“From Molecules to Behavior: Role of Nicotinic Acetylcholine Receptors…”-Marina Picciotto, PhD

“From Molecules to Behavior: Role of Nicotinic Acetylcholine Receptors…”-Marina Picciotto, PhD


So now I’d like to introduce dr. marina
Picciotto doctor dr. Picciotto is the Charles B Murphy professor of
psychiatry and professor of Neurobiology and pharmacology at Yale University she
received her BS from Stanford University and her PhD from Rockefeller Rockefeller
University under the supervision of dr. Paul Greenberg she went on to conduct
postdoc postdoctoral work at the Pasteur Institute in Paris and she joined the
Yale faculty in 1995 so the goal of dr. potatoes lab is to understand the role
of single molecules and complex behaviors related to psychiatric illness
more specifically she has focused on understanding nicotinic respected colon
receptors which I believe she’ll be talking to you about today she’s used
multiple methods and multiple approaches to studying these receptors including
knockout and transgenic mice viral vector mediated gene transfer
pharmacology and behavior analysis more recently some of her work has focused on
the interactions between stress and nicotine and the role that those
nicotinic receptors play she’s been able to model many of the behavioral effects
of nicotine and mice and demonstrate the links between individual nicotinic
receptors and stress-related effects of nicotine in addition she’s also focused
some of her research on the neuropeptide Gowan in’ in opiate reward and
withdrawal and overall these studies have helped explain the molecular basis
for complex interactions that lead to addiction stress interactions and may
help target new medications dr. Picciotto oops sorry I’m behind
slide dr. Picciotto is also or was also the
senior editor for The Journal neuroscience from 2006 to 2012 and she
sat on many NIH and other study sections she’s been the recipient of numerous
awards including several NAR sacks and most recently was an elected fellow at
the Institute medicine this is a very important
position as the Institute of Medicine asks and answers the nation’s most
pressing questions about health and health care it’s an institute of
medicine if you don’t know is an independent nonprofit organization that
works outside of the government sorry I’m in pain there we go
works outside of the government provide unbiased and authoritative advice to
decision makers and the public members are elected for their excellence and
professional achievement for those at the top of their field membership at the
Institute of Medicine reflects the high professional treatment and commitment to
service additionally she has fostered and mentored many students throughout
their achievements such as National Institute of Mental Health research
awards and training grants she has over 174 publications and six
more currently impressed as funa peoc grows its own commitment to neuroscience
and seeks to attract new students I particularly admire dr. potato’s unique
ability to make science accessible to wider audiences this is evidenced by her
role in bio step which is an intensive summer research program for students and
underrepresented groups who are interested in biomedical research we’re
extremely pleased to have dr. Picciotto speak to us today and so please join me
in welcoming and welcoming her perfo talk so first of all thank you for that very
generous in introduction I am actually relieved now that I’ve seen pictures of
myself that I chose to wear a different jacket than the ones that are in that
those pictures that would been highly embarrassing so before we start I’d like
to thank the organizers for putting on this incredible meeting so let’s thank
them because this is an incredible amount of work great job and second I
would like to welcome you and thank you for coming out on a Sunday morning which
is pretty awesome it’s not not every profession can get a whole bunch of
people out on a Sunday to come listen to neuroscience so go neuroscience and
other I think Randall applause so I’m going to talk to you today about an
overview of basically our philosophy of how we can get from very basic science
to really important questions for human beings and overall although I’m going to
be presenting the research that we’ve done what I really want you to pay
attention to is the fact that we can use molecular approaches we can use
techniques that are really asking fundamental questions about how
molecules and neurons work and then we can actually broaden our approach to
take in behaviors that are important and ultimately to investigate in human
subjects how we can use this information to develop new treatments or to go
forward in understanding human disease so I’m gonna give you one problem but in
fact of course this is applicable to to many many of the projects that you’re
probably working on now and I’m happy to talk more about this approach later
after the talk so here’s the problem that I’m going to talk about today and
that is that there are still despite the rise of obesity the the number one
preventable cause of death today in the developed world is still smoking we
still volunteer ourselves to do a neuro science
on the effects of nicotine on our brains and up to 350,000 people died in the
United States alone every year due to smoking related illness and of course
this is a medical problem lung cancer is something that we can investigate we can
investigate chronic obstructive pulmonary disease but wouldn’t it be
easier to understand the neuroscience of the behavior that drives that addiction
and that keeps people smoking despite the fact that every one of us in this
room knows that it’s a really really bad idea so the majority of smokers actually
would like to quit and I was at a conference years ago when I was
listening to a panel of teenagers who all said yeah I started to smoke
somewhere between the ages of 13 and 16 and then I got to 18 and I really
thought this was stupid and I decided to stop and I couldn’t I made that decision
before I could actually figure out that later on it would be a regret and when
it came time to stop I actually could not change my behavior there’s the idea
that this is a failure of will that it’s a problem with a habit and that all you
have to do is really make the decision to stop but of those who really choose
to stop if they do it without any help you know if they just say I’m gonna go
cold turkey only about six to eight percent actually can still remain
abstinent from smoking a year later so this is still a current problem it used
to be that probably around 60% of the population smoked now it’s about down to
about 25 or so maybe somewhere around 23 percent of the US population so the
people who were able to say okay well you know I just do this for fun have
probably stopped and those who are still smoking are very dependent on nicotine
so let me outline now the molecular biological neurobiological problem that
we’re working with so nicotinic receptors in the brain and I’ll talk a
lot more about this are distributed very widely this is an image through a human
brain showing you that here’s a structural MRI showing you the the brain
areas and here’s an overlay of binding of a
nicotine like molecule to various brain regions and you can see actually that
here there’s a very high level of binding there’s a little bit lower here
these lighter these darker blue areas are still actually significant areas
where nicotine binds in the brain with high affinity and in fact for human
imaging of these receptors it’s quite remarkable it’s very hard to get a
negative control there isn’t a brain area you can go to
and say well let’s subtract that because these nicotine receptors aren’t almost
every neuron and then there’s this other problem there’s an incredibly wide
variety of reasons that people say that they smoke and I’ll again go into some
of this in greater detail but if you ask one smoker they may say well I just
enjoy it and what does that mean that means that the reinforcement the parts
of the brain that are in charge of drug reward are being activated by nicotine
there are others that say well I smoke too to manage my anxiety I smoke to
manage my depression it’s another set of brain areas in another set of behaviors
and finally there are those that say well I’m afraid to gain weight when I
quit and I will talk a little bit more about all three of these and in general
what we want to know is our people smoking for the same to activate or
inactivate the same nicotine receptors are they smoking to activate or
inactivate the same brain areas and are they smoking to activate or inactivate
the same behaviors and the answer to all of those is no it’s not the same there
are a lot of reasons and finally here is the big problem that we are actually
interested in in an approaching and that is that there are an awful lot of
nicotine receptors so this is a family tree of all the nicotine receptors you
can see that they’re about 15 different subunits that have been identified in
human and mammals in general this family up here are those that are at the
neuromuscular junction that is that all of your significant communication
between your nerves and your muscles actually go through nicotinic
acetylcholine receptors and they use the normal neurotransmitter acetylcholine
nicotine does not act very well at these muscle receptors which is good because
then in fact we would really have trouble with movement but it’s
these family this family here these receptor subunits here that are
expressed in neurons and throughout the brain that we think are absolutely
critical for the behavioral effects of nicotine so here’s a little bit more
about these receptors those of you who are molecular biologists will probably
recognize these because they’re sort of the poster child for excitatory expert
for amino acid gate I’m sorry but neurotransmitter gated ligand gated ion
channels so here’s what they look like they span the membrane they’re large
proteins and they have a pore right down the middle and when astral choline the
normal neurotransmitter binds to these receptors the pore opens ions flow into
the neuron and the cell is depolarized nicotine binds at exactly the same site
as acetylcholine well exactly as in the same part of the molecule not
necessarily exactly the same amino acids and it does the same thing it opens
these receptors but what is really important to remember about these
receptors is that whereas nicotine can open these receptors transiently it can
then desensitize or turn off these receptors for a much longer time so
although the initial thing that nicotine does is to activate neurons to open
these channels and defend depolarize neurons over a longer period of time
what it does is to interfere with the normal signals through Astle choline
so when Astral choline is released if nicotine has been around and
desensitized these receptors it can no longer do what it normally should do
these are sort of head-on diagrams of what the nicotinic receptors look like
they’re like staves around a barrel five subunits are arranged around that
central pore and what’s important to remember is that a shtetl choline and
nicotine binds at the interface between these subunits and what’s important
about that is when you have a large family of subunits that combine in
different organizations each of those interfaces is going to have slightly
different properties each one is going to bind a little more tightly or a
little less tightly to nicotine or to acetylcholine each one of those is going
to make the channel open for longer or a shorter period of time and maybe make
the channel more or less likely to desensitize after it’s
open so it matters which of these different subunits are arranged in that
receptor and it’s also important for drug development if each of these
interfaces is different then you can imagine that we could actually make
medications that would target different interfaces differentially and I’ll talk
a little bit more about that the end so here the family of receptors that are
important in the in neurons and you can see that there are three broad types
I’ve diagrams just three types but you can imagine that with with all of those
subunits this is really just a broad representation and the three families
are those that are Homo Merrick that is five different subunits are exactly the
same around the outside so they are going to have very different properties
from these where you need at least one alpha and one beta to make a functional
receptor and if the the nicotine binds at the interface you can see that in
these types of heteromeric receptors they’re going to be about there are
going to be two binding sites whereas in these home america receptors they’re
going to be five binding sites and that’s going to change how these
channels react to nicotine in the brain we have mainly these two families but
this family is represented in a smaller subset of neurons but in the
fight-or-flight response in the autonomic ganglia responsible for for
example the adrenaline response to to distress to fear this is the receptor
that’s really critical for the fast synaptic transmission for the ability of
Astral choline to drive those ganglionic responses in the brain these are more
modular Tory so they change behaviors that are already initiated to some
extent rather than driving behavior the way that the these autonomic ganglia
receptors do so in terms of where they’re expressed here’s where I showed
you before where nicotine binds in the human brain and in rodent and in other
mammalian brain systems it’s pretty similar you have a very high level of
receptors in for example here’s the thalamus which is the part of the brain
that’s important for relaying sensory information to the cortex and then there
are two families of receptors I don’t know
how well you can see this but you can get an idea that there’s some subunits
that are really broadly expressed and they follow this pattern of nicotine
binding and there’s some that are really localized like these here that are only
expressed in the in this case these are the dopamine cell bodies in the midbrain
that are important for the rewarding properties of drugs of abuse and that
just simply sets the stage to tell you that we can use
perhaps pharmacological agents that target these different subtypes to get
very specific effects on behavior or more general effects on behavior and
we’re going to now segue into the many reasons that people smoke so the first
really important reason is that people get addicted and despite the fact that
actually tobacco industry executives still say well you know people choose to
smoke it’s not an addiction there is obvious there’s evidence from human
studies there’s evidence from animal studies that in general nicotine
activates the same parts of the brain as other drugs that are abused by humans
including cocaine amphetamine morphine heroin and that that drives the
rewarding aspects of cigarette smoking and that those kinds of rewarding
aspects allow you to make associations between that reward and your environment
that drives a compulsive behavior habitual behavior which is not habit but
in fact the ability of cues in your environment to take over and drive your
smoking intake so I showed you this and now you can see a little bit larger but
here in the midbrain are the neurons that are activated in some way or
another by every one of those drugs of abuse any addictive substance that is
used by humans somehow increases dopamine signaling from this area so
here in the middle this is a mustache right it looks really very
characteristic if you see it you know you’re in the dopamine system and what
you can see is that here in this medial region is the ventral tegmental area
which is critical if we oblate that area or if we block it then drugs of abuse no
longer actually can control behavior and what you can
see is that there are a lot of nicotine receptors in that brain area if you look
here here those very selective subunit expression patterns alpha six and beta
three it doesn’t matter what their names are are almost exclusively expressed in
these dopamine neurons and then if you look these broadly expressed subunits
beta 2 and alpha 4 well they’re actually enhanced enriched in this brain area as
well and there are accessory subunits there may be more broadly expressed but
also seem to pull out this area and what that suggests is that these nicotinic
receptors really can control activity in this area essential for drug reward in a
way that is very effective so here is my diagram of a very very very highly
simplified brain it has actually 1 2 3 5 neurons in it and all of those neurons
can be controlled by nicotine receptors and what you can see here is those are
the dopamine cell bodies and I told you they’re sitting in the ventral tegmental
area they have nicotine receptors on them and when nicotine binds to those
receptors it increases the firing of these dopamine neurons very simple
increases the release of dopamine and that can result in the rewarding effects
of nicotine but that’s not enough because there also other ways that
nicotine modulates the circuit so for example the excitatory inputs to these
dopamine neurons have nicotine receptors on their terminals as well so that
nicotine drives not only the the direct response of the dopamine neurons but
actually and gives it an extra punch by increasing glutamate the excitatory
neurotransmitter released onto these neurons and on the terminals there are
nicotine receptors that are enough to induce dopamine release even without
action potentials coming down the axon so it can hit it here it can hit it here
it can excite it here what’s interesting is that when you’re naive to nicotine
nicotine can also increase inhibition on to these dopamine neurons so at baseline
you have this mixed excitation and inhibition so you have one set of
responses and then over time in a slice nicotine can do
sensitizer inactivate these receptors while these receptors stay active so you
can go from a mixed excitation and inhibition to a more unmixed drive of
this circuit and that’s thought to be one of the reasons that despite the fact
these receptors can turn off or desensitize prolonged exposure to
nicotine like the kind of prolonged exposure you would expect over years of
smoking can actually go from a mixed excitation inhibition to a more unmixed
drive of this circuit so in fact over time the nicotine signal gets stronger
in this circuit so what does that do in a in a whole organism well if we take
slices through those dopamine cell bodies and we look at what nicotine does
to the firing rate normally it increases the firing rate of those dopamine
neurons and we can start to identify which molecules are essential because if
we take away one subunit the beta 2 subunit which is the one that’s
responsible for this broad pattern of general binding to nicotine throughout
the brain what you see is that broad binding goes absolutely away and
knockout mice that lack that subunit and so does the ability of nicotine to cause
the firing of those neurons and if we just look more broadly at the ability of
nicotine to stimulate the dopamine system by evaluating the release of
dopamine in the terminals of those ventral tegmental areas what you can see
is in a normal animal we have a dose-dependent increase in dopamine
release when we give nicotine to these peripherally and that completely goes
away if we take away one subunit out of those 15 and that goes along with the
ability of nicotine to be rewarding we can actually evaluate this by asking and
I call this the bar office experiment if I gave you permission to to go to the
bar and smoke and drink as much as you wanted or if I told you when you go to
the office you can to smoke or drink at all and then on Saturday I asked you to
choose where do you want to go do you want to go to the bar or to the office
those of you who actually do smoke or drink
or who who have other rewards let’s say food
would probably go to the area where you could get those rewards and you would
avoid the area where you are forbidden from having those rewards and that’s the
same as this place preference experiment what we do is we pair exposure to
nicotine with one compartment of a chamber and exposure to saline just a
random non rewarding stimulus and the other compartment and after training we
ask the individual to choose between those compartments and if they liked the
nicotine they’ll go spend more time exploring the compartment where they got
the nicotine and that’s what you see here at baseline both compartments are
exactly preferred the same way and after training with nicotine there’s more
exploration of the nicotine paired compartment less exploration of the
Saline paired compartment and when we take away one subunit of the nicotine
receptor that completely goes away nicotine is no longer preferred and in
fact saline and nicotine are equally boring so there’s no change in behavior
and we use this as a measure of nicotine reward and what I can tell you is that
we can also start to identify the cell types that are involved the brain areas
involved as you can see from this picture if you take away the beta 2
subunit all of the nicotine binding goes away but we can put it back using
conditional methods to express the nicotine receptors only in particular
brain areas and what you can see here is that in this particular transgenic we
put the nicotine receptor back only into the mustache into the VTA
and into the terminals here’s in the forward part of the brain and the
nucleus accumbens where these neurons actually project and release dopamine
and when we did that what we could do is to rescue behaviors that are associated
with psychostimulants and so in this case I’m just showing you one behavior
as a field this has been done for reward it’s been done for reinforcement for
habitual behavior in this case I’m just going to show you psycho stimulant
effects so you know psycho stimulants like cocaine and amphetamine can
actually arouse a person or an animal and we can measure that by increased
locomotion and what you can see here is that nicotine does the same thing when a
normal individual gets nicotine they’re actually more active if we take away
these receptors throughout the brain there’s much less where there’s no
effective nicotine on a psychostimulant response and if we put it back only into
the dopamine neurons we can rescue that so that means that although these
receptors are expressed throughout the brain you actually only need them here
for at least some of the behavioral responses to nicotine so as it field
this has been a huge success story many many different laboratories have been
working out on this over the last about 15 years and using various combinations
of molecular techniques and various behavioral tasks we’ve been able to
narrow down exactly which receptor is involved in these let’s say reinforcing
or psychostimulant properties of nicotine we’ve been able to identify the
cell types in the brain area and we’ve been able to actually target these
receptors not as it as a drug company has done this and treat smokers and help
smokers who would otherwise be unable to quit to quit and I’ll just give you a
quick summary here we can actually make some of the nicotine subunits
hypersensitive and we can show that that is actually sufficient to drive a
preference for nicotine that reward experiment I showed you the transgenic
expression of one subunit in the VTA and the ventral tegmental area and that
rescued psychostimulant effects you can use a virus – just to reexpress a
particular subunit in that brain area and show that that rescues the
willingness of the animal to work for nicotine and we can actually knock out a
nicotine receptor only in those dopamine neurons and show that that is sufficient
to get rid of nicotine reward and altogether this has given us the name of
the receptors alpha for alpha 6 beta 2 it’s given us brain area ventral
tegmental area and shown that that is sufficient for nicotine reward
reinforcement and psychist so here’s our diagram again of our
different nicotinic receptors groups here’s the receptor that has been
identified here’s the brain area that’s been identified and it’s this interface
right here that’s targeted by chantix varenicline i have no association with
the drug at all but Pfizer developed that with an intelligent drug design
method to target this interface and find a way to stimulate it a little bit but
block the effects of nicotine at that interface and that partial agonism is
what makes this actually currently the most effective way to help smokers who
want to quit get abstinent so I think that’s a success story because in
psychiatry we’ve been trying to find receptors for various psychiatric
illnesses we’ve been trying to find drugs that very selectively target those
receptors and to see if we can actually help people who are otherwise
unresponsive or or not medicated by the current treatments we have and here’s
one for perhaps our most important killer in the in the psychiatric world
and that is nicotine addiction so you can imagine that decreasing smoking by
about 10% would already be enough to save many many lives okay so that’s one
it’s very obvious it’s an addiction story but why else do people smoke well
another thing that people report is that they smoke because they’re afraid to
stop smoking and gain weight and even scarier one group of smokers teenage
girls report that they start smoking specifically to try to be lean to to
lose weight and if weight control is the number one reason that a particular
group of smokers are saying they start that’s really particularly tragic so is
it possible that it’s the nicotine and smoking that drives this overall
decrease in weight in in smokers you can imagine there’s a lot of reasons right
it could be just that you have something in your mouth something in your hand
it could be one of the other 4,000 constituents of tobacco smoke that
changes metabolism or changes weight so we have to ask is it the nicotine in
tobacco that’s important well there are some clues from animal studies and for
example the laboratory of neil greenberg was a pioneer in this area and he showed
that in rats at least nicotine alone can decrease food intake and there’s
interesting sex differences female rats are more sensitive to this appetite
effect than male rats and Michaela Zoli and his group also showed that you can
model the the fact that nicotine withdrawal can increase food intake
decrease metabolism and increase weight he showed not only that the behavioral
consequences of nicotine withdrawal matched between rat and human he also
showed that he could find molecules that were associated with changes in
metabolism that were induced by with by smoking with drove by nicotine
withdrawal and that all leads to the idea that perhaps it is indeed the
nicotine and tobacco that controls appetite in humans however in general
the neurobiological effects of these mechanisms are not really well
understood so that’s what I’m going to tell you about now so first we have to
show that we can model these effects in a model in an animal model in this case
in mice and what you can see is that mice in general gained weight over time
and this is from very late adolescents about let’s say eight weeks of age all
the way through until adulthood and if we give nicotine to these animals they
actually decrease the rate of weight gain and you can see that there’s a
dose-dependent so that at the slightly higher doses there’s even a little bit
of dip in their weight before they start a whole new curve of weight gain and
these kinds of doses are very very low they’re not very different from what you
would expect if there was heavy smoking so what can we do we can now start to
use pharmacological agents that selectively target different nicotine
receptors and ask which of these receptors might be implicated and
luckily there are some pharmacological agents that are at
there that we can try so here’s two here’s a nicotine I’ve already showed
you that that is effective at decreasing the growth curve and what you can see is
that there all of the heteromeric receptors in fact all of the receptors
for nicotine not surprisingly are responsive to nicotine and you can see
that here in a slice preparation in neurons in the brain nicotine causes an
inward current regardless of which receptor is on that neuron but here’s
another nicotinic like molecule it’s called cytosine
it’s an extract of a plant called laburnum which actually grows outside is
probably starting to bloom and give you allergies here on campus and this
extract is interesting in that it is used in a smoking cessation aid in
Eastern Europe that is called I don’t remember however that that smoking
cessation aid is basically just cytosine which acts a lot like that chantix drug
that I told you about already it is a partial agonist of the receptors that
are important for nicotine reinforcement and what does that mean that means that
nicotine causes a big opening of those receptors cytosine causes a little
opening but if you combine the two it blocks the effects of nicotine at
another set of receptors these are the ones I told you about that are
responsible for the fight or flight response nicotine and cytosine act
exactly the same so dis cytosine have the same or a different effect on weight
gain it has the same effect as nicotine so that gives us a clue if it acts the
same on those alpha 3 beta 4 receptors first of all that suggests the receptors
that are important for nicotine reward are not the same receptors that are
important for appetite and it also tells us that now we can perhaps target the 2
receptor subtypes independently so here we’ve got another picture of our our
receptors not this time it’s not the this header america receptor that’s
important but in fact this receptor that has the alpha 3 and the beta 4 subunit
that may be important for appetite so the autonomic ganglia are not in the
brain so if we think that perhaps it is the fight-or-flight response it’s
important for this effect Daan appetite and you can imagine that
it might be you wouldn’t want to go eat if you were being chased by a tiger then
it might be that this effective nicotine is actually happening somewhere else in
the body not in the brain and so what we can do is we can block the peripheral
receptors those in the ganglia those in the body with a drug that doesn’t get
into the brain and we can say does this block the effect on feeding and what you
can see here is that it does not so here you see that if we give animals this
drug that decreases their their appetite they eat a little bit less and if we
block try to block that effect with a nicotine blocker that doesn’t get into
the brain it doesn’t actually alter that that appetite suppression so in fact
this is probably not happening in the body it’s probably happening in the
brain so where in the brain might that be well we know a lot now about the
parts of the brain that control food intake and what’s really cool is that we
can now as a field drive the firing of different neurons in the hypothalamus
and show that there’s one population of neurons that actually causes animals to
eat and one population of neurons that causes animals to stop eating and
they’re both sitting in the same brain area and this is work by many people but
most recently by Scott Stern s’en and his colleagues here is diagrammed the
arcuate nucleus of the hypothalamus now the hypothalamus is a part of the brain
that really tells you to match your behavior to the needs of your
environment so what what does it do it tells you when you’re thirsty tells you
when your body needs calories it tells you actually it’s also involved in
stress responses it tells you when it’s less important to get calories than it
is to escape a predator and all of those signals converge on the hypothalamus and
somehow the hypothalamus balances those signals and the output says eat if you
stimulate your npy neurons stop eating if you stimulate your pom C neurons so
we were very naive we said okay well maybe what nicotine is doing is somehow
it’s activating these pom C neurons so how do we test that
well one way that you can test it is look at a molecular marker of the
history of whether those neurons have fired when you give a challenge with
nicotine that actually decreases appetite and the way that we do that is
by looking for immune acetic chemistry for an antibody response that recognizes
a marker called cephas now cephus is simply a gene that is induced by high
activity in a particular neuron so what we can do is we can give animals
nicotine we can evaluate whether or not that decreases their appetite and then
we can actually look for C fossa mean or activity and Costain for pom see the
marker of the neurons that tell animals to stop and humans to stop eating and
what you can see here is images through the arcuate nucleus of the hypothalamus
the dark brown stain is C phos and this purple stain is pomf C and when we have
value and we count the number of neurons particularly throughout the hypothalamus
that are marked with C Fuss after a nicotine challenge you can see that
cytosine and nicotine overall don’t cause a change in the C fossa mean of
reactivity but when we co label only for those pom C neurons the appetite
suppressing neurons you can see there is a big induction with both of the
nicotine drugs that decrease appetite and the pom C neurons and what’s
interesting about that is probably what you’re getting if you get no overall
changes you’re getting a decrease in the activity of the neurons that say eat and
an increase in the activity of the neurons that say don’t eat and what you
can see that’s very important for the validity of this finding is that that
doesn’t go away over time whereas many of the effects of nicotine are
desensitized that is they decrease over time this effect stays even if you treat
for 28 days nicotine still site in this case
cytosine still decreases food intake and it still increases the activity of the
neurons that say stop eating all right so this is a
showplace that nicotine is acting this is by no means proof this is pure
correlation but it gives us a hint of where to look look at some more so the
first thing that we’re going to do is the same thing that I told you was done
for evaluating the effects of nicotine on reward and that is let’s look at a
slice through the brain in the area where those pom C neurons sit and we can
do this because we can express a fluorescent marker GFP only in pom C
neurons and when we record from pom C neurons what we see is that nicotine
increases their firing this should look familiar
and that gets blocked by a nicotine blocker it’s dose dependent mecha
melamine is the nicotine blocker it completely blocks it and it suggests
that yes at least in this slice we can stimulate those neurons directly with
nicotine so now what we want to do is is it in to ask is it enough to eliminate
those pom C signals to get rid of the ability of nicotine to control appetite
so how do we do that well we can look at a knockout nice mouse that doesn’t have
pom C at all and ask in those knockout mice does nicotine have any effect on
appetite and I’m showing you here wild-type mice here’s a dose-dependent
decrease in food intake when we treat with cytosine in this case and that goes
away in these pumps you knockout mice so that’s a hint okay good pom C neurons
may be essential for this effect but because these this is just one node in
the pathway and because these neurons are so essential for food intake in
general it’s important to interrogate this another way so we asked is can we
intervene in another point in this pathway and get the same effect and so
what you note what we know is that these pom c neurons have their cell bodies
here the neurons project upward in the hypothalamus to the paraventricular
nucleus and there they signal through mc4 receptors to send that signal to
stop eating go do something else you’ve got you’ve had enough food to other
places in the brain so if we can now intervene here by decreasing the
activity of these mc4 receptors is that now sufficient to block the ability of
nicotine to decrease feeding it’s the way that we did that was to
just design viral vectors that target a very specific gene using something
called a small hairpin RNA an SH RNA that finds one particular target in the
genome binds to the RNA and both blocks its translation and causes its
degradation it’s a very nice way in cells or in in the brain to target a
particular gene without disrupting the rest of the genome and to do it in
adulthood and in very specific neurons using a viral vector to deliver these
shrnas so we delivered these shrnas to the paraventricular nucleus of the
hypothalamus and we targeted the pom c receptor mc4 so what we could show is
that we could decrease the expression of MC for very efficiently and that when we
put this decrease this sh RNA into the paraventricular nucleus that we could
block the ability of nicotine to decrease food intake so here’s gfp
control so just to control for the viral vector infusion into the into the art
starting to the paraventricular nucleus we still get the ability of cytosine to
decrease food intake and that’s highly blunted when we knock down MC for so we
have a circuit we know where we want to look for nicotine’s effects on apatite
but we have this strange receptor it’s expressed mostly in the autonomic
ganglia and here we’re proposing that it’s actually acting in the arcuate
nucleus of the hypothalamus to change the activity of pom c neurons and to
decrease food intake so what is the evidence that there is actually any
nicotinic receptor of that type in the hypothalamus it wasn’t known so what we
did was to take those pom C GFP expressing sections and to isolate the
neurons that Express GFP using a laser capture of microscopy procedure to take
those cells and to prepare RNA from them and then to use quantitative PCR on the
on the RNA expressed in those cells to ask the question is the beta for subunit
even expressed in those neurons and what you can see here is that yes those
neurons have the RNA here I’m showing the cDNA for the pom C gene that’s good
we’ve got the right identified neurons they also Express the beta 4 subunit and
we know that’s selective because if we do this in a beta 4 subunit knock out
that transcript is gone so indeed to our surprise in the arcuate there is this
important subunit that controls the response to stressful events in the
environment and in the brain this particular subunit is important for the
ability of nicotine to decrease appetite now I’ve shown you it’s expressed but I
haven’t shown you that it’s necessary for this effect of nicotine so then what
we wanted to do was exactly the same we wanted to use shrnas targeting only the
beta 4 subunit in the arcuate nucleus and to ask if we knock down the beta 4
subunit can we abolish these appetite effects of nicotine and so as a nice
control here we had another subunit the one that we know is involved in nicotine
reward it’s also expressed in this part of the brain we could knock that down
and say if our hypothesis is correct we shouldn’t have an effect of knocking
down beta 2 but we should have an effect of knocking down beta 4 and that’s what
we’ve done so I’m showing you here again validation that we can actually knock
down either beta 2 or beta 4 using these small hairpin RNAs that we deliver with
a virus here’s the arcuate nucleus of the
hypothalamus our control is a scrambled shrna delivered with an AAV vector here
is paid a to knock down here’s beta 4 knock down in each case we challenge
with cytosine and you can see that here we have a decrease in food intake in the
normal animal we have a decrease in food intake when we knock down beta 2 in the
arcuate but that completely goes away when we knock down beta 4 in the arcuate
nucleus so really this is good evidence that the pathway that we’ve outlined is
actually essential in the arcuate nucleus for the appetite suppressing
effects nicotine and here’s a sort of summary of
our circuit and that is that in the arcuate nucleus we have mingled pom c+
neurons that decrease appetite and npy positive neurons there are nicotinic
receptors containing the beta 4 subunit on these pom si+ neurons when nicotine
comes into the brain they’re activated they go up to the paraventricular
nucleus where they activate the mc4 receptor on paraventricular nucleus
neurons and this signal is essential for the ability of nicotine to decrease
appetite decrease food seeking shift behaviors presumably to others that are
not involved in in this particular behavior and to cause weight weight the
weight control aspects of nicotine nicotine signaling so back to this
picture you’ve seen it before now we’re targeting these receptors
right here we know they contain the beta 4 subunit
we don’t actually know what the Alpha is we know that in the ganglia in the
fight-or-flight response itself of 3 but in the arcuate we’re now actively trying
to ask what are its partners so that we can target this interface and find
pharmacological agents that really work selectively to to suppress appetite and
we know the brain area we know that the arcuate is absolutely essential as are
its projections higher in the brain so that’s another reason that people smoke
but it is again only one of many so I’m going to ask now one more behavior why
do people smoke despite the fact that they know that it is highly toxic
the reason is another reason is that people smoke to control their symptoms
of mood and depression so one thing that we know from epidemiology studies is
that people who are depressed are much more likely to smoke I told you about 20
to 25% of the general population smokes those who have depression are about
double have about double the likelihood of smoking and we have a paradox and
I’ll tell you what that is in human studies a number of investigators have
delivered nicotine different ways and phan found that they have very
different effects on mood so Paul Newhouse and his colleagues in Vermont
were able to infuse nicotine very rapidly in an IV paradigm and he found
that his patients reported that they felt depressed and anxious another set
of studies have used nicotine patch to actually treat depression symptoms in
people who are never smokers so in those studies nicotine delivery is
antidepressant so how can that be why would two forms of nicotine delivery
have very opposite effects on mood well as molecular biologists as people who
work in model systems we can actually answer that question there are
hypotheses that we can make so first of all we know about the channel properties
of these receptors we know that high levels of nicotine delivered rapidly
should activate these receptors should open them we also know that low levels
of nicotine that are delivered constantly over time are much more
likely to desensitize these receptors and actually keep them closed
when you give a challenge with nicotine or when you signal with Astle choline so
based on those human studies we can make a hypothesis our hypothesis that is that
maybe it’s not opening nicotinic receptors that is antidepressant maybe
it’s actually blocking them and if that’s the case then smokers who
transiently open and then desensitize their receptors over the period of the
day may in fact be increasing their depression symptoms during the time that
those nicotinic receptors are open and then solving their problem that they’ve
just created by smoking by desensitizing their receptors during the course of the
day and as as basic scientists we have a way of testing this hypothesis because
there are non nicotine ways of blocking these receptors so I told you about
mekka melamine mekka melamine is a channel blocker that sits in the pore of
nicotinic receptors and blocks their activity and we can ask if we treat with
MECA melamine does that actually act like a classical antidepressant and the
answer is yes it does so we can evaluate the antidepressant like properties of
molecules I’ll never tell you whether we can say that
is depressed or not depressed but we can ask whether it changes its behavior
based on drugs that we know should actually cause a alleviate depression
symptoms and the way that we evaluate them very they’re generally responses to
mild stressors in the environment and in this case I’m showing you what happens
when we give the animal the we put the animal in a environment that it can
escape from a small beaker of water and if you give that animal Prozac or other
drugs that are used as antidepressants they will actually try to escape for
longer and if you evaluate mecha melamine you can see that it works the
same way so here is the amount of time that the animal stays in mobile and what
you can see is that if you give a nicotine blocker the animal is a mobile
for much less time and what you can see is that if we knock out these high
affinity nicotine receptors remember those that are responsible for smoking
that that effect is completely gone mecha melamine no longer changes the
likelihood that the animal will be immobile but what you can also see is
that even at baseline with no chain treatment with methylamine these animals
without the nicotine receptors are more likely to keep trying to find a way out
of that inescapable environment than animals that have normal nicotine
receptors they look like their nicotine receptors have already been blocked so
that gives us a hint that perhaps blocking these nicotine receptors might
be antidepressant in humans so as a result of these studies and others we
have colleagues Tony George and colleagues who tried this in a human
subject population and what Tony George did was that he recruited patients who
were already on Prozac or some other serotonin regulating antidepressant and
they had not responded and this is actually quite sad and that is that
about somewhere between 30 and 50% of individuals with depression don’t
respond to our current medications so he recruited a subset of patients he kept
them on Prozac or whatever other and pressin they were on and he added Mecca
melamine and he was able to do that because it had been used for blood
pressure treatment in the past and what he saw was that in this small group he
could actually improve their depression symptoms and if he gave a placebo to
these individuals who had been on Prozac it had no effect so at least in some
small studies Mecca melamine can also be used as an addressing in humans and we
wouldn’t have tried this if we didn’t know that there was a rationale from
these basic science studies and if we didn’t have some data that it might work
in a very very large trial recently Mecca melamine has not been shown to be
the greatest add-on drug so it may not be that this molecule is the one that is
pursued for for depression but it does give an idea for a future drug
development so the question is same one I’ve been asking where in the brain is
this happening and which receptors so we went back to see Foss again we looked at
a marker but this time of a decrease in neuronal activity because we’re looking
at the ability of nicotine blockers to cause this behavior and we used both
cytosine which is also antidepressant like in these models and Mecca melamine
and we saw that the place where we saw the best convergence in this case was
the basolateral amygdala and the basal lateral amygdala is important because
it’s absolutely critical for stress responses it’s an area that’s been shown
to be hyperactive in subsets of depressed humans and so we asked if we
block nicotine receptors only in the amygdala by giving a local infusion or
if we knock down receptors only in the amygdala can we recapitulate these
antidepressant like effects and the answer is yes we can block nicotine
receptors with Mecca melamine only in the amygdala and we get increased let’s
our decreased immobility and if we knock down the excuse me the beta 2 subunit
locally it has effects across models of depression like sorry of antidepressant
efficacy so that leads to the idea that there’s
something about the signalling of the normal neurotransmitter acetylcholine in
this brain area that’s really important for regulating mood so what we can ask
is can depression actually result from too much acetylcholine and what we did
was again to collaborate with our colleagues who are clinical
investigators and to ask if we can see if there were any changes in estill
choline signaling in the brains of patients with depression so if if
depression is in is associated with an increase there’s a ghost trying to come
into that door it’s been coming in and out if depression is associated with
increased festal choline what would we see in an imaging study so we can use a
nicotine receptor binding tracer and we can say if there’s a little bit of
acetone choline signaling in the brain and we give this tracer to two humans
that there should still be sites for it to bind and other receptors and we
should be able to image them however if Astle choline signaling is elevated in
the brain it should occupy more of those receptor sites and now when we put in
the tracer there should be fewer sites for it to bind to and so that’s exactly
what we saw in depressed patients who were actively depressed at the time that
they were scanned we saw less binding of this tracer throughout the brain than we
did in the brains of people who had never been depressed we have to do this
in non-smokers because nicotine occupies exactly the same sites as Astle choline
so we can’t actually we can’t image people who are actively smoking but this
was suggestive that perhaps throughout the brain there was more Astle choline
occupying those receptors now those of you who do these kinds of experiments
could also say well hey this could be a competition with a stole choline but it
could also just be that they have fewer nicotine receptors and that would be
counter to argument that’s exactly what we thought so what we then did was we
tried to we scoured the country for people who had brain banks of people who
had of individuals who had died who were actively depressed mostly from suicide
and individuals who had died at the same age but from accidents or other
other events that did not have to do with depression and we asked did they
have any difference in the number of nicotinic receptors we used exactly the
same tracer that was used in the human imaging studies and in those post-mortem
brain studies there was absolutely no change in the number of nicotine
receptors when we washed out all of the Astle : and you can see we used also
another psychic another psychiatric illness schizophrenia as it as a third
control and what you can see here is that it suggests that in the imaging
studies that the decrease in binding to the tracer was likely as a result of the
competition with the tracer and I’m going to close even though I have a few
more slides to show you with just a hint to all of you that really most of our
ideas have in some way or another come up before and so in the 1970s a
psychiatrist Janowski was able to increase acetylcholine levels in the
brain by challenging with a blocker of the breakdown enzyme for Astle choline
physostigmine and he showed that when he challenged people with fisa stigman if
they had a history of depression or even if they did not he could increase
depression like symptoms in humans and he suggested that that meant that if you
have too much austell choline you’re more likely to be depressed and based on
the medications that were used at that time for depression that having too much
norepinephrine might make you manic and so that this balance between
norepinephrine and Astle choline would be essential for depression
unfortunately for dr. Janowski he proposed this right about the time of
the prozac revolution and Prozac as those of you who are pharmacologists to
know actually works on the serotonin system and because it became the
dominant paradigm serotonin was really pursued as the neurotransmitter in
charge of depression and dr. Jonas keys theory sort of got shunted aside and I
would like to argue that in fact we are not a bag of single neurotransmitter
that the balance of neurotransmitters across many systems are likely to be
important for the kinds of complex behaviors that are really important for
psychiatric illness and so in fact it’s quite likely especially based on these
human imaging studies that we be looking again at other systems
particularly the acetylcholine system and asking how does this regulation of
these other systems actually contribute to to depression and I’ll close with one
more slide of data to show you that actually we can block a stole choline
breakdown and particularly target a still calling breakdown to a particular
brain area the hippocampus and we can see exactly the same thing that dr.
janeski saw when he challenged individuals with physostigmine but we
can now identify the particular brain areas and the particular receptor
subtypes that target that response and in the interest of time I’m now going to
skip to my conclusions which are that now we’ve got another area where we know
the symptoms of a particular psychiatric illness can can decrease the likelihood
that folks can quit smoking we have a same the same receptor that we’ve
actually talked about with respect to nicotine reward and reinforcement but in
this case instead of active activating that receptor in order to get
antidepressant responses now you want to block that receptor and particularly we
want to block it in the amygdala as I showed you earlier and if we do that
then we get potentially another insight into why some people smoke even though
they we know that they’re going to have a bad outcome
so I started by introducing the problem which was the very large molecular group
of targets for nicotine in the brain and the broad expression of those nicotine
receptors as well as the large number of reasons and behavioral consequences of
smoking and then I have good news the good news is that a combination of
molecular genetics and of pharmacology both in animal models and in humans as
actually as a field made very good progress in dissecting the nicotinic
receptor subtypes and the brain areas that are responsible for these specific
nicotine dependent behaviors that contribute to smoking so targeting this
these receptors is already a success story for rational drug design
particularly for smoking cessation and it has resulted in an effective
treatment for smoking but we have a big challenge
we know that existing pharmacological agents seized in humans for the most
part don’t target specific nicotinic receptor subtypes and in fact agents
that are somewhat selective like cytosine and mecha melamine have
different selectivity for human receptor subtypes so we really have to do a
better job of coming up with new safe molecules to target these different
subtypes and our hope is that medications that are targeted to these
highly specific nicotinic receptor subtypes could be useful not only in
smokers to help them quit or to motivate themselves to quit even despite the fact
that they’re afraid that they might be in wait or they’re afraid that they
won’t be able to control their depression symptoms but maybe they’ll
actually be useful for individuals who don’t smoke at all to treat depression
or to help those who struggle with obesity to control their appetite and I
just like to close by thanking our funders particularly the National
Institute on Drug Abuse and the National Institute on mental health NAR said and
tobacco Center funding as well as tobacco center as in tobacco treatment
center not from the tobacco industry no tobacco industry here as well as our
many collaborators in other laboratories and our current and former lab members
who really contributed not only to the experiments that I spoke about here but
all of the planning of those experiments and the concepts and the design of those
experiments thank you very much for your attention you

2 Replies to ““From Molecules to Behavior: Role of Nicotinic Acetylcholine Receptors…”-Marina Picciotto, PhD”

Add a Comment

Your email address will not be published. Required fields are marked *