cc  >> Welcome everyone to Wednesday  Nite at the Lab here at the UW  Madison Biotechnology Center.  And tonight we have Meyer  Jackson who will be speaking  about biophysics and  neuroscience today.  He is Kenneth Cole Professor in  the Department of Physiology, as  well as the director of the UW  Biophysics Ph. D. program.  He got his BA from Brandeis and  his Ph. D. from Yale.  And he's also had positions at  the NIH and UCLA before this.  And he's also the author of a  textbook called Molecular and  Cellular Biophysics.  So please join me in welcoming  him to Wednesday Nite at the  Lab here today.  (applause)  >> well, can you hear me?  Thank you, thank you very much  for inviting me to talk to you  about the research that's going  on in my lab.  I have fun doing it and I have  fun talking about it.  So I decided for this kind of  lecture that it would be  interesting to show you how two  very different fields of  research can be combined in an  effective way to solve some  classes of problems. 

 So here you have biophysics and  neuroscience.  Two fields, there's a important  journal, the "Biophysical  Journal," an important journal  called the "Journal of  Neuroscience."  There are many other journals in  these fields.  And there's actually a very  important intersection between  the two where you can apply  biophysical techniques and  concepts to study the nervous  system, how the nervous system  works.  And from my perspective the  nervous system, how the brain  works, is one of the great  scientific challenges that's  still remaining, still largely  unsolved.  And physics, I've always been  fascinated by physics, and so I  approach a problem from a  physicists' point of view.  And I'll show you how physical  measurements, physical  experiments, physical techniques  can be used to solve basic  problems in the nervous system,  how the nervous system works.  So we'll look at this  intersection with a few selected  problems, selected approaches  that my lab has undertaken to  illuminate how understanding  physics can also help understand  how the brain works.  So the brain, first of all,  obviously one physical approach  that should work is some kind of  electrical measurement.  Because the brain runs on  electricity, the nervous system  is basically a complicated  electrical signaling machine.  I don't like a say it's a  computer because it's much more  than a computer, but there's  still a lot of electronics  that's involved. 

 And a lot of the scientists, the  biophysicists, who study the  nervous system know electronics  very well.  They design and build fancy,  sophisticated amplifiers and  analyzing circuits and things  like that to study the nervous  system.  So electronics is an obvious  place where knowing physics can  help you study the nervous  system.  Now the nervous system is built  up on circuits.  There is fundamental elements,  neurons, nerve cells that are  electrically active and that  form these complicated networks  that generate a very rich  variety of activity.  And this is an example of a  model circuit that my laboratory  studied many years ago in hopes  of understanding how epilepsy is  generated.  But we can also focus on the  single cells and the junctions  between cells.  And really one of the most  fundamental objects within the  nervous system that we studied  at a very basic level to try to  understand those circuits is the  synapse.  So here is your generic synapse,  a chemical synapse.  One nerve cell signals another  nerve cell by releasing a  chemical. 

 It releases a chemical from  this cell here.  The upper cell is the  presynaptic cell.  The chemical is contained within  these little bags called  vesicles.  They're released into this  region called the synaptic cleft  and the molecules that are in  these vesicles, the  neurotransmitter molecules, then  bind, attach, they recognize a  specific site on the cell  surface of the postsynaptic  cell, the receiving cell, and  that initiates an electrical  event in the postsynaptic cell  that sends the message on its  way down the postsynaptic cell  to other elements within the  circuit.  And so the synapse is one of the  most elementary objects within  the nervous system.  Studying the synapse at a very  basic level then tells us things  that we can go back and study  networks.  So first I'll tell you about  some studies of how these  synapses work, and then I'll end  the talk by talking about how we  study networks of synapses,  clusters and computers formed by  neurons to always thinking about  how the synapse works and using  the ideas of synapses to  understand how networks work.  So first I'll talk about the  postsynaptic cell and some  studies that can be done on  these synaptic receptors, the  proteins that rest in the  membrane of the postsynaptic  cell and which respond to the  release of neurotransmitter by  opening a channel, as is  illustrated here.  So this is a cartoon sketch of  what we know these postsynaptic  receptors can do.  They are large protein molecules  and they switch, they flip  between a closed state where  nothing can get through, ions  cannot get through, they are  electrically high resistance,  they don't do anything. 

 And then they open, sorry, they  open a molecule, the  neurotransmitters binds to these  sites that are shown empty here  and full here.  And when that happens the whole  protein undergoes a global  change in structure, it  rearranges and forms an open  channel.  And these recordings down below  here, this is the current  through a small piece of  membrane, a small patch of  membrane, and when that channel  opens and that piece of membrane  has these channels in them, and  when the channel opens the  current slips down, as ions can  get through, and when the  channel closes again, the ions  stop flowing through.  And this flipping back and  forth, this opening and closing  occurs as long as there is  neurotransmitter molecule around  to bind and unbind the receptor.  And these measurements in fact,  they're quite old now, but this  was an important observation,  the fact that you could see  these single molecule  transitions, these single  proteins undergoing this  transition from a closed state  to an open state.  That really established the  existence of these ion channels  in the membrane.  It was a very important  breakthrough made about 30 years  ago that really established the  ion channel as the basic  signaling molecule, the  electrical molecule of nerve  cell membranes.  And I should just point out that  these steps that are shown in  this trace here are actually the  activity of a single molecule.  As good as our instrumentation  is it's still very hard to watch  one molecule do something, and  this is a measurement of one  molecule.  So if you look at the magnitudes  of these signals, this  calibration bar here, one  pico-amp here, one pA, that's  one pico-amp here, that is about  a trillionth of a current that  flows through a typical light  bulb. 

 It's a very, very small signal,  and you have to build a good  amplifier.  You have to know your  electronics well, and you have  to know a lot about the  underlying physics of how to  measure things to see these  kinds of things.  But nevertheless, we can see  them and we can see them very  well and we've used methods like  this to elucidate an enormous  amount of basic mechanistic  information about how these ion  channels open and close, how  they do their job, how a  receptor for a neurotransmitter  does its job of sensing the  release of neurotransmitter and  transducing that release into an  electrical signal in the  postsynaptic cell.  Now this would have been a  great, this single channel  recording approach would have  been a great thing to give a  general talk about 20-30 years  ago, but as remarkable as the  methods are, to me it's old-hat;  I've been there, done that.  And I want to talk about a newer  approach.  So I'm going to look at these  proteins, these proteins that  form the channels, and I'm going  to see if we can work out  something fundamental about this  movement takes place, how these  channels make this transition  from a closed state to an open  state when this neurotransmitter  molecule binds to it.  And here's an example of what we  know about the molecular  structure.  These proteins have all been  cloned.  We know their DNA sequences.  We know what they're made of. 

 And in some cases we have some  real detailed atom by atom,  amino acid by amino acid  structure how they're put  together.  So I'll take one example of one  of those proteins and that's the  protein that is the receptor for  GABA.  GABA stands for gamma  aminobutyric acid.  There's its chemical structure  up there.  It's a pretty simple molecule.  If a student who's just taken  and organic chemistry class  hears gamma aminobutyric acid,  they know exactly what it looks  like.  It looks like that.  It's got an amino group on one  end and a carboxyl group on the  other.  It's a small molecule.  And it is the major inhibitor  neurotransmitter in the brain. 

 Your brain is full of GABA.  Nerve cells are constantly  releasing GABA in your brain.  The GABA is then binding to GABA  receptors and actually quieting  down the nerve cells that it  binds to.  So GABA is an inhibitory  compound that reduces the  electrical activity in any cells  that it comes in contact with.  Now the receptor for GABA is the  GABA A receptor.  There actually is a GABA B and a  GABA C receptor for all of these  proteins.  They come in multiple forms.  Nature has found many ways to  get the job done.  Making proteins that do respond  to GABA, bind to GABA and do  something a little different.   But the major receptor for  inhibition in the brain is this  GABA A receptor. 

 It's a protein and here's a  structure of a very closely  related protein, and the GABA is  known to bind to these pockets  between the sub-units of this  protein.  So the protein is actually put  together from five different  peptide chains, they form a  pentamer like this, and there's  this binding site in the pockets  between them, and somehow GABA  binding in those pockets makes  the channel open and close.  Now the way we've been studying  it more recently is by a  technique called fluorescent  labeling.  Fluorescence is also quite an  old technique and fluorescent  labeling is quite old, but  applying it to channels has been  very exciting.  There were a lot of technical  reasons why it was hard to  study, to use fluorescent  labeling in these ion channels.  And in the last 10 years or so  those problems have been solved  and it's now a very powerful  technique for studying how these  ion channels undergo these  rearrangements as the channels  open and close.  So the idea is that you attach  your label somewhere, say here.  And it's in the specific site.  Now if it's fluorescent that  means that you shine a specific  color light on it, say blue  light or green light, and that  label absorbs the light and  because it's fluorescent the  light comes out a different  color.  If it absorbs blue light than it  might emit green or yellow  light. 

 If it absorbs green light, it  might emit yellow or red light.  Now the amount of light that  comes out from this label that  we've attached to a specific  site depends very much  on what is around the label.  If there's a lot of water around  the label you actually don't get  much light coming out.  The light goes in and it never  comes out again.  On the other hand if it's in an  oily environment, what we call a  hydrophobic environment or a  nonpolar environment, a region  where there's no water and where  the electrical twitter around  there is much weaker, then you  get a lot of light out.  In this cartoon here, in this  drawing here, a lot of light  comes out of the label because  it's buried inside the protein,  and when the channel opens much  less light comes out.  And we've used that method to  study how the GABA receptor  opens and closes.  What happens when you add GABA  and make the channel open.  Now the way we've done that is  by introducing this molecule.  This is a molecule that  fluoresces very well. 

 It's a very bright red compound  that histologists have been  using for many years.  And it has the important  property that its fluorescence,  the amount of light that you get  out of it, changes depending on  its environment.  There are some details up there  about it.  And this part that's circled  here, that is a part that's not  fluorescent but that is a part  that's targeted for specific  parts of proteins.  So by combining the protein  targeting chemical function with  the fluorescent chemical  function we can create a label  that will bind to the site that  we create in the protein and  tell us what that site is doing.  So another key step in terms of  understanding this experiment is  we know the DNA, we have DNA  that encodes to the GABA  receptor, and there's the  sequence up there, for whatever  it's worth.  I always tell students they  don't have to memorize that,  don't be scared, but it's  valuable information and we can  go through it very carefully and  find sites that we have a good  reason to believe there will be  some kind of change in the  structure, that something will  happen at that sight when the  channel opens.  So we make a mutation in the  wild type protein.  We introduce a different amino  acid that we know we can target,  and then we inject it into a  frog egg.  This is a frog egg here. 

 And these cells were very  popular for expressing proteins.  You can inject the RNA or the  DNA into one of these cells and  a few days later the cell will  make the protein that you've  told it to make.  And so you can have a frog egg  producing the same kind of  chemical responses that a nerve  cell produces.  You can almost turn a frog egg  into a nerve cell from the point  of view of making an experiment,  studying how the protein works.  And this has been a very  valuable approach for  genetically engineering proteins  and figuring out how changes in  those proteins will change the  way it behaves.  So we've taken that approach.  We've made our GABA A receptor  mutant so that we could target  it.  We've injected it into frog  eggs.  And then a few days later we  would record the current and we  would record, not only would we  record the current but we would  record the fluorescence  simultaneously.  So we have this little frog egg  that's big enough to see with  the naked eye. 

 It's about a little less than a  millimeter in diameter but it's  one cell.  It's sitting on the stage of a  microscope, and there's a strong  light being shined at that frog  egg and the light at a different  wave length, a different color,  is being measured all the time.  And at the same time with these  electrodes here that are drawn  here, this electrode and this  electrode, we can control the  voltage and measure the current.  And so if we apply GABA through  this solution exchanger, we can  see both the channels open and  close by the electrical  measurements, and we can see the  change in the fluorescence of  that one site of that label and  find out what's going on at any  part of the protein we choose.  So we've made mutations in some  selected sites near the binding  site between these red and  green, yellow sites were sites  we selected.  We had reasons to believe that  something interesting would  happen there.  And we attached our label at  those sites, and then we made  some measurements.  Now in the upper panel here, the  eye, that's the current.  That tells us the channel is  indeed opening.  Yes?  >> So are you injecting DNA that  codes for the receptor?  >> We're injecting RNA that  encodes for the receptor. 

 >> You're not injecting the  receptor itself?  >> No, that is not possible.  We wish we could do that, but  this is the only way we know at  present to make a genetically  engineered protein in a membrane  that we can study its electrical  behavior.  So here's what it looks like.  The current tells us the channel  is indeed opening.  And the fluorescence tells us  that the site that we labeled is  actually going, in this case,  from a watery environment to an  oily environment.  So this site here is becoming  more exposed to water, I'm sorry  more exposed to oil when the  channel opens.  And this site here, very nearby,  we also get channel opening when  we label that site but now the  fluorescence changes in the  opposite direction.  So there's a very interesting  rotation that occurs around  these two sites allowing one of  the sites to become more exposed  to water and the other to become  more exposed to oil.  Now this site here is on what we  call the alpha sub-unit which is  very near the binding site, very  near the site to which GABA  binds.  But we've also labeled a site on  the beta sub-unit which is very  far. 

 And interestingly that site also  gives a similar fluorescence  change.  So it's reasonable that close to  the binding site the protein  undergo some kind of structural  rearrangement.  It's not surprising.  It seems reasonable or something  we would have expected.  But it is surprising that quite  far away from the binding site  the protein is also undergoing a  roughly qualitatively similar  change in structure.  That tells us that this protein  is really undergoing a global  change.  It's not just a local sort of  deformation around the binding  site.  The whole protein makes a  movement and that there's a  rough symmetry to that movement.  That homologous or parallel site  within the same protein but far  from the binding site make a  movement that's similar to a  site to places that are close to  the binding site.  So going to some basic ideas  about how these big proteins  respond, how a little molecule  makes a big protein change,  there are these two very old  theories that were introduced 40  or more years ago. 

 The Monod-Wyman-Changeux theory  is a very famous theory and the  Koshland-Nemethy-Filmer theory  is, they're both very famous  theories and introduced by very  famous people.  And they differ in terms of the  idea that the transition is  symmetrical.  Koshland, Nemethy and Filmer  said that the change in  structure occurs only around the  binding site, only around the  place where the molecule binds,  the neurotransmitter molecule  binds, and Monod, Wyman and  Changeux said it was symmetrical  and you'll get changes in  structure even at very distant  places.  And so the result of this  fluorescent labeling experiment  suggested that the picture is  closer to the  Monod-Wyman-Changeux model and  that when neurotransmitter  receptors respond to  neurotransmitter they undergo  fairly large global changes in  order to allow their ion  channels to open.  So that's an example of how  we've used a very physical  approach to ask a very basic  question about one side of the  synapse, the postsynaptic side  of the synapse.  Now I'm going to go back to the  synapse and ask about another  part of the synapse.  So we're going to look at the  ion channels that are in the  nerve terminal, in the releasing  ends, because this whole  process, the release process is  set in motion by the work of ion  channels.  In particular calcium gets in  through calcium channels here  when they open, and the calcium  then goes in and makes the  vesicles merge with the plasma  membrane.  So now I'll ask something about  how the ion channels work in  nerve terminals.  Now that is also very difficult  to study. 

 It's actually more difficult to  study than the postsynaptic  side.  In fact I like to tease people  who work on postsynaptic  receptors say it's a solve  problem, the real challenge is  understanding how nerve  terminals work.  And the reason it's been such a  challenge is illustrated in this  picture.  This is a picture of a nerve  cell grown in culture, an  incubator, taken out of a mouse  once upon a time.  And it was stained, that is  treated in a way that allows us  to visualize, to see an enzyme  that makes GABA.  So the GABA making enzyme are  these brown spots, these brown  pepper-like spots that cover the  surface of this nerve cell.  Those are nerve terminals.  Now the point I want to make  with this picture is those nerve  terminals are tiny, and the  electrical measurements that we  use require a minimum size.  We can look at something that is  a hundredth of a millimeter,  which is on the order of this  nerve cell here, the cell body  here.  This is probably about  two-hundredths of a millimeter. 

 But these little spots are less  than a thousandth of a  millimeter.  So an electrical measurement  from those little nerve  terminals that are covering this  postsynaptic cell, those  electrical measurements are  impossible.  So what us neuroscientists have  done in order to find ways to  study nerve terminals is we've  looked through the organisms,  looked in different brain  regions, looked in different  animals and found some rare  examples of nerve terminals that  are very large, and we found  that there's a kind of nerve  terminal called the mossy fiber  nerve terminal that's in  hippocampus, and that is  involved in learning and memory.  We found a nerve terminal in the  posterior pituitary and that's  an endocrine organ.  And I'll tell you what that  function is soon, but I think  it's more important to learning  and memory.  You'll soon see why and you'll  be able to decide for yourself  whether it's more important than  learning and memory.  Finally, I'll jump down and tell  you that there's a nerve  terminal that controls hearing  in the auditory brain stem  called the Calyx of Held.  It's involved in the timing of  signals from the different ears.  The way you localize sound is by  determining the time that the  sound arrives at your two ears.  If there's a little bit of delay  then you look in the direction  where you heard the sound first. 

 You can't hear that timing  difference but there are parts  of your brain that can, and they  have large nerve terminals that  help them do that.  Finally, there's also a cell  line, something called PC12  cells.  These are really engineered  cells created in a laboratory,  and they're good for studying  the release process but they  have no function.  Sometimes I take some heat for  that.  Why am I working on cells that  have no function.  Sometimes a scientist has to  make a compromise and they can  sometimes ask a basic question  in a system that very remote  from the biology but still  they'll learn something about  how those molecules work in a  model system.  And so we've used this model  system, these PC12 cells, to  elucidate basic aspects of how  nerve terminals work, and we're  always thinking about the real  live synapses between neurons  and we sometimes can come up  with questions, once we've  learned something in PC12 cells  we can then go back to the  synapse and ask a similar  question but from a better  informed point of view.  So first I'll tell you about the  posterior pituitary.  It's this endocrine structure  just below the brain.  It's actually formed by nerve  terminals. 

 So the nerve cells in the  endocrine part of the brain  called the hypothalamus, the  hypothalamus sends these axons,  these nerves down to the  pituitary gland and those axons  form these very large nerve  terminals.  And here's some pictures of  nerve terminals in posterior  pituitary slices.  So this is a giant nerve  terminal.  It's about two or three  hundredths of a millimeter.  So we can make good recordings  from that.  And we've been doing that.  It sort of looks like this.  We make a slice of the  pituitary, and we can put an  electrode right up against the  surface of one of these nerve  terminals and study the release  process.  So now one of the nerve  terminals that released by  oxytocin is, I like to call it  my favorite hormone because it  is a hormone that's involved in  sexual desire, sexual arousal.  It's been called the cuddle  chemical or the hormone of love. 

 And it also is a motherhood  hormone.  It's responsible for maternal  behavior.  For many years endocrinologists  only knew that oxytocin was  involved in lactation, the  release of milk during feeding  when an animal is suckling.  And it's also involved in the  delivery of a fetus.  When a mammal is born oxytocin  is the hormone that's released  and it makes the uterus  contract.  But then about 20 years ago it  was discovered that oxytocin  also has these important  behavioral functions.  So it is interesting.  But I started working on the  posterior pituitary simply  because it was a nerve terminal,  and it was actually before the  involvement in the sexual  functions of oxytocin were  learned.  So I considered myself very  fortunate that after deciding to  work on these nerve terminals  such an important function was  found.  Now I'll tell you about how the  release of these hormones is  regulated, and then I'll tell  you some important insight that  was gained that specifically  relates to the role of oxytocin. 

 So we can record from these  nerve terminals.  And this is an example of a  simultaneous recording of  electrical activity.  These spikes, which are called  action potentials, are the basic  units of information in the  nervous system.  And here down below we've  measured the calcium that gets  into a nerve terminal.  We measure the calcium by  filling the nerve terminal with  a fluorescent molecule that  becomes more fluorescent when  calcium binds to it.  And sure enough when the  electrical activity starts here,  we stimulated it electrically,  the calcium goes up and calcium  going up in a nerve terminal is  very important because that's  the first step in triggering the  release of either the oxytocin  or the neurotransmitter in the  synapse.  So that's all making sense.  And we found that a molecule, a  signaling molecule called cyclic  GMP, I won't show you a  structure but suffice it to say  it's a small molecule that's  produced inside cells and that  changes things.  It's a small molecule that binds  to proteins and changes what  they do.  And we found that when there was  cyclic GMP inside a nerve  terminal there was a lot more  activity. 

 And so this was a good thing to  study.  We did a lot of good experiments  where we applied blockers and  did manipulations and worked out  a basic signaling cascade.  Now in general when a student  studies cell biology or  biochemistry they learn about  these control cascades where a  second messenger is produced,  one molecule binds to a protein,  that protein makes another  molecule and so on and so forth.  And finally you get an output  which is in this case the change  in one of those ion channels.  So we worked out, we did all the  basic stuff here and we worked  out that calcium goes in during  those action potentials and that  generates something called  nitric oxide.  Nobel Prize of 1999 was nitric  oxide.  Nitric oxide then binds to an  enzyme that makes cyclic GMP.  Cyclic GMP binds to a molecule,  another protein that sticks  phosphates onto ion channels,  and when it's all done those  action potentials get more  frequent, you get more action  potentials.  So that's all how this basic  signaling process works.  And an awful lot of what goes on  in understanding cell function  involves elucidating these basic  signaling cascades. 

 There are a whole bunch of  different signaling cascades  that are found in nature, and  we're all interested in how they  work.  Usually when you get to the  bottom of one of these signaling  cascades you've learned  something pretty important about  how the cell works.  But all signaling cascades have  a break.  They have something that stops  it, that turns it off.  And in this case the break is a  molecule called  Phosphodiesterase type 5.  It's an enzyme that chews up the  cyclic GMP.  So nitric oxide binding to --  makes cyclic GMP.  Cyclic GMP then does something,  and when it's time to shut the  system down, cyclic GMP is  degraded by this enzyme.  Well it turns out as we were  working on this it dawned on us  that there is a very important  drug that blocks  Phosphodiesterase type 5.  Does anyone know what it is?  Well, I'll tell you what it is. 

 It's Viagra.  Viagra is actually a simple  inhibitor of an enzyme, and the  chemical name is Sildenafil.  So Sildenafil is the molecule  that's making drug companies $5  or $10 billion a year.  And it blocks Phosphodiesterase  type 5.  So we naturally wondered  oxytocin, that has a control in  sexual function.  And the cyclic GMP cascade,  that's the target for Viagra.  What does Viagra do in the  posterior pituitary nerve  terminals?  Well, it does something, it  enhances the electrical  activities.  So we found that when we measure  these action potentials under  very controlled conditions with  no drugs added we didn't get  much enhancement of these action  potentials.  But if we put a little bit of  either Viagra or a Viagra-like  drug into the nerve terminal, we  got a lot more action  potentials, we got a lot more  electrical activity.  So it does work the way we  expected. 

 It enhances excitability.  And when we actually measured  the release of oxytocin with a  chemical assay we found that the  presence of Sildenafil enhanced  the activity evoked by  electrical stimulation.  So there's always a little bit  of release going on in the  background down here but when we  stimulate we get a lot more.  And this is what we get when we  stimulate in the absence of  drug, and this is what we get in  the presence of Sildenafil.  So these results show that  Sildenafil has an action in the  release of this key hormone.  So we think this might be the  basis for why Sildenafil has  been used so much as a  recreational drug.  It's obviously being used by  thousands of perfectly healthy  people without erectile  dysfunction, and people have  been reluctant to acknowledge  that there could be this  recreational or psychological  component, and I think at least  at the basic level we now have a  mechanism that we can put  forward to explain the  widespread recreational use of  Viagra and other  Phosphodiesterase type 5  inhibitors.  So on to other questions about  the synapse.  That was an example of how the  ion channels out here can be  controlled and do something to  control the release of a  neuropeptide hormone and also  presumably neurotransmitters as  well.  And now I'll turn to the actual  fusion process, these vesicles  that contain neurotransmitter  calcium coming in through these  channels does something to this  process here and makes these  vesicles fuse with the cell  membrane and the insides, the  contents of those vesicles then  come rushing out. 

 So how does that happen?  We're working on that by  studying the molecules.  Now 20-30 years ago that fusion  picture, that picture of  vesicles fusing with the cell  membrane, was worked out in an  electron microscope.  These scientists -- and -- did a  very important experiment where  they took pictures of nerve  cells and they then stimulated  the nerve cells and took  pictures again.  And if they took the pictures  very quickly after stimulating  they saw that these vesicles out  here did indeed fuse with the  cell membrane.  And since then scientists have  been searching for the molecules  that are in the vesicles and now  we have a complete molecular  description of those vesicles.  And here's an example of what we  think a vesicle looks like.  All of the proteins, those  vesicles are filled with  proteins, many different kinds  and we know their names, we know  their DNA sequences and, in some  cases, we know their  three-dimensional structures.  But what we're still trying to  do, just knowing the molecules  doesn't solve the problem, what  we're still trying to do is  figure out how these molecules  that are in the vesicles and  also in the plasma membrane, how  they make these vesicles here  fuse with the cell membrane.  And that we're starting to work  out too.  So let me tell you a little bit  about that. 

 First there's a class of  proteins that are very important  in holding cell membranes  together.  And they've been names SNARE  proteins.  SNARE is a catchy name for these  proteins, it's an acronym based  on some completely technical  starting point soluble NSF blah,  blah, blah.  I won't finish.  It's very easy to remember the  name SNARE.  And there are three particular  SNAREs that are present in  synapses: Syntaxin, SNAP-25 and  Synaptobrevin.  Those are proteins that are in  the vesicle membrane and in the  cell membrane and they form this  very tight bundle to pull the  two membranes together.  And we know that those proteins  form this complex, you can put  them into a test tube and  they'll bind to one another and  form this complex crystal  structures are known, a lot of  is known about the SNARE complex  but we still don't know how it  makes this fusion occur, how it  makes the two membranes combine  into one.  And so I'll tell you a little  bit about how we think that  might be happening.  We think that the first step is  what can be called a fusion  pore. 

 A channel or a hole that doesn't  just go through one membrane, it  goes through both membranes.  It goes through the vesicle  membrane here and then through  the cell membrane here.  And we think that the SNAREs can  bundle together and form these  channels by forming these rings  where each channel in each  membrane we have a ring of maybe  six or seven or eight of these  membrane spanning segments  coming together to form a  channel.  And the reason we think that  that's happening is we've done  experiments with a method called  Amperometry where we push a  carbon fiber close to a cell,  and the carbon fiber doesn't  measure anything when it's far  from a cell, but when it's near  a cell and that cell releases an  oxidizible molecule like  Norepinephrine, the  Norepinephrine gets oxidized,  the two electrons that are  produced by that oxidation  reaction are gobbled up by the  carbon fiber and we measure  something.  And each one of these spikes in  this long record here is the  fusion of one vesicle that's  filled with Norepinephrine.  So one vesicle contains about  50,000 Norepinephrine molecules.  That's enough to produce a  fairly large spike with this  recording method.  And if we look at those spikes  closer up, we see that they're  not just spikes there's  something that happens  beforehand.  And we think this foot at the  start of the spike is the  opening of one of those fusion  pores that I drew in the last  slide.  So here's what we think is going  on. 

 During the Amperometry record a  closed fusion pore, the SNAREs  hold the vesicle close to the  cell membrane but the pore's  closed so there's no current  coming through being detected by  that carbon fiber.  Then the fusion pore opens.  And then we see a little bit of  Norepinephrine dribbling  through, just trickling through  the hole.  And then the pore dilates, it  expands.  And then the rest of the  Norepinephrine comes gushing  out.  So why do we think that story  holds up?  Well, if we imagine that we  could change those  membrane-spanning segments,  those segments that go through  the membrane and form the walls  of the pore, if we stuck  something bulky into those  segments we might be able to  make things go out the hole a  little bit slower.  So we tested the idea by making  mutations in the  membrane-spanning section of  Syntaxin, Syntaxin was one of  those SNAREs.  And we measured the feet for  many of those spikes, for each  of those mutations.  And we found that there were  three sites in the  membrane-spanning section of  that protein that reduce the  amplitude of the foot current.  So the foot current is normally  about two and a half pico-amps,  that's not that big but we can  still measure it. 

 And sometimes it goes down to  almost two.  And those measurements are  statistically significant,  they're small but we know  they're real.  And when we look at where those  sites are, where I269 is, where  G276 is, I283 is, when we put  those into a model of the  membrane-spanning segment of the  protein they're all on one side.  So they're right where they  should be.  They're all in the same face.  And that's what you would expect  if that was the part of the  protein that was the liner of  the pore, that actually formed  the walls of the pore.  So we put together models like  this where the membrane-spanning  segments of Syntaxin and this  one protein form the fusion  pore, and that at least is the  first step in the fusion  process.  We still don't know what the  next step is, but we're making  progress.  One step at a time.  This is the first step. 

 This is probably one of the most  hotly pursued questions in  molecular neuroscience now is  the mechanism of fusion, how do  nerve cells release  neurotransmitter?  And we're actively engaged in  trying to work that out.  So here's our model of the first  step of exocytosis.  And we think the SNARE proteins  come together, form a fusion  pore and somehow this dilation  process starts.  So that's all I want to say  about synapses, and I'm going to  spend the remaining 15 minutes  or so talking about networks  because really synapses are just  the basic building block.  We want to know how networks  work.  We want to know how synaptic  communication in many cells that  are interconnected can generate  complicated forms of activity,  ultimately generate behavior,  generate the response to sensory  inputs.  So we have networks like this.  We imagine the nervous system  has different kinds of neurons  that are connected together.  And electrical activity runs  through these neurons from layer  to layer to layer through the  different courses.  And depending on whether perhaps  this cell and this cell and this  cell are activated, you might  see follow-up cells get  activated. 

 And the output, what that neural  network produces, will depend on  the specific nature of the  input.  In order to work something like  that out we need techniques that  can really study not just one  nerve cell at a time with an  electrode but make a movie,  follow the activity of many  hundreds or even thousands of  nerve cells simultaneously.  So the technique we use for that  is an imaging technique.  We would like to make a movie of  how the nervous system  electrical activity courses  through an isolated piece of  intact neuro network.  And the way we do that is we put  the piece of tissue on to the  stage of a microscope and have a  detecting device that records  what is going on in each region  independently.  And to make a signal that's  voltage sensitive, that tells us  what the voltage is doing, we  actually add a molecule, in this  case a molecule that we call  RH-482, there are so many of  these that people have run out  of names and they give numbers,  but this is a molecule that it's  a very bright strong blue  molecule and when you put a  piece neurotissue into a  solution that contains this  molecule, that molecules enters  all the membranes, it stains the  whole tissue and your light  brown piece of rat brain comes  out dark blue or deep blue.  And then we put that onto the  stage of this microscope and we  measure the light, and when the  electrical activity changes,  when there's a voltage change in  the membrane that contains that  RH-482, the amount of light  coming through changes.  So we actually have an optical  signal, light, that tells us  what the electrical activities  in that particular location is  doing.  So this is a part of the brain  that we like to study, the  hippocampus which is involved in  learning and memory.  And we can make slices of them  that look like this. 

 They have this very  characteristic pattern where  these slightly clearer  translucent regions are cell  bodies and we know that there  are other parts of cell,  dendrites, these are what  dendrites look like in a cell.  They are this part of the slice.  And there are axons that course  through in this direction and  that direction forming synapses  so that the cells of the  hippocampus form synapses with  one another.  And when we stimulate it, when  we put an electrode in and apply  a shock, these little blue  squiggles, each one of these  upstrokes is an electrical  event.  And we've recorded the light  coming through these regions,  464 of these regions at the same  time, and we can actually see  the electrical activity going  through the slice in response to  a particular pattern of  stimulation we might choose.  So we have a method, really what  turns out, as you'll see, as a  very crude method for  essentially making a movie of  the electrical activity in a  piece of brain.  So here's an experiment that we  did with that technique.  We stimulated here and we  converted those squiggles, the  size of those squiggles from the  previous slide into color.  So red means that the squiggle  was large, blue means it was  hardly visible at all, and we  apply a shock at an instant in  time and we see that the signal  spreads a bit in this direction,  a bit in this direction and a  bit in this direction.  And then we did something to the  slice, we applied, instead of  one shock, we applied hundreds  of shocks in a very short time. 

 A lot of electrical activity.  And then we went back to  applying one shock at a time, we  saw that the same shock produced  more activity, bigger responses  that spread further.  And when we subtracted this map  of electrical responses from  this map, we had a difference  map that mapped out the changes  in synaptic function.  So that high frequency  stimulation changed the  synapses.  Now synapses are at the heart of  the nervous system and a change  in how strong a synapse is is  thought to be what happens in  the brain when information is  stored.  So we believe we were looking at  a map of the basic information  storage events that occurred  when this particular slice of  brain was stimulated very  vigorously.  And we've mapped that out very  carefully and it's interesting  that the map is complicated.  We think that might be an  interesting and informative  result, and we're going to study  it more.  And understanding how this  enhancement of synaptic  strength, this of potentiation  of synaptic strength, how it's  distributed, what makes some  parts of the slice potentiate  more and some less, I think that  will help us understand how  information is actually stored  in a part of the brain that's  involved in learning and memory.  Now we've done a few experiments  to try to figure out what it is  that dictates one region will  get potentiated more and one  will get potentiated less. 

 And one thing we've done is look  at our signals very carefully  and see there's a fast part, a  spike which is what we called an  action potential.  It's a process that the nerve  cell uses to send signals to the  next stage in the network.  And there's this slower part,  the synaptic potential which is  just the inputs.  So we see that as we increase  the amount of input, we see  these spikes.  And we can separate by looking  at these signals we can see  these spike parts and measure  that quantitatively and we can  measure the synaptic part and  measure that quantitatively.  And when we map that out, when  we measured the size of these  spikes during those vigorous  high frequency stimulation  patterns that induce the  enhancement, when we map that  out we see that regions of the  slice which produce more spikes  during these high frequency  trains are the regions which  will be more potentiated, more  enhanced by that potentiating  stimulus.  And here's an example of a map  of the spikes during the third  high frequency train.  And it looks very similar to the  map for the potentiation.  So the potentiation follows the  production of spikes but not  spikes just during a simple test  pulse but it does follow the  spikes during these trains of  activity that actually make the  enhancement occur.  We've also studied  epileptic-like activity, what we  call epileptiform activity that  can be produced in slices of the  hippocampus, and we see that in  three different examples we  always see the epileptic  activity start in one region of  the hippocampal slice called the  CA3 region. 

 We then see very little activity  in the CA2 region, but we see a  big surge of activity in the CA1  region.  And it's interesting that the  hippocampus no matter how you  start the epileptic activity,  the discharge, it always  progresses through the  hippocampus with this very  stereotypical pattern.  And more work is ongoing on  that, but voltage imaging in  particular is a very good way to  study epileptic activity, it's  very easy to see with these  methods.  Now, I'll finish, the last part  of my talk is I'll tell you a  bit about what we're trying to  do to make voltage imaging  better.  So voltage imaging has not been  very popular in neuroscience.  Neuroscientists have not flocked  to the technique.  They haven't bought the  instrumentation, they haven't  built the set-ups.  And the reason is the technique  is not very highly regarded.  The signals are not very good.  The neuroscientists don't want  to see that there's more  activity of this hundred micron  area of brain which has maybe a  hundred nerve cells. 

 They want to see the activity in  a single nerve cell.  So the kind of things I just  showed you that's a bit of a  teaser.  That doesn't really get at the  big questions.  And we would like to have a more  powerful method of imaging that  can get at some of the bigger  questions.  And so we're just at the start  of trying to develop that method  and I'll tell you a little bit  about how that works.  So it starts with using a  fluorescent protein, and, in  fact, the latest Nobel Prize in  chemistry was for fluorescent  proteins.  They called it green fluorescent  proteins but by now they've got  perhaps a hundred different  colors because once you find one  protein that has a particular  color you can then make little  variations and make lots of  different colors.  So the idea is you attach a  fluorescent protein to the inner  surface of the cell membrane.  That's not so hard to do.  And then you treat the cells  with another molecule, this  molecule which we call DPA,  dipicrylamine, its picture is  down here, and it has a negative  charge so voltage makes it move,  but it's also very oily so it  will go into the oily interior  of a membrane. 

 And when the voltage changes the  DPA will go from being far away  from the fluorescent protein to  near the fluorescent protein.  And the DPA, it's close enough  so that it can absorb the  molecule, the fluorescent light  from the fluorescent protein.  So instead of seeing a lot of  light coming out you'll see very  little light coming out.  So it's a way of turning  fluorescent proteins into  voltage centers.  We use fluorescent proteins to  create an optical signal that we  can then measure in a microscope  and make a movie of voltage.  So this is what it looks like.  We've produced the fluorescent  protein.  We've expressed it in a cell, in  PC12 cells again that are easy  to use.  And you can see that the  fluorescent protein is mostly on  the surface of the cell.  And we've added DPA and we've  applied, we've changed the  voltage of the cells by using  the same techniques we used to  study the nerve terminals. 

 We stepped the voltage up to  plus 30 millivolts, down to  minus 70.  And you can see that the  fluorescence changes.  The light we record from one of  these cells changes as the  voltage does.  So we have a technique, a method  that gives us fairly large  changes in light associated with  changes in voltage.  And we're just starting to use  that.  So here's an example of that  technique applied to a  hippocampal slice where we can  see squiggles in our optical  recording that correspond very  nicely with voltage changes.  And here those squiggles blown  up so they're bona fide changes  in light that are actually quite  a bit larger than the changes we  saw with that other dye RH-482  that I spent a few minutes  talking about before.  So we still don't have single  cells here but it's a start,  it's a better technique which,  in principle, should tell us,  give us the ability to see  single cells.  And we've also applied this  technique to a spinal cord slice  and seen the same kind of  electrical activity, optical  recording of electrical activity  in the spinal cord.  Again fairly large signals  compared to those we saw with  RH-482. 

 With this technique we may  eventually fulfill the dream of  voltage imaging, of actually  seeing the electrical activity  sweep through a nervous system  cell by cell.  We're not there yet but this is  a very promising future  direction.  So with that I'll end.  I'll mention that I have some  great collaborators, friends,  students and colleagues that I  work with.  And they've done most of the  work.  In fact, my lab is a graduate  student lab.  The graduate students do almost  all the work.  I've done a few experiments but  they've done a lot more and  they're much better at it.  And I also have faculty  collaborators in the university,  in particular Ed Chapman, Cindy  Czajkowski and Baron Chanda who  are all in my department and who  I collaborate with on various  aspects of this project.  Finally, I'll just mention that  I think doing science is a great  idea and going to graduate  school, our graduate program is  a real future in that so I would  urge you all to encourage people  in college now or thinking about  starting careers to consider  doing graduate work in  neuroscience or biophysics. 

 So with that I'll end.  Thank you.  (applause)