 Welcome back to the NPTEL lecture series on bioelectricity. So, today we will be starting our tenth lecture. So, in the last lecture we talked about in details about microelectrode array the planar microelectrode arrays. So, we talked about how this planar microelectrode arrays could be used in drug discovery, understanding the neuronal circuit and understanding cardiac action. So, yesterday I showed you a circuit in that where I showed that you know you can make a small circuit with defined number of neurons. Similarly, you can place there are few other modifications what you can do you could recreate say for example, we know that the cardiac myocytes or the heart cells are being regulated by sympathetic and parasympathetic neuron. So, do the muscle cells are being regulated by motor neuron. So, what you can do by the same same patterning techniques you can rebuild those circuits. So, something like this say for example, if I call this if these are the cardiac myocytes you know the beating cardiac myocytes and we know these cardiac myocytes are being inner weighted by the sympathetic and parasympathetic neurons. So, what we can essentially do we can really study. So, if cardiac myocytes and these are the parasympathetic sympathetic circuit. So, these sympathetic parasympathetic circuit actually regulates the cardiac myocytes. So, what essentially do you can translate this whole thing on in a microelectrode array to study the exact mechanism what is happening. So, in order to do that what you have to do say for example, you have an array like this out here and you have these electrodes sitting all over the place like this. You can make these arrays custom made you can I am just drawing a limited number of arrays in order to and say for example, you have a physical barrier like this. So, that the cells on the other side cannot move to this side whereas, the only the processes can move something like a physical barrier like this. So, what you do see take the cardiac myocytes and you start growing the cardiac myocytes on this other side. So, these are the cardiac myocytes which are growing these cardiac myocytes cannot move on to the other side because you have created a barrier. So, that these cells cannot move whereas, you put your sympathetic parasympathetic neuron likewise but the process can really travel to this side. Similarly, you have so essentially what you are doing you are recreating that circuit of control of sympathetic and parasympathetic to the cardiac myocyte or in other word you can study the heart physiology cardiovascular physiology on a small chip or a microelectrode chip and these are the power of these kind of simple system where you can. So, what you essentially can do you can say for example, you can stimulate this you stimulate this and stimulate this electrode out here and you could see the response out here you will be able to see the see the kind of response which is taking place on this electrode on this electrode or wherever. So, you could have a 1 to 1 connection between it. So, this side is showing just for you the parasympathetic I am showing sympathetic or parasympathetic neurons and on the other side you have the cardiac system and these kind of devices are called biohybrid devices and these biohybrid devices are nothing but where there is electronic component are being integrated with the biological component and that leads to a marriage of these 2 diverse system in order to extract meaningful information out of it which could be used for several purposes. I mean look at this system this could be used for I mean wide array this could be used for drug discovery one of the major areas this could be used for a chronic studies a long term effect of drugs or something like that this could be used for understanding network which is a very challenging task in a real life situation you really cannot and basically what you can do you can add component by component into these kind of circuits to do similarly you can build another circuit like you could have. So, we know that most of this motor neuron from the spinal cord are the ones which are innervating all your skeletal muscle these are your skeletal muscle just sitting out here like this with. So, how to build these kind of circuits. So, again the similar way you can translate them on a microelectrode array the way I was showing you if this is the array and again you create partition you can really custom made all these things and you have the electrode sitting like this like this the electrodes are sitting all over the place and here you are going growing the muscle cells like this and the gap is. So, much that it would allow really and you can actually pattern these substrates in such a way that they will only go grow in a certain fashion as I have already discussed to you with you people about the pattern and here the cells will and here they are forming the neuromuscular junction something like this. So, these are the power of these kind of systems. So, basically what you can do out here you can stimulate the individual motor neuron and you can see the response out here in the muscle. So, you have in your control now the individual synapses. So, you can really access the individual neuromuscular junction or N M J or neuromuscular junction synapse. So, these are the reductionist approach to study bioelectrical events which are responsible for our very core of our survival and these not only helps you to understand circuit as I was mentioning you are a whole path of other application this is an upcoming rising area by hybrid devices which can change the way we think. Another approach is now what is the next level of development which is taking place whenever you look at these kind of arrays these are flexible arrays. So, these are flexible arrays. So, now what you can do essentially is these flexible arrays now there are materials by which you can make this non flexible arrays you can make them flexible arrays flexible micro electrode arrays M A's these flexible M A's could be implanted directly into the nervous system or any other excitable systems of the body. So, you can really roll them up you can put it there and they have they are finding in future they will rather they will find applications in terms of prosthesis in terms of wide range of applications which as of now is kind of out of like you know this stuff to comprehend at this time, but these are the areas which are going to change the way we study biological systems though these are very reductionist approach, but they give very profound information very clean information with least amount of noise in them. So, with this understanding of micro electrode arrays I will move on to the patch clamp technique. So, before I start the patch clamp technique. So, let us get some of the time line right what happened when during last. So, if you go back and if you look the way if this is the I am drawing the x axis and I am showing you the time somewhere 1700 or 1800 this is the time when there were preliminary understanding. So, people have understood by electricity was pretty much there all over the place and somewhere down out here somewhere out here by electricity was discovered followed by that people were starting to do a whole bunch of started doing sharp and during this time of course, this gives birth to the whole field of electrical engineering. If you go back to the time of Volta Galvani during the time well there was no formalized discipline called electrical engineering and people were discovering charges and all these kind of things and it is during that time that by electricity was discovered as a matter of fact some of the bioelectrical phenomena were discovered much earlier than formalizing the whole field of electrical engineering. So, after that during 1800 there were people were doing sharp electrode they were trying to you know pushing electrode and with voltmeter they were trying to make recordings and everything during early 1900. So, this is the time when late 1800 and 1900 this is the time plant by electricity was discovered and plant and will be of course, will be dealing with this whole section there is a whole section in this course will be dealing with it and the pioneer in this was Sir J C Bose. He worked extensively on MIMOSA touch me not plant and several other related plants which show electrical activity and published is seminal contribution and pretty much proved beyond doubt these plants have plants exhibit bioelectrical phenomena. So, in one way it can be said those were the systematic beginning of understanding bioelectricity early half or later half of 18 century and as a matter of fact, the that is the same time the pretty much out here just for later half of the 18 century when the discovery of Galena as semiconductor and as a matter of fact semiconductor concept of semiconductor came even earlier than that by the first such evidence which could not be described was shown by Michael Faraday he really could not explain he saw some deviation from the known ohm's law you know there was a very unusual deviation non-linear relation started picking up he could not explain it was after that the semiconductor was being discovered and I am forgetting the name of the individual I will get back to you with that and then it was the discovery of Galena as a functional device. So, this is basically what he showed is but Bose showed is a functional device, but after this he moved on to the plant and made seminal contribution in understanding plant bioelectricity and as a matter of fact he the first one to propose that plant have life actually that is the time it was kind of you know out of the world and how it could be said that plants have life he pretty much showed that they generate action potential like impulses and those are recorded and that was pretty much a systematic beginning then on during this part 1900 to 1950 I should say the time when there was enormous work happened across Europe on animal bioelectricity a systematic study mind it these studies were going on it is not that systematic studies were being done some of the I should say some of the very hallmark was the discovery of action potential by Hodgkin and Huxley there are several other people whose name I am not mentioning here, but that does not mean their contribution or any less it is just I am just trying to build up the story how the patch clamp came into being, but even Hodgkin and Huxley they use sharp electrodes which I have shown you before earlier. So, you have this cell and you have a sharp electrode and you are making recordings as a voltmeter or whatever, but based on their action potential traces they did a complete mathematical formalism of what they believed is something like when the membrane is kind of you know becoming short, but then the suspicion that there are entities like ion channels. So, there was no ion channels which was known and it binded this is the time when not a single protein was kind of crystallized it was the time when Max Barouds was trying to crystallize hemoglobin today you see of so many proteins which have been crystallized and all those things and forget about membrane protein those are not even clear and it is the time when the most famously celebrated membrane model of the fluid mosaic membrane model that was not I mean it was kind of it was not really clear at that time what is the structure of the membrane. So, essentially what we did not know at that time was if this is a cell the structure of the membrane was not clear. So, this is I am talking about 1940's membrane structure was not clear the ultra structure was not known proteins crystallization has not taken place. So, really we had no idea much idea about protein just recently DNA was discovered. So, this is I am talking about 1940's and 50's DNA was discovered by Watson and Crick it is a time when action potential was discovered, but nothing was clear that there are ion channels and all those things which we talk so easily nowadays was like you know could be an could be at that time could be a like you know very daring dream and that is what pretty much Hodgkin actually did the in their mathematical formalism came up with you know there are possibilities there are specific channels through which the ion passes and likewise when and so forth. It was purely mathematical based on certain experimental data then came the next breakthrough in 1970's with the discovery of a technique called batch clamp and so if I go back in the timeline chart. So, the next discovery was here 1970 and in between there is one discovery out here which is very significant though nothing to do with biology though, but it played a significant role out here that is this this is the time when semiconductor was discovered and they go all hand in hand semiconductor discovery by Bardeen, Britain, Shockley. It was during that time that opened up a flood gate of developing silicon based devices mind it semiconductor sorry I am wrong here just let me correct it crystalline semiconductor. So, semiconductor was known much earlier than that, but what was what was the breakthrough out here was the crystalline silicon using crystalline silicon and this was the discovery which was made in Bell Labs New Jersey. So, this discovery change the way we perceive the world there are certain discoveries we totally change everything and this is one discovery, but how this discovery has to do with what we know today about ion channels or bioelectricity. So, the connection is fairly straight forward because of the discovery of semiconductor this leads to development of electronic devices which are much more profound as compared to previously when people are using germanium and all those kind of you know where the signal at signal occurring fidelity is fairly low and all those, but here there was a scope that you could really acquire very profoundly good signal. So, that leads to the development of very high precision during this period it opened up a flood gate for the development of very high gain electro meter electro meters and amplifiers this. So, those of you have forgotten electro meter if you remember that you could major charges getting a gold leaf foil that is why they use electro meter. So, this was the time which was a really very amazing time where like you know some of the path breaking discovery took place and the whole field was kind of rocking and with the discovery of very high gain amplifiers. So, one of the challenge you have to realize that you are majoring current at nano ampere pico ampere dimensions it is not easy it is very noisy you really have to have a very good hold or understanding about the phenomena otherwise you will be recording noise. So, it was during that time this discovery of these high end amplifiers and simultaneously it was during that time pretty much around 70s on this is the time which saw the next development I will come back to this and they all have played very significant role to what use people see today is with computers it is the time when computers were slowly coming into existence. So, with the discovery of crystalline silicon as a material semiconductor material for device development followed by development of very high end amplifiers for these kind of recordings the stage was set for the next discovery and the next discovery was I started when you start is that the discovery of patch clamp, but then when you shifted all the way to Germany two gentlemen two Germans Irving Nihar and Bert Sackman they were instrumental in developing patch clamp technique what essentially patch clamp is all about. So, if this is a cell so we are talking about fine this is a cell and these are the iron channels on the cell this green millions of such iron channels all over the cell. Now, what you wanted to understand is you want to access or you want to measure the current flowing through these iron channels it could be a potassium channel it could be a sodium channel it could be a calcium channel it could be a chloride channel. So, your goal is this you want to see the movement like this or you want to measure the movement like this or say like this or this. So, in to measure these ionic fluxes so in order to understand that you have to really come close. So, that you can access an individual iron channel how to do so. So, what was being done by Nihar and Sackman is Nihar essentially do is they develop this nice glass biped. So, how they did it let us understand it first. So, you have this you all have seen a capillary. So, here you have a capillary tube now I take the capillary tube and keep it in without touching and keeping it at a very hot coil they keep it in a hot coil. So, this is whose temperature you can control and on these two sides you have two clamps by which. So, for example, these are the clamps by which you can pull this in two direction on either direction. So, now what will happen when you will expose it to a very high temperature this will start to melt like this and then if you give a very strong pull on either side this for your understanding sake. So, you have this coil now if you give a very strong pull what you will be essentially landing up with is this depending on the strength of the pull will be landing up with a narrow bore or a thick bore glass biped like this. This is what I was trying to draw here which is essentially like this this is what you are going to land up with. Now, the if you look at the diameter of these kind of glass pipet they will be approximately around 1 micron or micrometer or maybe 0.5 micrometer likewise and mind it if you look at the cell is compared to this which is 20 to 30 micron or even sometime may be 35 to 40 micron the bigger better of you are in terms of picking up signal. So, now what essentially you can do with that small tip you can access at least a 1 micron patch of a cell and mind it what is the name and the name is patch of a cell patch clamp. So, in other word what you are trying to do by this word what this word says you are taking a small patch of the cell like this and you are clamping it or you are you know holding it. So, you are holding a small patch of a cell and in that small patch of a cell you are manipulating the membrane properties along that small patch and that is why it is called patch clamp and what all you can hold there it only two parameters I will come to the whole electronic configuration, but let us understand it there are two things you can hold here. If you go by the most fundamental of v is equal to i r you can either. So, where v is your voltage i is your current and r is your resistance. So, there are only two things you can hold here you can either hold the voltage or you can hold the current when you clamp in that zone you can hold the voltage this is called voltage clamp because you are holding the voltage or the voltage is in your control you can change the voltage you can clamp it at different level minus 80 minus 70 minus 60 minus 50 minus 40 minus 30 minus 20 minus 10 0 likewise you can hold the voltage or you can clamp the current which is called current clamp. So, current clamp is essentially you can inject a finite amount of current to the preparation through that electrode. Now, what we will do we will. So, essentially you would see patch clamp with a. So, I kind of give a graphical summary of this. So, you have patch clamp and under patch clamp you have voltage clamp or you have current clamp. Now, with this background I will move on to the next phase where we will be talking about how these circuits are being built how these are being dealt and all other details. So, today we will close in here and the next class we will move on and we will talk about in depth about the circuits. Thanks a lot.