GCaMP | GCaMP calcium indicator | How GCaMP works? | GCaMP application in neuroscience

This video will talk about a genetically encoded calcium sensor GCaMP. GCaMP is a genetically encoded calcium indicator (GECI) initially developed in 2001 by Junichi Nakai. It is a synthetic fusion of green fluorescent protein (GFP), calmodulin (CaM), and M13, a peptide sequence from myosin light-chain kinase.

Hello in this video we’ll talk about a genetically encoded calcium sensor which is known as g camp and we’ll talk about its application in biomedical research gcamp which is a genetically encoded calcium sensor was first reported in 2001 by junishi nakai in this video we’ll talk about what is gcamp how gcam works an advantage and disadvantages of using gcamp

In neurobiology research from 2001 the neurobiology research has really changed a lot gcam is a big discovery in neuroscience and let me tell you why so gcamp is kind of like a gfp molecule which is attached with a calmodulin residue and calmodyline is a protein that can binds to calcium also there is a m13 peptide that is binding to the gcamp moiety now when

Calcium binds to these g camp then there is a conformational change generally gcamp in calcium unbound state does not fluoresce because the chromophore moieties are protonated now when calcium binds there is a conformational change and the fluorescence is now shown notice the m13 helix is now in a different location and this results in the conformational change

And fluorescein of the gf gfp molecule now so gcamp can fruitfully report the calcium fluctuations inside the neuron and calcium fluctuations in the neuron can work like a proxy of neuronal activity so gcam can can reproducibly report the neuronal activity but what’s the big deal about it because neuronal activity can be recorded using patch clamp technique and

That has been reported several decades ago so what we have achieved so far using g chem what’s the added advantage and that can be understood if we try to look at the applications of gcam first of all activity from several neurons can simultaneously be recorded using gcam based approach so under the fluorescence microscopy in a field of view let’s say there are 100

Neurons and all of their activity can be recorded simultaneously which was not possible using a patch clamp recording in bachelor recording one neuron has to be recorded at a time and that’s the added advantage now using g camp and a combination of two photon microscopy scientists are able to record neuronal activities from behaving animals such as this particular

Mouse they can really drill a hole on its skull and can record the record the activity if the mouse expresses g camp in its particular brain region in this example we can see how the mouse brain region is fluorescing over time and because they are marked by gcamp another advantage of gcamp is we can utilize these approach combined with two photon microscopy to

Image neurons which are located in a deeper location in the brain now using conventional patch clamp technique it might not be so easy to get access to those deeper neurons but light can access these neurons easily right so obviously the fluorescence light that is coming from those deeper layers can be recorded using a two photon microscope now let’s see how the

Data obtained from gcam recording can be analyzed generally scientists record the delta f by f value and plot it over time so what is delta f by f so it is relative change of fluorescence over baseline fluorescence so delta f is a quantity which says fluorescence at time t minus fluorescence at time 0 and baseline fluorescence is given by f0 so what is these

Parameters so you can see if this is the activity profile of g camp the ft is a particular time point where we have recorded the fluorescence and f0 is basically the baseline fluorescence actually after recording the fluorescence over time scientists can get several time frames like these so here we can see the fluorescence intensity as a function of time so

Fluorescence intensities are shown in each frame which are like 2.5 milliseconds apart and we can see at time f equal to 10 the fluorescence intensity reach its peak so it’s kind of like a peak activity time and again it falls down to near baseline so overall we get any time point and compare it with the baseline fluorescence and thereby we generate these delta

F by f curve now the biggest advantage is we can draw roi or region of interest around specific neurons from a particular live imaging movie and we can calculate the fluorescence over time and that would give us an idea about how the neurons are firing over time period so you here we can see at least the this particular um neuron which is marked by the orange

Traces has three peaks where the violet traces has much more peaks so these kind of informations that means special temporal dynamics of a neuronal activity can be captured using these approach moreover we can also get an idea about the overall circuit activity on the right hand side you can see the circuit is functioning in a synchronous manner that means all

The neurons in these circuits are firing in a synchronous fashion whereas in the left side we can see the neurons in these circuit are functioning or firing at an asynchronous way so they are not firing all together these kind of informations can be obtained using gcam based approach and live imaging approach but what are the disadvantages of this kind of live

Imaging so if we record and image the calcium activity from the same neuron one thing might be very clear that the g camp is a proxy readout for neuronal activity it does not really resolve the neuronal activity in time so much so let’s say this is a fast spiking neuron and it’s burst at a very fast rate and that can be seen from this image now if we record

In a whole cell configuration the voltage fluctuation shows there are like multiple peak of activity burst of activity but that is not nicely resolved in this calcium imaging that means temporal resolution is slightly low in case of gcam now in order to circumvent these kind of problems scientists are now improving gcamp they are making gcamp f which is which

Can possibly capture these kind of fast dynamics so there are also many different type of g cams they have also generated r cams which is in a rfp range so overall in this video we understand how gcamp is a very useful tool for the neuroscientists i hope this video was useful if you like this video give it a big thumbs up don’t forget to like share and subscribe

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Transcribed from video
GCaMP | GCaMP calcium indicator | How GCaMP works? | GCaMP application in neuroscience By Animated biology With arpan