>>Edmund Griffin: Good evening. I'm very excited to share with you today some
recent findings from my research on the molecular basis of
drug addiction vulnerability. Let's start with the question at hand. Why is it that some people just can't get
enough? As a clinical psychiatrist I do get this question
a lot. And I think that the very wording of this
question actually captures some of the frustration and also, to an extent, some of the stigma that's associated with
substance use disorders. But the question also captures one of the
most confusing and frequently misunderstood aspects of addiction,
which is this; when it comes to substances: alcohol, cocaine,
exactly how much is enough? Where do we draw that line in the sand? For alcohol, is it two drinks a night, three
drinks a night, four? Or for cocaine, is it two lines of cocaine
a night, four lines of cocaine a night? Or does any type of routine cocaine use mean
that you are addicted to cocaine? That you just can't get enough. How much is enough? Now, to be fair, as a psychiatrist, I can
tell you that this problem with precision, it's not
something that's unique to the diagnosis and treatment of addiction
disorders. Sometimes, I will get questions about this
with regard to anxiety or depression. A person might say, "Dr. Griffin, I can't
stop worrying about this mole on my arm. Do you think I have an anxiety disorder?" A little bit of anxiety and worry about the
mole is a good thing. But how much anxiety is enough? How much is too much? Or, similarly, a person might ask me, "Doc, I've been sad for months since my company
failed. Sure, a little bit of sadness and self-reflection
might be good. It's my fourth company to fail. But, wait, Doc, how much depression? How much sadness is enough?" How much is too much? Often, in the diagnosis of psychiatric disorders
- this is including addiction - the answers to these questions lies not in
the quantification, but rather in the function. How are these factors affecting your life? And which direction are they moving the needle
with regard to your relationships, with regard to your work function or your
time at school or to your physical health? So, when we start to think of substance abuse
from the functional perspective, things start looking differently. In medicine - one of the most important lines
in the sand to distinguish addictive drug use from recreational drug
use is compulsivity; continued drug use despite negative consequence
to your physical health, to your work life, or to your relationships. And when we look at addiction from this view, as opposed to the arbitrary definition of
enough, something very interesting emerges. We find that only a small percentage of people
who use a drug of abuse will transition into addiction. For alcohol, that percentage is 12 percent. For marijuana, it's 6 percent. And for cocaine, it's 15 percent. Think about that for a second. All right, when you consider the social and
economic cost that addiction takes on our society, these numbers
are strikingly low. What it tells us is that if we could make
even a small dent into these percentages we could be looking at a very, very important
impact in our society. So, what is it? What are the factors that make one person
resilient to cocaine and another person vulnerable to addiction? Well, the first step to answering these questions
is to look at the risk factors. And some of these are listed here on the left:
history of trauma, early onset of use, poverty, history of depression,
personality disorder, early use of alcohol, marijuana or nicotine,
marital status, race and ethnicity. So, if you look for cocaine addiction, and
you Google any one of these terms, you'll find a study that looked at some connection
between the two. But when you do the meta-analysis or you look
to see what is the most strongly associated factor, what
factors are most reproducible in the epidemiological studies, the two factors
that stand out most strongly associated with subsequent cocaine addiction
are prior use of alcohol, prior use of nicotine. This observation was actually first made as
far back as 1975. It's often referred to as the Gateway Hypothesis. The Gateway Hypothesis is a population-based
observation that describes the sequential pattern in drug abuse progression. Use of alcohol and nicotine usually precedes
the use of marijuana, which typically precedes use of illicit drugs
like cocaine. Most of the public policy debate, as you guys
are all aware, will tend to focus on marijuana as the quintessential
gateway drug. But the epidemiology is clear that any one
of these three drugs, either individually or in sequence, is a very
strong predictor of subsequent drug use and abuse. But we have to tread lightly here because
this is an epidemiological study. It's a correlation. As all of you know, correlation does not equal
causality. In fact, in the 40 years since the Gateway
Hypothesis was first described, we still didn't know whether or not the order
of the different drug classes is socially determined or if it's biologically
determined. For instance, it could be that smoking marijuana
and drinking a lot, you're more likely to meet somebody who happens
to have cocaine in their pocket. That's not biological. But if it is biological, what are the cellular and molecular mechanisms
that might mediate this process? Addressing these two questions could reveal
new insights into the developmental trajectory of addiction. And it could also help us identify new molecular targets for treatment. Fortunately, research or breakthroughs - actually
- in the biology or neurobiology of memory formation have provided an ideal
platform for us to begin to address these questions on a biological
level. What does memory have to do with addiction? Accruing evidence over the past 20 years has
actually pointed to the idea that addiction is a disease of learning and
memory. This view stems from the observation that
both the neuroanatomical pathways, as well as the fine-toothed molecular pathways
that mediate formation for a natural reward, such as food, water or mating, these same
pathways are exploited by drugs of abuse. So, let's look at the neuroanatomy first. This here is a cross section of a human brain. In the context of a natural reward - it's a nice big piece of turkey or a new love
interest - dopamine is released to different regions
of the brain that will then work in concert to help you remember both the environmental
queues, as well as the sequences of behavior that
helped you obtain that reward in the first place. Dopamine that goes to the nucleus accumbens
helps to consolidate memory to increase your motivation to perform certain
tasks in the context of environmental queues. Dopamine going to the dorsal striatum increases
habit formation. This is so that you don't have to learn how
to use chopsticks every time you eat sushi. Dopamine going to pre-frontal cortex provides
executive control over both motivation and habit formation. This makes it so you don't start eating your
sushi in front of a big tiger. This is a choice mechanism, and this is what's
used in the formation of natural reward. This is very important. That dopamine here is not the pleasure chemical that you might read about in the news. Dopamine isn't there to make you feel happy. Dopamine's role is to act as a reinforcing
agent to make sure that you do it again. It's critical for evolution that you eat,
that you drink water, and that you make babies. And dopamine is there to make sure that happens. What we know and what we've learned over the
past three decades that all drugs of abuse: alcohol, nicotine,
cocaine, including powdered sugar, all of those act by increasing dopamine in
each of these regions. The trick here is that whereas dopamine levels
will start going down over time with your new love interest, they don't go
down with cocaine. Cocaine hits the spot each time, essentially
tricking the brain into thinking that it's found new love. Now, most of what we know about addiction
centers on the nucleus accumbens, and that's what we'll focus most of our talk
today. But if you take the nucleus accumben cells,
take it out, crush it up and look at it on a plate, you find that some of the fine-tooth molecular
pathways that are used to form memory - for a natural reward - these
are also exploited by cocaine or addictive drugs. So, your brain - your neurons - adapt and
respond to the environment in a very unique way. Whereas, your muscles can get bigger when
you exercise or get smaller if you don't exercise, your
brain doesn't have that option. Because it exist within the confines of your
skull, it doesn't have the option of hypertrophy. So, your brain - your neurons - adapt to the
environment by growing synapses, The more synapses, the stronger the memory
connection. The formation of memory in a cell precedes
in two steps. These two steps are, one, a signal transduction
pathway that sends a signal into the nucleus. You can think of a signal transduction pathway
as molecule A hits molecule B hits molecule C,
kind of like a string of dominoes. But the point is to communicate something
that happens in the environment to the nucleus, which is the control center
of the neuron. Once that information is communicated, it
activates a molecule here called CREB, which binds to the DNA, which is the template
that holds all of your genes, and drives new gene expression, new proteins,
that then create a stronger synapse. This is the basic two-step formation of memory. Well, cocaine has learned this two-step formation
process. And they use exactly these two steps in the
brain when you're exposed to drugs. When you receive cocaine, it initiates a signal
transduction pathway that sends the signal to your nucleus. This creates new gene products, and those gene products go on to alter synaptic
connections. We now know several of the molecules that
are involved in this cascade that are critical to the formation of addiction. How do we use this information to understand
the Gateway Hypothesis with regard to addiction? One way to do this would be to translate the
epidemiological observation into a rodent-based study where we can do
things in the laboratory, where we can actually take their brains out. This would have several advantages. Number one, it would allow us to address the
Gateway Hypothesis in the absence of social factors that are
already known to influence the development of addiction such as availability
or your peer group. So, in this case, they're rodents. They're in a cage, and they're doing drugs. So, we can test the biology a little bit more
specifically. Number two, because they're rodents, we can
take their brains out. Can't do that with people. This allows us to test the cellular and molecular
mechanism of addiction to understand how sequential
exposure to different drugs might affect things on the biological level. And, finally, and most importantly, we can
do causality studies. We can use pharmacological or genetic interventions
where we change the animal's genes. We can use these types of interventions to
test specific molecular hypothesis with regard to how addiction is working. And this is something that you cannot do with
epidemiological studies or with humans in the lab. So, how do we create an animal model? Not as easy as it sounds. As many of you already know, we've seen a
revolution in what we know with neuroscience and how the brain works. But translating that revolution into actual clinical interventions in psychiatry
has been pretty slow. One of the reasons for the discrepancy in
progress is the absence of adequate rodent models. We can model something like stroke in a rodent. Just clamp the artery. You can model something like asthma in a rodent. But how do you model depression in a mouse? Or what does a paranoid delusion look like
in a rat? It's very difficult. In fact, the same difficulty exist in the
study of addiction. For the past 30 years, the gold standard model
for studying addiction in rodents has been rodent self-administration. So, this is a picture of it. This is a rat. And he presses a lever. It looks like he's snorting from the lever, but he's actually going to press it. When he presses that lever, it activates a
computer, which sends a signal to a pump. And that pump will inject a quick dose of
cocaine directly into his bloodstream. So, in this model, the animal chooses when
and how much cocaine he uses. And they will do the cocaine. They'll work for it, which is great. Looks like addiction. No, it doesn't. That's not addiction. As we discussed earlier, simply using cocaine,
simply liking cocaine is a big difference from being addicted to
cocaine. As we said earlier, 85 percent of people who
use cocaine will not become addicted. And that's not a recommendation for anybody. But those are the numbers. Eighty-five percent will not become addicted. So, in order to study addiction, we need to
improve the model. So, when I was a resident in psychiatry I
started working in Eric Kandel's lab, and I started trying to develop a model to
see if we can get the animal to press the lever for cocaine even under aversive consequences. To do that, what we did is we inserted a mild
foot shock of increasing intensity every 40 minutes during his cocaine self-administration
session. During the first 40 minute session there's
no consequence. He presses the lever one time, two times,
three and four. Now, on the fifth lever press he gets an injection
of cocaine. One, two, three, four, five, cocaine, and
then he kind of runs around in a circle, looks high for a while and he'll pause. And about three minutes later, one, two, three,
four, five, then he'll run around again in a circle. He'll do this for 40 minutes. Just a regular day of cocaine. Everything's fine. After a time-out, the next 40 minute session
is different. He presses the lever and a blue warning light
comes on. He looks at the warning light; means nothing. But he keeps pressing, but on the fourth lever
press he gets a mild foot shock of 0.1 milliamps. It's not an intense foot shock, just to let
you know. It's your finger in a lightbulb. Even less than that. He gets the mild foot shock. He'll slow down, but if he continues pressing he'll get his cocaine injection. What this does is it forces the animal into
a conflict. He wants to use the cocaine, but the cocaine
comes with a consequence. And our hypothesis is that prior exposure
to a gateway drug like nicotine or alcohol will make some animals more compulsive in
this phenotype. To test that hypothesis, we use the following
paradigm: we have the animals receiving either alcohol
every day or a water control. After 21 days of these two treatments, we
introduce cocaine self-administration, which happens concurrently with their alcohol
treatment. Now, importantly, the alcohol is given for
only two hours a day. We want the alcohol to be fully metabolized
by the time that they do cocaine self-administration. And that's important because we don't want
the two drugs to see each other, and also because if they're drunk they get
in there and they don't even press the levers. It's a very difficult study. So, they do not have alcohol in their system
when they're doing cocaine. And we'll get back to that a little bit later. So, does prior exposure to chronic alcohol
increase compulsive cocaine self-administration? The answer is yes. If you look at the water group - these are
our alcohol-naive group - even with the small challenge of 0.1 milliamps
they decrease their lever pressing for cocaine by 85 percent. That's a huge drop off. And then as you increase the severity of the
punishment, or the consequence, to 0.2 milliamps the majority of animals start
dropping out of the study. They feel that shock once, done. By the time you get to 0.3 milliamps, they've
stopped. In contrast, animals who have been previously
exposed to alcohol stay in the study, pressing five times more with the mild foot
shock of 0.1 milliamps. And as you increase the intensity to 0.2 milliamps,
they're still pressing it 20 percent. And then when you get to 0.3 milliamps, they're
still in the study. This substantiates our hypothesis that prior
exposure to alcohol does increase compulsive cocaine self-administration. But, wait a minute, alcohol's a potent neurotoxin,
as many of you might know. It kills neurons. It could be that being exposed to alcohol
made it so that the animals couldn't even feel the shock on their paws. Or maybe the alcohol had some other type of
cognitive effects, so that they can't interpret the blue warning
light. To test for these types of non-specific effects,
we use the same paradigm - one group receiving alcohol daily and the
other group receiving water- but instead of having them press for cocaine,
we asked them to press for a sugar pellet; a natural reward. And we asked does prior exposure to alcohol
enhance compulsive pressing for a sugar pellet? And it did not. Alcohol treatment did not enhance compulsive
lever pressing for a natural reward in food restricted animals. As you can see here, as you increase the intensity of the foot shock from 0.1 to 0.2 and 0.3,
they decrease their lever pressing by 50 percent at the 0.2 milliamps. And by the time they get to 0.3 they're down
to about 10 percent. But, importantly, there's no difference between
the alcohol pretreated and the water control. What this tells us is that alcohol is acting
as a gateway drug, and that it does not have non-specific effects
with regard to natural reward. There's another take-home point here. If you look at the gray line, just look at
the water control. These guys have never been treated with alcohol. These are just regular, every-day rats. And see here that they stay in this - for
natural reward, when they're food restricted they're actually pretty compulsive. They'll stay in the study and tolerate a foot
shock way more than they do even for cocaine. And this is important because it gets back
to what we were talking about earlier that evolution has equipped the mammalian
brain with the machinery to navigate this type of conflict. The machinery can navigate a conflict between wanting something important, like
food when you're hungry, and the consequences. So, if I told you about my friend Joe who
lost three fingers and sustained third-degree burns on his body while going through a fire to
save his daughter, you'd say Joe's a hero. He did the right thing. But if I told you that Joe lost three fingers
and sustained third-degree burns while saving his cocaine from a big house
fire, you'd say Joe had a problem. Joe has a problem with cocaine. And what we see here is that like the human
condition, simply using cocaine does not put you in that category. Whereas, the same rats have a capacity to
make this decision when they're hungry. What tips them over here is prior exposure
to the gateway drug alcohol. So, what we have here is a full developmental
trajectory from a risk factor - alcohol use - all the way to cocaine addiction. This puts us in a very unique situation where
we can now ask what's going on in Joe's brain. What's happening there to tip him over towards
addiction? Well, we can begin by looking inside the nucleus
accumbens, which, again, we have a large baseline of
information from previous studies. Now, as I mentioned before, cocaine exposure
alters gene expression in the nucleus accumbens. Now, for several decades we thought this was
the end of the story. If you have the genes for cocaine and you
use cocaine, that's a wrap. But we now know that DNA is not destiny. That there is many other aspects controlling
your genes. So, you might have the genes to be tall, but
that doesn't mean you're going to be tall. You can have the genes for diabetes. Doesn't mean you're going to get diabetes. You can have the genes to live to be 100. Doesn't mean you're going to live to be 100. There are other layers that control our genes. And what we found over the past very recent,
maybe decade or so is that all of your genes exist in an inactive or
active state, depending on these epigenetic modulators. Epigenetic is above the genes. It's another layer of control. So, your DNA isn't just sitting there waiting to be activated by the environment. There's several layers that control or decide
whether or not your genes will exist in a silent form or in an active form. But two big controllers that we'll talk about
today are the HATs and the HDAC. The HATs are called histone acetyltransferases, and what they do is they add these negative
charges to the histones. Your DNA is tightly wound around these balls
called histones. Adding a negative charge causes the DNA to
repel against itself because DNA has a negatively charged backbone. So, histone acetyltransferases cause the repelling
and put the DNA in an unwound or permissive state. Histone deacetylases do the exact opposite. They rank these negative charges off, causing
the DNA to go back into a coiled inactive form. Every cell in your body exist in some dynamic
process between these two classes of molecules. Amir Levine a post-doc in Eric Kandel's lab,
used this information to ask a question about nicotine, which is
one of the other gateway drugs that is strongly correlated with subsequent
cocaine use. And he found that nicotine inhibits histone
deacetylate activity, which essentially releases the brakes, causing
the DNA to become increasingly permissive, which makes
it more acceptable for cocaine; basically opening the gates, so to speak,
for cocaine-induced gene expression. So, using this information we're intrigued
by the possibility that alcohol might have a similar mechanism. After all, epidemiologically speaking it is
a very strong gateway drug as well. So, we wanted to know does chronic alcohol
exposure enhance cocaine-induced gene expression. To test this hypothesis we gave the animals
10 daily exposures to alcohol. And the other group received just water control. After 10 days of alcohol exposure, we gave
them a one-time injection of cocaine. And then we asked does that prior exposure
enhance cocaine-induced gene expression? And if it does so, does it do this by increasing
histone acetylation by decreasing HDAC activity? Let's go through each one in turn. We found that chronic alcohol use does indeed
enhance cocaine-induced gene expression. And what we're looking at here is DeltaFosB. This is a protein that's already previously been shown to be one of
the molecules that goes on and is involved in later synaptic transformation. What we found, though, is that animals who've
been previously treated with alcohol have enhanced cocaine-induced
gene expression almost twofold above our alcohol-naive group. So, our next step is to start working backwards. Okay, if it's increasing cocaine-induced gene
expression, how? So, first we asked is it doing this by adding
these negatively charged molecules to the histones? So, we did an assay to measure these histone
acetylation aspects, and we found that prior exposure to alcohol
does indeed increase the production of these negatively charged
molecules on the histones around the DeltaFosB gene that we were just
talking about. Then we asked does it do this by inhibiting
histone deacetylase? And, yes, the answer was there true also. Chronic alcohol use decreases histone deacetylase
activity in the nucleus accumbens. But which HDAC? I initially had HDAC as one little ball. It turns out there are 11 of them. If we could figure out which histone deacetylase is inhibited, this would be a very important step towards
understanding what could be a specific target for treatment. So, we actually went through several steps
to determine which one of the HDACs is being involved. We actually focused a lot of our attention
on HDAC4 and 5. We found that chronic alcohol use is acting
by decreasing nuclear accumulation of histone deacetylase 4. So, again, we used a 10-day exposure protocol, and we took the animals' brains out at different
time points after their last sip of alcohol. As we talked about earlier, exposing animals
to alcohol causes a decrease in histone deacetylase activity. And as you can see here, as alcohol's leaving the blood stream there
is a precipitous decline in overall histone deacetylase activity. If you take these same lysates - these same samples - and run them on a gel
and look at the actual amount of protein for histone deacetylase 4, you see that HDAC4
is declining precipitously during the withdrawal phase. So, this tells us that we have a specific
target that alcohol is acting on to release the breaks towards cocaine addiction. This is very important. It says that HDAC4 could be acting as a node
to increase vulnerability to cocaine addiction. You may have heard the term that your genes
load the gun, but your lifestyle pulls the trigger. This has been a statement that we've heard
in all forms of medicine. Not just in brain science. Liver, lung, kidney. This is us in medicine moving away from this
idea of genetic determinism and starting to accept and acknowledge the
fact that your genes are being regulated on so many different levels, and that these
levels are exquisitely sensitive to the environment. What we're proposing here is that the lifestyle
choices chronic alcohol use, chronic nicotine use, and that the trigger
is HDAC4. But to test that hypothesis, we would need
to inhibit histone deacetylase 4 independently. We want to pull the trigger independently
of that prior exposure. So, one way to do that is to use a molecule
called MC1568. I was very happy when we found this molecule. It was actually produced by chemists with
specific intention of decreasing HDAC activity - HDAC4 specifically. It's a selective histone deacetylase inhibitor. So, we said if we give the animals this HDAC
inhibitor, if our theory's correct, giving them this
molecule - no alcohol, no coke, no nicotine - it should recapitulate the cocaine addiction
phenotype. And that's exactly what we found. That selective degradation of HDAC4 enhances compulsive cocaine self-administration. This first slide here is just looking at natural
reward. Again, we want to check to see if this drug
is causing non-specific effects. And we found that MC1568 does not affect compulsive
lever pressing for a sugar pellet. The animals at 0.1 milliamps, there's no effect. And by the time you get to 0.2, they decrease
by 50 percent down to 20 percent. But there's no difference between these groups. But when they're lever pressing for a cocaine
reward, you see a split. At 0.1 milliamps, there's no difference. They've decreased their lever pressing by
about 15 percent. So, they're both sensitive to the shock. But as you get to a more intense shock, the two groups start to divide. Our control group has decreased down to 40
percent. Whereas, our other group is still at 75 to
80 percent. And as you get to a lower intense foot shock,
our control group starts to drop out of the study. Whereas, our guys who've had selective inhibition
of HDAC4, they're staying in even at a very intense
foot shock of 0.4. So, this completes the loop. This is the causality experiment that I was
describing earlier. So, in conclusion, we found that animals with
a history of chronic alcohol use have enhanced compulsive self-administration, which is used despite negative consequence, which is the line in the sand that we use
to distinguish recreational drug use versus addictive drug use. We found that alcohol, like nicotine, inhibits
histone deacetylase activity in the nucleus accumbens - the region that
controls motivation. And this results in a permissive environment
for the genes, which allows for more or enhanced cocaine
induced gene expression. We found that chronic alcohol use promotes
this decreased nuclear accumulation of HDAC4. And, finally, we show that selective degradation
of HDAC4 with the inhibitor MC1568 recapitulates the compulsive
phenotype. Thank you very much.
