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Measuring depolarizations over the membrane

Measuring depolarizations over the membrane


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Measuring the rest potential of a membrane is relatively easy and straight forward: put one electrode inside the neuron, the other one (the "counter electrode") outside the neuron.

Because everything is at equilibrium (steady state), it's not important where exactly inside the neuron you place the first electrode, and even less important where outside the neuron you place the counter electrode: in the extracellular fluid next to the neuron or inside a container filled with extracellular fluid placed anywhere. One may even connect the other electrode to the ground (a lightning rod), if one does it consistently (over all measurements).

But now consider you want to measure the spread of depolarization along the membrane by measuring the membrane potential at two different locations of the membrane: You place one electrode underneath the membrane at point $p_1$ (and its counter electrode to the ground). And you place another electrode underneath the membrane at point $p_2$ (and its counter electrode to the ground).

Now it seems possibly important where exactly inside the neuron you place the two electrodes: the distance from the membrane might make a significant difference. (But maybe not.)

What probably would make a significant difference: Since the ionic compositions of the extracellular fluid in the vicinity of $p_1$ and $p_2$ will differ, it might be important to connect the counter electrodes not to the ground (or to some volume of normal extracellular fluid) but to corresponding points $p_1'$, $p_2'$ directly opposite to $p_1$, $p_2$ on the other side of the membrane.

I emphasized might be important because in practice it may be not important. That's what my question is for:

Is it OK, even for the measurement of the dynamics of depolarizations, to connect the counter electrodes to the ground (if only consistently). What would be the difference in the measured voltages, and can these be determinstically be cancelled out?


It is extremely important where you put your electrode when measuring such delicate structures as dendrites (which is when you want to compare voltage changes between two parts of a neuron). That is why you put your electrode inside the dendrite. Each recording electrode has its own ground that goes inside the bath, i.e. outside the neural tissue. Alternatively, you can observe fluorescence changes of a calcium or voltage dependent indicator that you have introduced inside them.

https://www.janelia.org/sites/default/files/Labs/Spruston%20Lab/Stuart_Spruston_2015.pdf


Measuring depolarizations over the membrane - Biology

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed a novel, genetically encoded probe that can be used to measure transmembrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive K + channel so that voltage-dependent rearrangements in the K + channel would induce changes in the fluorescence of GFP. The probe has a maximal fractional fluorescence change of 5.1%, making it comparable to some of the best organic voltage-sensitive dyes. Moreover, the fluorescent signal is expanded in time in a way that makes the signal 30-fold easier to detect. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism noninvasively and targeted to specific developmental stages, brain regions, cell types, and subcellular compartments.


The action potential – not so spikey.

The ins and outs of the action potential are taught to every neuroscience undergraduate. Neurons, when excited, generate an action potential, which is propagated along the axon to the nerve terminal, where it causes the quantized release of neurochemical transmitters from vesicles into the synaptic cleft. The transmitter molecules diffuse across the synapse, binding to receptors on the post-synaptic cell and causing it too to generate an action potential.

It was taken for granted that the action potential is all or nothing. In other words, a neuron will only generate an electrical impulse if its membrane is polarized beyond a certain point (the &lsquothreshold&rsquo) otherwise, it will remain in its resting state.

Vertebrate neurons are therefore thought of as something like digital switches they are either on or off, generating action potentials or resting. It was thought that the main determinant of neuronal activity was the frequency of electrical signals received by a cell.

Two recent studies, however, indicate that neurons may also use analogue signalling.

Alle and Geiger and Shu et al now show that neurotransmitter release from the nerve terminal can be influenced by small, sub-threshold fluctuations in the membrane potential at the cell body.

One study looked at mossy fibres in the hippocampus, the other at pyramidal cells in layer 5 of the cortex. Both show that the length over which a sub-threshold membrane event decays as it is propagated from its point of origin is much longer than was previously thought.

Shu et al demonstrate that 10mV depolarizations of the cell body membrane potential can increase transmitter release by up to 30%, probably because of spike broadening (a slower return to resting potential). In the experiments performed by Alle and Geiger, depolarizations of the cell body were shown to increase the amplitude of action potentials produced in the post-synaptic cell.

Physiologists have not, until now, noticed how these graded changes in membrane potential can affect neurotransmitter release. This is mainly because the electrical activity of neurons is usually investigated by using microelectrodes to take extracellular recordings, and this method does not record the sub-threshold fluctuations in membrane potential.

It is well known that invertebrate neurons can release neurotransmitters in response to minor changes in membrane potential. The threshold is however, much closer to resting potential in these cells than in the vertebrate neurons investigated in the current experiments.

These studies show that vertebrate neurons can function like those of 'lower' organisms. Furthermore, the vertebrate neurons in question are involved in the processing of information for complex functions such as memory and cognition. Could it be arrogance that led us to assume that vertebrate neurons function differently from invertebrate neurons?

The basics of brain function, which were thought to be well understood, are actually far more complex than was previously thought. Undoubtedly, there remains much more to be discovered. The mechanism of the action potential was discovered 50 years in a classic set of experiments by Hodgkin and Huxley, who used microelectrodes to measure the movements of electrical charge across the membrane of the giant squid axon in response to electrical stimulation.

Neurons have a low concentration of sodium ions and a high concentration of potassium ions with respect to the outside of the cell. In such a situation, ions tend to move down their concentration gradient (i.e. from an area of high to an area of low concentration). The nerve cell membrane prevents this diffusion, (although there is some leakage), and instead can precisely control the movement of ions in both directions. It is this controlled movement of ions across the nerve cell membrane that underlies the action potential.

In its resting state, a neuron is said to be polarized &ndash that is, the inside of its membrane (the neurolemma) is negatively charged with respect to the outside, due to a high concentration of negatively-charged ions on the inside of the membrane. Textbooks usually give this &lsquoresting potential&rsquo an average value of &ndash70millivolts (9mV).


CENTRAL PATTERN GENERATION OF FORELIMB AND HINDLIMB LOCOMOTOR ACTIVITIES IN THE CAT

Intracellular recording of forelimb motoneurons / Fig. 3 /

Membrane depolarizations of motoneurons, accompanied or not by firing, were related to the variations of activity in the corresponding nerves. They gave more precisions on the timing of locomotor commands, since they revealed the subthreshold excitations which could precede or outlast nerve discharges by 200 msec or more, and thus confirmed that transition periods between F and E bursts have significant durations. In F /n = 10/ and E /n = 20/ motoneurones / Fig 3A , B /, membrane potential variations consisted of one depolarization within each locomotor cycle, with opposite time courses. In motoneurones of shoulder muscles / Fig. 3C,D /, membrane potential changes were more complex. In sSc motoneurones /n = 20/, there were two main depolarizations, one before and the other at the end of the F burst, separated by a clear membrane potential increase during this burst depolarization persisted during the E burst. In T maj motoneurones /n = 10/, variations were opposite: depolarizations occurred during the F burst and at the beginning and the end of the E burst.

Fig. 3 . Intracellular recording of forelimb motoneurons /mn/. Membrane potential calibration: 20 mV.


Cell Biology Chapter 22: Signal Transduction Mechanisms I

One extension is multiple, branched dendrites that receive and integrate electrical signals.

For neuron-to-neuron junctions, synapses occur between an axon and a dendrite, but they can also occur between two dendrites

The resulting electron potential is called resting membrane potential (Vm)

Cells diffuse from high concentration to low concetraion (ex. potassium ion concentration gradient favors outward diffusion)

Electroneutrality - cells in a solution must be in pairs of neutral charge, counterions (K is counterion for anions chlorine is the counterion for sodium)

The very large squid giant axon has been used for studies of nerve transmission since the 1930s

Its large size allows for easy insertion of microelectrodes to measure and control electrical potentials

The resting membrane potential (RMP) can be measured
- Electrodes compare the ratio of negative to positive charge inside and outside the cell
- because inside of plasma membrane is negative there is a negative potential
- The RMP is about -60 mV (millivolts) for the squid giant axon

When a channel is inactivated, it cannot reopen immediately, even if stimulated to do so

Inactivation is caused by part of the channel called the inactivating particle that inserts into the opening of the channel. For channel to reopen, the particle must be removed. Proteases that inhibit the particle causes the channel to remain open.

Epilepsy is a Na+ channel dysfunction. Ataxia (muscle coordination disorder) is a K+ malfunction.

Nernst equation describes relationship between membrane potential and ion concentration

Goldmann equation described combined effects of ions on membrane potential


Small molecule fluorescent voltage indicators for studying membrane potential

Optical voltage sensors promise high-speed tracking of membrane potential in neurons.

Several classes of small molecule voltage indicators exist.

Voltage indicators using electron transfer as a trigger provide speed and sensitivity.

Electron transfer voltage indicators can be tuned across a range of colors.

Photoactivatable voltage indicators improve labeling in heterogeneous system.

Voltage imaging has the potential to unravel the contributions that rapid changes in membrane voltage make to cellular physiology, especially in the context of neuroscience. In particular, small molecule fluorophores are especially attractive because they can, in theory, provide fast and sensitive measurements of membrane potential dynamics. A number of classes of small molecule voltage indicators will be discussed, including dyes with improved two-photon voltage sensing, near infrared optical profiles for use in in vivo applications, and newly developed electron-transfer based indicators, or VoltageFluors, that can be tuned across a range of wavelengths to enable all-optical voltage manipulation and measurement. Limitations and a ‘wish-list’ for voltage indicators will also be discussed.


Toxins and disease

Many natural toxins target ion channels. Examples include the voltage-gated sodium channel blocker tetrodotoxin, which is produced by bacteria resident in puffers (blowfish) and several other organisms the irreversible nicotinic acetylcholine receptor antagonist alpha-bungarotoxin, from the venom of snakes in the genus Bungarus (kraits) and plant-derived alkaloids, such as strychnine and d -tubocurarine, which inhibit the activation of ion channels that are opened by the neurotransmitters glycine and acetylcholine, respectively. In addition, a large number of therapeutic drugs, including local anesthetics, benzodiazepines, and sulfonylurea derivatives, act directly or indirectly to modulate ion channel activity.

Inherited mutations in ion channel genes and in genes encoding proteins that regulate ion channel activity have been implicated in a number of diseases, including ataxia (the inability to coordinate voluntary muscle movements), diabetes mellitus, certain types of epilepsy, and cardiac arrhythmias (irregularities in heartbeat). For example, genetic variations in sodium-selective and potassium-selective channels, or in their associated regulatory subunits, underlie some forms of long-QT syndrome. This syndrome is characterized by a prolongation in the depolarization time-course of cardiac myocyte action potentials, which can lead to fatal arrhythmias. In addition, mutations in adenosine triphosphate (ATP)-sensitive potassium channels that control insulin secretion from cells in the pancreas underlie some forms of diabetes mellitus.


Measuring osmosis and hemolysis of red blood cells

Since the discovery of the composition and structure of the mammalian cell membrane, biologists have had a clearer understanding of how substances enter and exit the cell's interior. The selectively permeable nature of the cell membrane allows the movement of some solutes and prevents the movement of others. This has important consequences for cell volume and the integrity of the cell and, as a result, is of utmost clinical importance, for example in the administration of isotonic intravenous infusions. The concepts of osmolarity and tonicity are often confused by students as impermeant isosmotic solutes such as NaCl are also isotonic however, isosmotic solutes such as urea are actually hypotonic due to the permeant nature of the membrane. By placing red blood cells in solutions of differing osmolarities and tonicities, this experiment demonstrates the effects of osmosis and the resultant changes in cell volume. Using hemoglobin standard solutions, where known concentrations of hemoglobin are produced, the proportion of hemolysis and the effect of this on resultant hematocrit can be estimated. No change in cell volume occurs in isotonic NaCl, and, by placing blood cells in hypotonic NaCl, incomplete hemolysis occurs. By changing the bathing solution to either distilled water or isosmotic urea, complete hemolysis occurs due to their hypotonic effects. With the use of animal blood in this practical, students gain useful experience in handling tissue fluids and calculating dilutions and can appreciate the science behind clinical scenarios.

Keywords: handing tissue fluids hematocrit osmolarity tonicity.


Measuring depolarizations over the membrane - Biology

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Chemical biology is a burgeoning field that has rapidly risen to prominence. This surge of interest has been fuelled by chemical biology’s applicability to understanding critical processes in live cells or model organisms in real time. This success has arisen because chemical biology straddles a nexus between chemistry, biology, and physics. Thus, chemical biology can harness rapid chemistry to observe or perturb biological processes, that are in turn reported using physical assays, all in an otherwise unperturbed living entity. Although its boundaries are endless, the multidisciplinary nature of chemical biology can make the field seem daunting we beg to differ! Here, we deconstruct chemical biology into its core components, and repackage the material. In the process we build up for each student a practical and theoretical knowledge bank that will set these students on their way to understanding and designing their own chemical biology experiments. We will discuss fluorescence as a general language used to read out biological phenomena as diverse as protein localization, membrane tension, surface phenomena, and enzyme activity. We will proceed to discuss protein labeling strategies and fusion protein design. Then we will discuss larger and larger scale chemical biology mechanism and screening efforts. Highlights include a large amount of new data, tailored in the lab videos, and a large number of skilled presenters.

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Amazing course, the best one i enrolled in so far here, very well build and got used to the simple fantastic teaching of Dr. Marcus Long, glad to have spent time studying this topic

Very challenging yet interesting topic. This is 21st century science towards revolutionizing Systems Biology and other science fields

A ruler over time and space! Fluorescent assays to measure complex parameters in real time

Here we will discuss how different fluorescent techniques have found fantastically useful applications to understand specific biological regulation processes in vitro and in live cells. We will focus on chromatin regulation and regulation of membrane tension as these are two systems which, without such techniques, we would not have the level of understanding we have today.

Преподаватели

Robbie Loewith

Marcus J. C. Long

Текст видео

Membranes are made of lipids and proteins tightly packed together through hydrophobic interactions. This packing can evolve in many different ways. In particular, lipid tension can generate more spread lipids. As you can see in the darker part of this membrane, the lipids are tightly apposed together. These lipids are representing an area of low tension. In the clear part of the membrane, you see that the lipids are more spread apart and this is an area of high tension. How can we think about this? The lipid membrane are closely put together through cohesive hydrophobic forces from the acyl (fatty acid) chain that pack the lipid together. These forces are globally invariant through the membrane, so there must be another force that will spread the lipids apart. This is tension. This tension essentially combats the hydrophobic forces of the membrane to spread lipids apart. The question is, how can we measure this tension? The classical technique of measuring membrane tension is shown here. It is a mechanical technique where a thin tube of membrane is pulled out of the cell plasma membrane using a bead in an optical trap. This bead is actually coated with a reagent, in this case, concanavalin A that binds to the plasma membrane of the cell. When hold into the trap and pulled apart, it extracts a small tube from the plasma membrane and the tube resist the displacement of the bead by applying a force that is proportional to the bead displacement in the trap. Now, that provides a direct way of measuring tension through the equation that you see here where B is the bending rigidity, the resistance to bending, T, the tension, and F, the force we measured., Now, this technique is very classic, but has some caveats and to explain a bit better, the caveats, I first want to show you one picture of such an experiment where you can see the bead, on the top left corner, the tube that is seen in the fluorescence image and the cell that is a globally fluorescent. This is technically challenging to extract such a small tube from such a small cell with such a small bead. The first caveat is that it's technically challenging to achieve one measurement. The second caveat is that it needs best specialist equipment because you need an optical trap and needs not common thing to have in the lab. The third caveat is that it's very low throughput because you will need a few minutes to pull one tube and it will take a few hours to get tens of measurements as statistically required. Now, the last caveat is that it's not very good for time-life measurements just because the time resolution is pretty poor. One question we ask is, can we find a more versatile method to measure tension? That said, we thought of, can we measure tension using a molecule that would be sensitive to lipid packing, knowing that lipid packing is affected by membrane tension? Such a molecule is shown here, and it's composed of what we call flipper, which is essentially two aromatic systems. One electron-rich, which is shown in yellow, one electron-poor, which is shown in red and when they are flat, they are conjugated, and as we have seen from Module 2, this provides a typical fluorescent dye where fluorescence will be emitted through the electrons transfer from the yellow system to the red system. This provides a red absorption and a fairly long lifetime of fluorescence. What is interesting in this molecule is because of the single bond that binds the two aromatic system, they can twist and in the twisted conformation, you uncoupled the two systems and they are not conjugated anymore. In this case, they fluoresce independently, and in this case, you get a blue-shifted absorption and shorter lifetime. Now, the question is, what is this molecule bring you? Well, first it's bringing two conformations from the same molecule with different fluorescence parameter that can eventually change with time. The question is, how can membrane tension affect the confirmation of this molecule? Knowing this, we have to ask the question, how does lipid packing will change the conformation of those molecules? Now, if you consider those two conformation independently, you can see that the flat conformation requires relatively low space because it's non-twisted. On the contrary, the twisted confirmation will require more space just because the lipid groups will be spread apart. In the low tension case, in a membrane with low tension where lipids are tightly packed, the flat conformation will be favored and on average, all the molecules will be flattened. On the contrary, in high tension membrane where lipids are spread apart, there will be more space for the molecule to twist, and it will acquire more often this conformation. That gives a direct link between tension, conformation of the molecule and its fluorescent properties. We expect as shown here, that in low tension conditions, we will have a long lifetime and in high tension conditions we would have a short lifetime. What does this bring to membrane tension measurement? Well, it brings technique that will be useful for everybody in every lab because we use microscope and microscope is a common technique in every Biology lab. Second, it's much more high throughput because we can measure many cells at the same time and we can then increase dramatically the statistics of our measurements, and third, it has very good time resolution because all measurements are done through light emission. Now, I've shown you classical and modern techniques of membrane tension measurement and in the next lectures, you will see how modern techniques can be put into action in different ways. [MUSIC]


Talk Overview

This lecture about photobleaching and photoactivation describes how fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) and photoactivation of fluorophores can provide information on the dynamics of molecules in cells. Uses of these techniques include studying the movement of proteins through membrane compartments, the diffusional behavior of molecules in the cytosol and membranes, the dynamics of cytoskeletal polymers, and protein turnover.

Questions

  1. Which technique would be most suited for quantitating protein turnover?
    1. Two photon fluorescence microscopy
    2. Photoactivation
    3. Fluorescence Recovery After Photobleaching (FRAP)
    4. Fluorescence Loss In Photobleaching (FLIP)
    1. Two photon fluorescence microscopy
    2. Photoactivation
    3. Fluorescence Recovery After Photobleaching (FRAP)
    4. Fluorescence Loss In Photobleaching (FLIP)
    1. Exposure with infra-red light converts from red to green fluorescence
    2. Reversible on-off fluorescence with cycles of light
    3. Exposure with blue light converts from green to red fluorescence
    4. Exposure to blue light shifts the absorption spectrum
    1. The turnover of the protein is high
    2. Active transport mechanisms are occurring as well as diffusion
    3. A fraction of the bleached molecules are immobile and non-exchangeable
    4. A fraction of the fluorescence has photoconverted to another wavelength

    Answers


    Experiments on Osmosis (With Diagram)

    The below mentioned article includes a list of four simple experiments on osmosis.

    1. Experiment to demonstrate the osmosis by using sheet of cellophane or goat bladder:

    Beaker, thistle funnel, goat bladder or sheet of cellophane, thread, water and sugar solution.

    1. Cover the lower opening of the glass tube with the goat bladder or sheet of cellophane and tie it with the thread.

    2. Fill in the interior of the tube with molasses, a concentrated sugar solution in water.

    3. Place the whole apparatus in a beaker containing water, preferably distilled water.

    4. Note the level of the water in the thistle funnel and keep the apparatus to note the results.

    Level of the water in the thistle funnel increases (Fig.2).

    1. Movement of water through the goat bladder or cellophane sheet into the thistle funnel takes place.

    2. Water concentration in beaker is 100% while in the sugar solution it is less than this, and, therefore, the water from the region of higher concentration moves towards the region of lower concentration. The movement is through a semipermeable membrane and so the experiment shows the phenomenon of osmosis.

    3. The force, with which the solution level in the tube increases, arises from the pressure exerted by the diffusion of water molecules into the tube. This pressure is called osmotic pressure.

    4. Stability of the water level in the funnel indicates that water concentration in both the beakers as well as funnel is same and thus osmosis stops.

    2. Experiment to demonstrate osmosis with the help of potato osmometer:

    Petri-dish, water, potato, sugar solution, cork and capillary tube.

    1. Take a potato tuber, remove its outer covering from one end and cut the same end flat.

    2. Scoop out a cavity from the other end of the tuber running almost upto the bottom.

    3. Fill the cavity with the sugar solution and fit an airtight cork fitted with a capillary tube on the upper end of the cavity (fig. 3).

    4. Place the capillary- fitted potato tuber in the water- filled petri-dish.

    5. Mark the solution level in the tube and watch the experiment for some time.

    After some time the level of the solution in the tube increases. Mark the level of solution when it stops to move.

    The level in the capillary tube increases because of the fact that osmotic pressure of the sugar solution is higher than that of the water, and the water moves through the semipermeable membrane of potato from petri-dish into the cavity. So the experiment shows that phenomenon of osmosis.

    3. Experiment to demonstrate the osmosis by the egg osmometer:

    Egg membrane, dilute HCI, water through, graduated tube, sugar solution and stand.

    1. Prepare an egg membrane by carefully removing waterproof shell of egg with the help of dissolving it away in dilute HCI.

    2. Remove all the fat and protein-containing yellow material of the egg by making a hole on its one end.

    3. Fill the sugar solution in the egg membrane through the hole and fit a graduated tube in the hole.

    4. Place the complete apparatus in a water-filled trough (Fig. 4).

    5. Note the level of sugar solution in the graduated tube and keep the apparatus undisturbed for some time.

    Level of the sugar solution increases in the tube.

    The level in the tube increases because of the fact that osmotic pressure of the sugar solution in the egg membrane is higher than that of water, and so the water from the trough passes through the egg membrane into the sugar solution thus increasing its level. Egg membrane is a semipermeable membrane.

    4. Experiment to demonstrate the phenomenon of exosmosis and endosmosis:

    Potato tubers (2), knife, conc. sugar solution, water, pin, beakers (2).

    1. Remove the outer skin of the tubers and cut their one end flat with a sharp knife.

    2. Scoop out a cavity from the other end of the tuber running almost upto the bottom as in experiment No. 14.

    3. Fill the concentrated solution of sugar in the cavity of one tuber, and water in the other.

    4. Mark the level of the sugar solution and water in the cavities with the help of pins.

    5. Place the potato containing sugar solution in a beaker containing water, and the another potato containing water in its cavity in the beaker containing sugar solution (Fig. 5).

    6. Keep and observe experiment for some time.

    The level in the cavity containing sugar solution increases while the level decreases in the another tuber, i.e., in the cavity filled with water.

    The level of the sugar solution in the first tuber increases because of the fact that water moves from the beaker into the cavity through the semipermeable membrane of potato. Thus it shows the phenomenon of endosmosis.

    The level of the water in the second tuber decreases because of the fact that water moves from the cavity into the beaker through the semipermeable membrane of potato tuber. Thus it shows the phenomenon of exosmosis.