 Welcome back to the NPTEL lecture series on bi-electricity. So, we have finished 8 lectures, now we are into the 9th lecture. In the last lecture, we talked about the microelectrode array and I requested you to please go online and check the real image of microelectrode array. So, one of the things what people are currently trying to do, so we will briefly talk about the applications of microelectrode array what people are trying to do. So, and then we will move on to the intracellular recording. So, one of the things what needs bit of a visualization is that think of this whole brain, it is thousands and thousands and thousands of neuron making multiple circuits out there. So, something like that if you have to visualize the brain, it will be something like this. For example, if this is your brain and this is the brain stem and here we have the brain. Now, at any point if you pick up any particular point and if you magnify it, what essentially you will see will be series of neurons like this sitting out there, functionally dynamic state. It is a complex network something like this and in between you have the glial cells and likewise series of supporting cells something like this. So, at one point of time in this network, it has been estimated that one neuron receives could receive of the ability to receive signals from ten thousand other neurons. In other word what that translates down into at one point of time a neuron on its surface as ten thousand synapses something like this. So, for example, if this is single neuron and this is the axon and here you have the dendritic tree something like this, this is the nucleus. So, this neuron at one point can receive signal from say ten thousand different sources like this. I could only draw certain visible space I have I mean I have limited space I cannot draw all of them. So, just to show you. So, one of the critical challenge of the modern neuroscience as a whole is how really to understand how a network functions because in a complex brain it is exceptionally challenging most of the time when you insert an electrode what you record is a field potential what I meant by field potential is that say for example, I have a electrode say I am showing it by like this I have this electrode out there. This electrode will only measure something like this in this region the all the activities which are taking place out here in a broad region that is all it does. So, electrode recording like that cannot really pinpoint what is happening at a small loci like this or it cannot really pinpoint the cellular events which are taking place what you are getting is something aggregation of say ten thousand neurons at one spot or like you know five thousand neuron. Of course, it depends on how smaller is the size of your electrode how finer the electrode is you get a field study the summation of the electrical activities of a population of neuron that is essentially is helpful for several to understand the rhythms and several circadian activities and all those there are several things sleep and all those things, but that is not really the way you can figure out what exactly is happening at individual cells. Say for example, I have a drug which is targeted to specific kind of neuron or say for example, I have a drug which is getting into the brain I have no idea what is what it is doing at the individual cell for that you need different approach. So, most of these approaches depends on in vitro culture model what does that mean there are two ways how you can approach use the electrical power or the bioelectrical techniques for understanding biological phenomena. One is that you insert an electrode or your poke an electrode in a live animal this is one way where the live animal the animal is moving around and you are recording in a real time you are recording the events which are taking place that is one way which is nevertheless is the one of the most powerful profound way to do it, but that would not give you any idea as I was mentioning about individual cell what is happening. In order to understand individual cell you have to go down to the cellular level and that you cannot do in a live system then you have to either take out a part of the tissue outside the system you can make a slice you can do a slice on the slice you can keep the slice alive for 6 to 10 hours and on that slice you can do recordings there is another way where you please the slice on a micro electrode array what I have shown last time. So, in that situation what you are essentially doing is you are keeping the site to architecture intact. So, for graphical representation what I am trying to tell you. So, these are the different techniques so this is the live animals say for example, if this is these are recording techniques live animal recording. So, you have electrode either implanted like this or you have surface electrode like this and the animal is alive this is a spinal cord likewise you know and essentially what you are recording are the field potentials the other set of recording out here which is and the you cannot do it in the live animal. So, this will give you field potential of a population of neuron. So, the drawbacks if I had to say the drawback just putting drawback as drawback as d b do not provide in for information for cellular event then this takes us to the next level how to understand cellular event how to understand cellular event for cellular event you need to have what we call in vitro recording what we essentially call this technique as in v o or in animal recording. So, to the in vitro recording. So, in the next slide we are moving the different techniques of in vitro recording. So, say for example, if I look at all the excitable tissues. So, one of them is your brain and the spinal cord then you have the heart is the brain. So, you essentially you have two major techniques one is slice recording the other one is dissociated dissociated cell culture recording what we meant by slice recording. So, in the next slide we will talk about what we meant by slice recording. So, say for example, this is the part this is the brain you anesthetize the animal you remove the brain and you kill the animal and we know that this is one of the areas which is called hippocampus which is involved in learning and memory. Campus we will talk more about it we will be talking about learning and memory involved in learning. So, what you do essentially you take out that organ like this and then you make slices. So, something like you make slices like this. So, you essentially get you can make slices in a different in different ways. So, you essentially get are very thin tissue like this and then you put them in a chamber emulating the condition of the brain like the tissue is sitting like this. So, here is a extracellular fluid with different energy source to make this tissue survive and then you approach and already the site architecture is all maintained just to mention slice recording. Slice recording and advantage site to architecture is intact then on that what you are getting you are putting the electrodes like. So, like this then you start doing the recording. So, this is essentially is talking about the slice recording and slice recording is very popular because your architecture remains intact you can really hope there is the circuit remains intact you can really poke the electrode at a specific zone of the circuit and stimulate one circuit and say for example, in this diagram if I just highlight this further. So, it will be something like this if this is the part of the tissue. So, for example, this is the hippocampus which is almost like this and it is known that hippocampus has something like this. So, hippocampus has different circuits on its system something like this C A 1, C A 2, C A 3 these are the circuits within hippocampus will come in depth on this one after word. So, now what you can do in order to study these different circuits and connectivity you can poke electrode here you can stimulate here you can do a recording from here or you can poke electrode here you can do a simultaneous recording from here or you can do it from here you can from here likewise or you can stimulate here and you see how it is distributing on both sides likewise and you can put multiple electrode. So, this is another advantage and you can even do you can do two kinds of recording here you can do a sharp electrode recording which I have already talked sharp electrode and you can also do something called which I have not discussed here patch clamp recording. We have not discussed about it, but this I definitely discussed with people and some people even try to take this whole circuit and take this whole slice and put it on top of a micro electrode array on a planar in here this whole circuit is placed on a planar amion as if you guys have seen it something looks like this please again I request you kindly go online and check the structure that will help you and the circuit sits like this something like this. So, you see so these are different techniques which are being used to understand the functioning of the brain. By extracellular so this is what I showed you in a planar micro electrode array is a extracellular pattern of recording then you could have intracellular electrode and you could have patch clamp patch clamp I have not discussed with people what I am going to discuss after this. So, this is one way, but there is another way which is the third way and which is so you have to realize the drawback of slice recording is this slice recording cannot last the slice cannot last more than 6 to 10 hours. It is really tough because it is a three dimensional tissue out there which is already taken out from the system it is not really adapted so for may be you can make it last for 18 hours may be a day if you are exceptionally good, but the problem is that whenever we talk about drug trials chronic situation, chronic experiments the story changes why the story changes because it will realize that say for example I put a drug and drug will be acting over a period of months and may be sometime years and most of the animal trials are really costly whenever and a long term effects at the cellular level at times get lost get missed. So, say for example if you go back to the slides where I showing you the real animal situation say for example I inject a drug into this animal now this is circulating all over the body all over the place. So, it is really tough to know exactly what is happening at the individual cellular level and over a period of time how it works. So, chronic experiments are really tricky and really tough to do and on top of that with animal ethics and the cost of animal every drug discovery really takes a huge amount of funding huge and that is when the drug comes to the market it becomes so costly. It is not costly because this drug is out of the world it is costly because it has to go through all the different channels of screening and those screening takes enormous money enormous amount of funding is required. So, that capital investment essentially jack up the price of a drug when it comes to the market. So, now coming back what is the other technique and especially these kind of drugs are exceptionally costly when you talk about the nervous system or the cardiac system which are kind of you know pretty much your life line cardiac drug it is not easy I mean it is really tricky you have to go through all possible channels of hoops before the drug kind of gets into the system. So, coming back where we were about the what is the third technique. So, we talked about the slice recording now we will talk about the third set of recording which is fairly old yet fairly new also there are two aspects of it what you do essentially here. So, from this diagram itself let us start to draw what you do here say for example, I wanted to have understand about the hippocampus what I will do is that I will pull out the hippocampus I will break the hippocampus in the sense I will dissociate the cells of the hippocampus by doing by using different enzymes or different mechanical ways. So, if you look at this circuit now. So, at the cellular level if you try to look at this circuit this circuit is essentially nothing but a series of neurons sitting like this like this thousands and thousands of neurons are sitting like this and they are making circuit at different level and this is just the top layer I am showing multiple layers and likewise and they are arranged in an specific array specific circuit and everything. Now, what we do is that you take the hippocampus take this out take this whole thing out or you can take him in part of it if you are very good at the section or something then take this out and for example, you have collected that part of the tissue out here likewise in an extra cellular fluid then you break the tissue and this breaking of the tissue is called dissociation of tissue into single cell suspect suspension. What we mean by single cell suspension it means now you have all this individual component what I was drawing are separated out something like this in that process of course, the tissue undergo a lot of damage or something, but those which survive are important for you and now you take these neurons and the accessory cell depending on you have different modes that you can purify at this stage you can. So, if you have the neural tissue in case of say neural tissue you can neural tissue you can purify the neural tissue you can have the glial cells separated which are the supporting cells you have the neurons separated out and then you put them in a dish to grow of course, they would not grow just in the thin air you need to do a bit of a homework you have to coat this dish with something on which they prefer to go some kind of substrate on which these neurons will grow. So, this is the substrate and on top of the substrate you have the neurons growing like this. So, if you get a top view of this something like a top view and it will look like this neurons are all over the place likewise it will be a random connection between different neurons this is a dissociated neuronal culture. So, these dissociated neuronal culture now you can approach the individual cell with individual electrode you can have a sharp electrode like this you can approach the individual cell and you can monitor several events you could put x y z compounds out here say for example, compound a compound b likewise you know you have this another compound out here or a third compound out here likewise and we can figure out their figure out what they do. So, this technique gives you an access to the individual cell in a dissociated culture this is what it does, but it comes with a drawback drawback is that I told you in the previous slice preparation the cyto architecture is maintained in other word if you go back. So, the circuit is all maintained out here circuit is not getting destroyed, but out here what you did once you dissociate everything the there is a random connectivity you do not have really a control on their connectivity. So, there is a random connectivity. So, this network is forming in a very random manner you really cannot dictate that how many synapses are forming you really cannot have any control, but eventually it becomes really cumbersome to detect say for example, think of a practical situation if this is I label that is a and I label this as b, this as c, this is d, this is e, this is f, this is g, this is h, this is i and j likewise. Now say for example, a signal is getting originated from here and I am seeing the signal is all over the place. Now, I really do not know how the signal has moved I really cannot trace it because signal may move like this, signal may move like this, signal may move like this, signal may take a back turn and likewise signal may have a connectivity like this. So, there is no way I can figure out how the network exactly functions. So, network behavior is really tricky it is almost the same situation as when you do a field potential measurement you really have no control about the number of cells which will be involved in generating that signal. So, you really do not know and moreover you really cannot in a random circuit it is really difficult to keep a tab at the changes at individual synapses because at individual synapses it is again getting connectivity from multiple sources because it is random there is no way that you can control that connectivity because anything can form connection with anybody. So, that makes the story very complex that is something you do not wish to happen, but you need technology now if you need to have a directed or you know completely patterned growth you need some different kind of technology. Then starts within this dissociated culture the current technology which most of the people in the area of bioelectricity or bioelectrical recording are following. So, they are trying to develop built circuits out of this dissociated cell how they are doing so. So, coming back to the basics again. So, I drew that I told you that there is a substrate. So, for example, let us try to understand it. See for example, you have a culture substrate like this imagine this is the culture substrate all the cells will grow on it everywhere. On this culture substrate say for example, if you have a way that you could introduce some pattern. So, for example, you do something like this you ablate these part of the circuit. What I meant by ablation means I am removing that particular yolo color compound from here. So, if you remove the yolo nothing will grow there provided you backfill it with something which one promote essentially what you are getting now look at it. So, essentially now the cells. So, say for example, I backfill it what I meant by that on this on these zones you fill it with another something. So, on black I really cannot draw anything because everything will this is the backfilling agent. So, that is ensuring that those ablated surface. So, what we those who are not understanding ablation let me just explain what you do essentially you. So, what you essentially do is say for example, you have the substrate like this you take a mask and if imagine this is the substrate and I have the mask I keep the mask here. If I keep the mask here and I put a laser beam or something. So, at this part where the mask is covering it nothing will get ablated rest of the places will all get ablated does that make sense. So, that is exactly what I am trying to tell you what I say for example, I put a mask the zone which were exposed to the laser beam are the one which are black. So, it burns out those spots what is left with all the yellow but things will grow and now where you have the black spots there what you do is that you backfill it you dip it in such a solution which will only sit on top of those ablated region that compound would not sit on top of the yellow. So, then that is what I am trying to do. So, this is that next compound by which you ensure this black compound is the one which will not allow any cellular growth at that particular black surface likewise. Now, you have a pattern situation this pattern situation will allow the neurons to grow like this. So, the neurons could grow if their thickness is like this something like this if this thickness is good enough for a neuron to grow they and may be connectivity coming out like this possibilities are there, but there are ways to you know control that. Now, what you are seeing essentially is you are trying to control the motion the position of the neuron and you really can do it in a very interesting way there are several geometries which you can follow. So, this is what geometry I showed you. So, this is done by a technique which is called laser ablation you ablated using laser. There is another way you can do it you can make these circuits using your older style inkjet printers what you do is that you print the circuit. So, for example, you on your word document you draw the circuit narrow it down and on the cart rates where you fill the old inkjet printer you fill the fill the ink you throw away the ink sterilize it and on top of that you put inside that you put the either the substrate you want to do. So, what you will do the jet printer will make say for example, a circuit like this. So, for example, I want a circuit like this I want the cells to grow like this. So, it will make a circuit like this or it can even make a circuit like only lines or it can make a circuit like or it can make a circuit like this. So, there are several ways you can make circuits and these are some of the different ways what I am trying to highlight and you can control the dimension. So, for this I would recommend you and we go through some of these extra materials which I expect you to see the papers of for micro contact printing one of the pioneering person micro contact printing you should refer to the work of professor Thomas Boland currently he is in university. University of Texas at El Paso professor Tao Zhu these people have done very significant of amount of work on micro contact printing and it is worth reading some of their work how they have done it using very very simple. Most of these work were published during 2003 to 2010 now also some of the work they are publishing and they are absolutely phenomenal I mean the way they have done all these things is just with very crude I should say very crude techniques around them they could really do very nice micro contact printing and some very well documented papers are there from their side. So, this is one group's paper I like you people to look at it for laser ablation and all these work I expect you please go through the work of there are few people whose work will be really looking one will be one second one paper by this is one of the very old paper Klein field in journal of neuroscience very seminal paper Klein fields then you should go through the work of James J. Hickman he has done extensively extensive work in that area then you should look through the work of Bruce Willer who also has done very significant amount of work in this area and then you have Gregory Brewer these are the people who have done significant amount of research in this area and it is definitely I will recommend you people please go through some of their work they have work in wide areas, but definitely they have made some seminal contribution in these kind of printing circuits. So, current status is like this with this I mean you can go to the other end of the world. So, there are techniques which are being used in laser ablation then there is something called photo lithography and this will get a lot of references in professor Hickman's work lithography and professor Brewer's work and professor Klein field's work and of course, professor Bruce Willer's work. So, we talked about the micro contact printing where you should look through the work of professor Thomas Boland and there are few other people who have done very significant work I will come back to that in the next lecture. So, these people have shown that they actually can guide the neuron in a specific trajectory a single neuron. So, you will see some of these circuits like you know you can nowadays you can develop like you know 2 neuron circuit like this these are you will see these kind of circuits are being developed single 2 neuron. So, now in these 2 neuron you can really approach with a single electrode you can have x y or z compounds all over the place you can really quantify the synapse. So, what all you can do you can quantify synapse one you can do chronic experiment for long period of time and this chronic experiment could be these circuits could survive for more than a month or so you know if you are really good at it. So, they may ok chronic experiment you can quantify the synapse you can do chief drug trials they reduce down the cost of drug trials on top of that you can introduce the supporting cells like you know the glial cells. So, you can study the glial cells dynamics and on top of that here is a control model where you can study learning and memory. So, these kind of control circuits and you can make series of them I mean as the authors whose papers I have mentioned you or the those who have made seminal contribution if you will read through these papers you will realize you can make series of such circuit to approach a single cell in a very elegant way and you can really understand the network behavior in a very I should say in a very simplistic reductionist approach. Of course, it comes with its drawback because you are rebuilding the circuit. So, you know there will be some error here and there, but the way biology works is that you start from whole animal you come at the single cell level and then it all has to merge. So, there is no one technique which is perfect and there can never be one technique which is perfect. So, the whole idea is you know having multiple techniques trying to tell you or trying to unravel the truth of nature. This is what we are always trying all throughout like you know we are trying different techniques. So, this is one approach. So, another approach in that same line which is in hybrid approach which is which is being followed I am going to do is I introduce you to the micro electrode arrays. Let me draw a micro electrode array and tell you what is that approach. It is a very interesting approach where say for example, you have these micro electrode arrays sitting out here like this. Now, electrodes like this. So, if you can pattern this say for example, I have a pattern like this say fine. I have a pattern like this. The cells will follow this trajectory something like this. Now, and I connect this. So, now rest of the places where you see yellow are the only places where the cells will grow rest of the places cells will not grow. So, I modify the surface of this planar micro electrode array in such a way that cells will grow all along those electrodes they are connecting the electrodes and the dimension of the electrode is say 20 to 30 microns and those lines. So, for example, the aspect ratio of you know 10 to 20 microns or maybe 10 microns and specifically except the places where electrodes are their aspect is slightly more. Maybe this is say 20 micron and the lines are say 5 micron thick. So, on a 5 micron surface it is really tough for a cell to sit, but the cells will preferentially will sit on top of the electrode because these electrode regions have more surface area. So, around 30 microns or 20 to 25 microns. So, when you put the dissociated cells into this chamber what will happen? So, for example, I put the if I represent the cells by you know if I represent the cells with red. So, now I am putting the cells into it. So, cells will preferentially will try to sit here because these are the zones where they will try to migrate to on top of the electrodes because that is where they will get the maximum surface area to grow, but these are all dissociated cells. So, once they will sit like this what they will try to do they will try to you know send out processes like this will try to send out processes like this to connect with each other. They can do it in like this like this several ways they can do you can even stimulate this circuit in order for this whole process to take place and they will form a very controlled network network which you can monitor in a real life something like this. So, they will start forming network inside you keep this whole system inside an incubator and you monitor it as they are forming the network once they are forming the network. So, what you can do you can give an external stimulation for network formation and you can register the electrodes. So, for example, I register them as e 1, e 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. So, I can register the electrodes and you really can monitor the activity at the individual channels and the individual channels of the amplifier you can monitor the activity what is happening in which electrode. Now, once the network is formed say for example, I give a signal out here I give an stimulation out here. Now, how this stimulation is moving along this circuit I can monitor in a real time how the how the synapses are forming out here how the synapses are forming here how it is forming here how which circuit is getting more strengthen how it is getting more strengthen I can study all these things now what and then based on that I can back calculate what is probably happening in the brain. So, if you look at it there are profound scopes which is open up with the advancement of modern microelectronics we are able to access a single neuron on top of an electrode and these are all could be done using extracellular recording. These are all extracellular planar MEA or microelectrode array recording. So, this is the advantage which microelectrode array offers in order to study the circuit from a very reductionist approach it is not an holistic approach it is a very very reductionist approach you are building the system from the base again from grass root you are break by break you are building the system. So, I will close in here for this class and in the next class we will talk about the other end of the intracellular recording where we will be approaching a single ion channel because once I will introduce the ion channel then I will talk to you about the structures and the details of the ion channels. Thanks a lot.