in real animals the behavioral phenomenon of "renewal" provides evidence that extinction is
probably at least in part due to active inhibition of fear that is not itself fully erased. There are in fact three ways to do a
renewal experiment. One is to train an animal in context A, extinguish it in the same context, and then switch to context B
and see whether there is some recovery of fear (if this occurs, it is described as "AAB" renewal). A second is to train in A,
extinguish in B, and then test in a third context, C ("ABC" renewal). A last is to train in A, extinguish in B, and then go
back and test in A ("ABA" renewal). In real animals, all three forms of renewal usually occur, but ABA is most extreme,
ABC less impressive, and AAB sometimes doesn't work very well. You should test at least for ABC renewal in FraidyRat.
Questions you should be able to answer after doing the above experiment. Is there renewal in FraidyRat? Does it
seem to you that it provides entirely convincing evidence that extinction is at least in part due to inhibition? In particular,
you should also try to decide whether ABA renewal, which in real animals is typically most pronounced, and ABC renewal,
which we are asking you to test here, provide equally convincing evidence for involvement of inhibition in extinction.
Do renewal experiments rule out the possibility that extinction is due to some sort of mix of inhibition and erasure? Be able
to explain the reasons for your answer.
Exp VI. Can effects of extinction be reversed with GABA receptor blockers?
Traditionally, behavioral experiments, such as those on Renewal have been used to try to assess whether extinction is
mediated at least in part by inhibition of conditioned responses that themselves persist despite extinction training. However,
another approach to investigating the possible role of inhibition in extinction is pharmacological: Can effects of extinction
be reversed with GABA receptor blockers? The purpose of this experiment is to look into that. The advantage of this
approach is that it is more direct and less inferential than that offered by Renwal experiments.
In Exp IV you discovered at least the most upstream region where extinction training reduced neural responses to the
CS, and you may or may not look for and find downstream places where responses seem to become still further reduced. In
trying to find out whether extinction can be reversed with GABA blockers, it probably makes sense to first use systemic
injections and to measure responses at some very downstream site where you feel sure you would see any signs of recovery
that there might be (for example look at freezing itself or neural activity at Grisham's nucleus). If you do get any recovery,
you should then probably infuse your drug into regions where it seems most likely to be having its effect when injected
systemically. One site you would certainly want to test would be the most upstream site of extinction-produced neural
response reduction. And then if (and probably only if) you have any evidence that further reductions occur at downstream
locations, you would probably want to see what effects infusion has there too [however, this is something that you will
probably not want to get into unless you do the relevant experiments in the "Going Further" section.]
Questions you should be able to answer after doing the above experiment. Were you able to reverse extinction with
GABA blockers, and if so, where does it look like the drug was probably having its effect?
Experiments that use GABA blockers to try to determine whether some specific process is dependent on inhibition are
complicated by the possibility that effects seen are due to GABA blocker-dependent increases in excitability that are
unrelated to the process of interest. In the present context, it would be possible that blockers could abolish inhibition that
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is constitutively operative and not related to extinction, and thereby cause extinguished (or perhaps partially extinguished)
responses to resume. Signs of such constitutive inhibition might not be noticeable in unconditioned FraidyRat because in
them there might be no excitation to be inhibited, and it might not be noticeable in well-conditioned, un-extinguished
FraidyRat because excitation or responses were too powerful to be successfully inhibited or so powerful that despite a
modest decrease of excitation, a very strong response is still observed (a "ceiling" effect). Therefore, what would be
needed would be to compare the effects of GABA-receptor block on responses that are weak or modest because of
extinction with responses that are similarly weak or modest for other reasons such as initially weak conditioning. We are
not going to ask for such experiments here, because our time is limited. However, one way to achieve the weak
conditioning that would be needed to do the experiment would be to simply reduce the strength of shock use as a US. An
adjustment for doing this is available on the program's front panel. This is discussed in more detail in Exp X of the "Going
Further" section below.
Exp VII. How is inhibition that contributes to extinction produced?
If extinction is really due to inhibition of fear that does not itself go away, one would like to know whether there are
inhibitory neurons that turn on in some way as the result of extinction training, and if there are, one would like to know
what it is that activates them, which could potentially be a complex chain of other neurons. As we have discussed, one
popular view for real animals is that there are neurons in PFC which drive neurons that inhibit amygdala cells that would
otherwise cause manifestations of fear. Of course even if extinction is due to inhibition, one might not be able to find
neurons that turn on as the result of extinction training, because it might turn out that the neurons doing the inhibiting fire
whether or not extinction has occurred, and what extinction training does is to make the inhibitory synapses themselves
more effective than they were before extinction.
The are a number of ways to go about trying get information that bears on the above matters. Two are the following:
Approach A. One would be to look for neurons that increase their activity as the result of extinction, either just in
response to the CS or perhaps whenever the subject is in the extinction context. The most obvious place to start such a
search would probably be in the region where neurons that would otherwise produce fear become less responsive during
extinction (as investigated in Exp IV). One might also look for neurons that might themselves be inhibitors or that might
drive inhibitors in other regions of the brain. However, one difficulty of such investigations is that if one fails to find what
seem like possibly relevant neurons in a region, it might be that they are in fact there, but just relatively few in numbers.
Approach B.. If one has reason to believe that inhibition might be driven from regions outside the fear expression
pathway, another approach would be to see if silencing candidate regions causes a return of extinguished responses. One
would of course want to focus ones investigation on regions that project to those parts of the fear expression pathway where
one thinks that neurons responding to the CS might get inhibited.
You should try both approach A and B.
Questions you should be able to answer after doing the above experiment. Can you find any neurons that seem to turn
on as a result of extinction training, and if so, where are they? Do they turn on and stay on to provide somewhere with a
tonic influence or do they only turn on when a CS is given? Can you find any regions outside the cued fear expression
pathway that seem as though they are possibly the source (or a source) of extinction-related inhibition (i.e. either are the
inhibitory neurons or generate activity that leads to the activation of inhibitory neurons)? If so, do they appear to be the
sole source?
Exp VIII. Where does extinction learning occur and is it due to NMDA-dependent synaptic strength changes?
Extinction is a kind of learning in which animals learn not to respond to former conditioned stimuli when those
stimuli are repeated without reinforcement. Given our general working hypothesis that learning in FraidyRat is all due to
altering the strength of existing synapses, one would suppose that it might be due to the weakening of synapses that were
previously strengthened by conditioning, or it might be due to strengthening of certain inhibitory synapses or to the
strengthening of excitatory synapses that help to make appropriate inhibitory neurons fire. Given our more specific
working hypothesis that FraidyRat learning is all due to NMDA-dependent synaptic change (LTP or LTD as studied at
Bliss-Lomo type synapses), one would expect extinction to depend on NMDA receptor-dependent synaptic change
somewhere in FraidyRat's brain.
Our goal in this experiment both see whether our hypothesis that all FraidyRat learning is NMDA receptor-dependent
applies to extinction learning and to try to find out what we can about where the synaptic changes that we presume underlie
extinction learning reside. There are various ways that one might approach investigation of these questions.
One would be to start by examining the effects of systemic injections of NMDA blockers on extinction. If they work,
then one would try them in specific places that seemed like plausible targets of their action. If they don't work, one might
then try systemic injections of drugs that affect other transmitter or neuromodulator actions that one suspects might play
roles in some kinds of synaptic change. As a guide to this, should it become necessary, there is a brief rundown on some
possibly relevant neuro-chemicals in at the end of this document. Having started with systemic drug injections, one would
then have to go on to locate the site or sites of their action.
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Another approach, if one thinks one has a pretty good idea of where the changes responsible for extinction learning
are occurring, would be to start right off with infusions into that locus.
Questions you should be able to answer after doing the above experiment. What, if anything, were you able to do to
prevent extinction and where did you have to do it?
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A Last Question. Let's see if you can put what you have learned about FraidyRat into some sort of coherent picture.
The cells in some important regions are pictured at the right. A single circle denotes a single cell type, not a single cell.
Amygdala Type 1 and 2 cells are shown.
The endogenously active neurons of Grisham's nucleus,
which drive exploration, are shown.
PFC cells are shown. You may or may not have recorded
PFC
from these and you may or may not have looked for effects of
altering their activity, but since they are thought to play a role in
extinction in real animals, they are included.
Posterior cortex cells are shown. You have not recorded
from them, but you can assume that some of them respond
Posterior
specifically to CS1 and others specifically to CS2.
Ctx
CS2
CS1
PAGd cells are shown. You may or may not have studied
their responses, but we know that PADd receives input from
amygdala and is on the cued fear expression pathway; assume
for the purposes of this question that there is only one cell type
in this nucleus and that all of them inhibit the spontaneously
Type
Type
Amyg
1
2
active neurons of Grisham's nucleus, which drive exploratory
behavior. We have not discussed reinforcement mechanisms,
but presumably there are cells somewhere that fire when a US
PAGr
occurs. These are in fact in PAGrostral, and they are included in
US
the diagram.
Based on your experiments and on what you know about
PAGd
conditioning in FraidyRat, which cell types do you think
synapse on which other cell types, and which of these
connections are excitatory and which inhibitory (use an open
Grisham's
triangle to denote an excitatory synapse and a filled circle to
nucleus
denote an inhibitory synapse)? You should draw in those
connections that you believe are or must be there, indicating
whether they are excitatory or inhibitory. And you should indicate which synapses you think change their strength, and in
what direction, when you condition CS1, and also which change in strength and in what direction when you extinguish CS1
after having conditioned it.
You need not have actually done experiments to get evidence for something you put on the diagram if logic tells you it
must be there. But you should explain your reason (data or logic) for each connection you draw and for anything you
indicate about how things change during conditioning or extinction.
Going Further [for future use]
Exp IX. Are increases of fear produced by GABA blockers in extinguished FraidyRat really due to reversal of
extinction-induced inhibition?
Exp X. Is extinction partially due to weakening of synapses that are involved in producing conditioned fear?
Exp XI. Test for neural response decrements produced by extinction in regions downstream of the most upstream site of
decrement.
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6. FINAL REPORT
1. How many kinds of neurons could you find in FraidyRat's amygdala, and what does each respond to? Illustrate
responses of each type of cell you found and explain fully your understanding of the circumstances under which each of
these types of neuron will be active (i.e. what stimulus will activate it, what responses depend on some sort of prior
experience, etc).
2. Suppose you conditioned FraidyRat to CS1 in context A and then extinguished him in context B. If you now compared
the response of an amygdala cell that before extinction responded strongly to the CS1 in context A and B, where would
you expect to get a stronger response of your cell after extinguishing in B:
In A or in a different context C?
Explain why you would you expect this?
3. Where can you better reverse extinction by infusing a GABA receptor blocker: Infuse in amygdala or infuse in PAGd?
Show an experiment that answers this question. Explain these results in terms of your current understanding of how
FraidyRat's neural circuitry works.
4. Suppose you conditioned FraidyRat with either (a) his posterior cortex, (b) his amygdala, or (c) his PAGdorsal
suppressed with a GABA receptor agonist during the training procedure, and you then tested his responses to the CS with
no drug present. In which of the three cases would you see responses to the CS during the test? Explain the reasons for
your expectations in each case.
5. A common view of many people working on real rats is that extinction is due to pre-frontal cortex-dependent inhibition
of neurons that would otherwise cause conditioned fear. Is this true in FraidyRat? Show the results of an experiment that
evaluates this possibility.
6 A. Regarding Exp VIII, what if anything, were you able to do to prevent extinction, and where did you have to do it?
6 B. Were your above findings regarding NMDA blocker and extinction consistent with the literature on real animals? Go
to PubMed and see what you can find there on this matter. You might start, for example, by searching on "fear, extinction,
NMDA". Look at earlier studies first, because that is where the basic studies will be. Later studies may be dotting i-s,
crossing t-s, discussing special cases and exceptions to basic findings, etc. You will probably find that just looking at
abstracts is sufficient (it will depend partly on how the abstracts are written), but definitely cite any studies that are crucial
to your answer.
7. Present a written answer to the "Last question" (asked after Exp VIII).
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