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What are negative and positive after potentials?

What are negative and positive after potentials?


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After depolarization: is the slow repolarization phase which follows a rapid fall in spike potential and extends up to attainment of the RMP level. It is called phase of negative after potential and lasts for about 4 ms.

After reaching the resting level (-70 mV) the potential further falls and becomes more negative (-72 mV).This phase is called after hyperpolarization or phase of positive after potential.

What is the reason for naming them as negative and positive after potentials?

My attempt: I think we are naming them based on the direction of current, i.e. during after depolarisation membrane potential is going further negative , so the name negative after potential, and during after hyper polarisation, membrane potential is going from hyper polarised state to resting state, so its named as positive after potential.

Am I correct?


Source:Textbook of physiology


Yes, you are correct. The negative current is hyperpolarising the axon and the positive current is returning it to baseline from a hyperpolarised state.

They are often referred to as inward/outward (negative/positive respectively) currents as well, due to the direction of the flow of ions. Which is further compounded when you start looking positive/negative ions.

Inward currents are inflow of +ve charge, or outflow of -ve charge. Outward currents are vice-versa.


Positive and Negative Afterimages

Sean is a fact checker and researcher with experience in sociology and field research.

An afterimage is a type of optical illusion in which an image continues to appear briefly even after exposure to the actual image has ended.   You have probably noticed this effect a number of times.

If you have ever stared for a long time at a fixed point and then suddenly shifted your gaze somewhere else, then you probably noticed a brief afterimage effect in which you continued to see the original stimulus. Learn more about what afterimages are and why they happen.


AFTER-CURRENTS, AFTER-POTENTIALS, EXCITABILITY, AND VENTRAL ROOT ELECTROTONUS IN SPINAL MOTONEURONS

The spinal cord constitutes a volume conductor. Potential changes are recorded therefrom only as current flows. During the period of the after-potentials current flows in significant density only if the after-polarization differs at different points of the active neurons. Thus one does not record after-potentials in volume one may record after-currents which are defined as the resultants of differences in after-potentials. Measurable excitability change during the period of the after-potentials, in the event no current flows, might be regarded as approximating the change of intrinsic polarization status at the region tested. In the presence of after-current flow excitability change would approximate the sum of intrinsic change and extrinsic change due to current flow. In giving rise by differences to current flow after-potentials come to act as agents, and events in one part of a neuron help to determine excitability in other parts. Since the intramedullary after-current flow is not the after-potential of the soma, it follows that ventral root electrotonus which results from axonal after-current flow cannot be considered the counterpart of somatic after-potential. Following conduction of an antidromic volley after-current flows between somata and axons. According to the signs of the recorded potential changes, after-current flow initially, and for approximately 45 msec., is in the direction from somata to axons. Thereafter, and for approximately another 75 msec., the direction of flow is reversed. During the period of after-current flow following antidromic conduction the excitability of neighboring motoneurons is altered in a manner that reproduces the phases of after-current flow. The initial phase, depression, was first described by Renshaw. The after-potentials of ventral root fibers have been studied. In a single action and in usual form, they consist of a negative after-potential of considerable magnitude and of some 35 msec. duration, and a positive after-potential detectable for approximately 120 msec. Variants and the influence of temperature change are described. The recovery cycle of ventral root axons in general compares with the after-potential cycle. Recovery of intramedullary motor axons differs from that of their extramedullary projections as ventral root fibers in a manner that is accountable to intramedullary flow of after-current. Since the intrinsic recovery process of the motoneuron somata cannot be measured in the presence of current flow it must be estimated by correcting the observed recovery for the influence of known current flows. When this is done the resultant in simplest form provides for intrinsic somatic recovery from refractoriness through a single phase of subnormality lasting some 60 msec. Conditions for the relatively undistorted recording of antidromic ventral root electrotonus are described. They include provisions that the proximal ventral root electrode must be within 12 mm. of the root-cord junction and that the distal electrode must be located in excess of 30 mm. from the distal severed end of the ventral root. Antidromic ventral root electrotonus is a counterpart of the current flows in the intramedullary stretch of the axons. Initially, during the phase of metadromal postivity of the intramedullary axons, electrotonus is negative. During the period of deflections Sp-An, that signify after-current flow into the axons, electrotonus is positive. Finally during the period of deflections Sn-Ap, that signify after-current flow outwards through the intramedullary axon membranes, electrotonus is negative. Electrotonic showing is not of sufficient magnitude to make the time course of ventral root electrotonus palpably different from that of the generating intramedullary currents.


MATERIALS AND METHODS

Cells, Reagents, and Plasmids

Human embryonic kidney (HEK) 293 T-cells were obtained from ATCC (Manassas, VA) and maintained in DMEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% Cosmic Calf Serum (Hyclone Laboratories, Logan, UT), 1% penicillin/streptomycin, 1% l -glutamine, and 100 μg/ml genicitin. HAb2 cells were grown in the same medium as HEK 293T cells, except genicitin was omitted. Huh7.5 cells were provided by Dr. C. Rice (Rockefeller University, New York, NY) and grown in DMEM supplemented with 10% fetal bovine serum (Hyclone Laboratories), 1% nonessential amino acids, and 1% penicillin/streptomycin. Poly- l -lysine, chlorpromazine (CPZ), and n-propyl gallate were purchased from Sigma Chemical Co. (St. Louis, MO). The fluorescent lipophilic dye, DiD, was purchased from Invitrogen (Carlsbad, CA).

Vectors expressing murine leukemia virus (MLV) Gag-Pol, and Gag-green fluorescent protein (GFP) were kindly provided by Dr. W. Mothes (Yale University, New Haven, CT). The SFV-pCB3-wt vector to express SFV E1/E2 was provided by Dr. M. Kielian (Albert Einstein College of Medicine, Bronx, NY) the pCDNA VEEd5 2 vector for VEEV E was provided by Dr. R. Davey (University of Texas Medical Branch, Galveston, TX) pHEF-VSVG was provided by the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program.

Electrical Measurements of Cell–Cell Fusion

As effector cells, we transfected HEK 293T cells by a standard calcium phosphate method to express SFV E1/E2, Venezuelan Encephalitis Virus (VEEV E), or Vesicular Stomatitis Virus (VSV G) (Samsonov et al., 2002). HAb2 cells were used as the target for the SFV and VEEV effector cells. Huh-7.5 cells, a human hepatoma cell line (Blight et al., 2003), were used as target cells for VSV G. Effector cells were loaded with CaAM (1.3 μM) target cells were not labeled. They were thus easily distinguishable by fluorescence. Effector and target cells were mixed and placed into the experimental chamber. The bathing solution was (in mM) 150 N-methylglucamine aspartate, 5 MgCl2, and 2 Cs-HEPES, pH 7.2. A target cell bound to a single effector cell was identified and patch clamped (Axopatch 200A, Axon Instruments, Foster City, CA) in whole cell configuration voltage was set at either −40 or +40 mV, and fusion pore formation and growth were determined by capacitance measurements as previously described (Markosyan et al., 2007). Fusion between selected effector and target cell pairs was triggered by using a closely positioned micropipette to locally apply a small volume of an acidic solution (pH 5.7) for 1 min around the cell pair. This micropipette contained four separate channels (150-μm diameter for the entire pipette Bioscience Tools, San Diego, CA), allowing up to four different solutions to be applied to the selected cells.) Using a focused IR laser, fusion was triggered by quickly raising temperature to 32°C for SFV E1/E2 and 37°C for VEEV E and VSV G (see section Virus-Cell Fusion for temperature-control details).

Determination of Infectivity

To optimize the choice of target cell for each type of pseudovirus, infectivity titers were determined for several different target cells. For infectivity measurements, pseudovirus was prepared by a standard calcium phosphate method, transfecting 293T cells with a plasmid containing the gene for the fusion protein of SFV, VEEV, or VSV and a plasmid containing a human immunodeficiency virus (HIV) gene in which a firefly luciferase gene was placed in the nef position as described (Connor et al., 1995 He et al., 1995 HIV luc, obtained from the NIH AIDS Research and Reference Reagent Program). The two plasmids were added in a 1:1 ratio, 3 μg of each, to the cells in 35-mm dishes. Two days after transfection, the media (which contained the pseudotyped virions) were collected, diluted 1:10, and added to target cells. After allowing infection to proceed for 2 d, cells were lysed, and luciferase activity was measured using a Luciferase Assay System (Promega, Madison, WI) and a plate reader (Wallac 1420 multilabel counter, Perkin Elmer-Cetus, Boston, MA). For each of the three fusion proteins, the type of target cell that exhibited the highest luciferase activity was used for the study of voltage dependence of fusion of pseudovirus to cells.

Preparation of Virus for Fusion Experiments

Pseudotyped viral particles were prepared with either SFV E1/E2, VEEV E, or VSV G as the fusion protein within the envelope, and with MLV Gag-Pol plus Gag-GFP as the core. Explicitly, these pseudovirions were prepared by cotransfecting, with a calcium phosphate method, 293T cells (60–70% confluent in 10-cm culture dishes) with MLV Gag-Pol (18.5 μg), MLV Gag-GFP (6.25 μg), and the plasmid containing a fusion protein (25 μg). [MLV Gag-GFP is cleaved, within the pseudovirus, into the smaller (∼37 kDa) nucleocapsid-GFP (Markosyan et al., 2005 Miyauchi et al., 2009).] One day after transfection, the membranes of the 293T cells were labeled with DiD, a fluorescent lipid, by incubating the cells for 3 h at 37°C in Opti-MEM (GIBCO-BRL) containing 2.5 μM DiD. The cells were washed and returned to their regular growth media. On the second day of transfection, the media were collected, centrifuged, and passed through a 0.45-μm filter. This material was separated into small aliquots and stored at −80°C. Each aliquot was thawed only once. For typical SFV E1/E2 and VSV G viral preparations, ∼30% of the labeled virions were double-labeled the other 70% of the fluorescent particles displayed only one of the dyes. For VEEV E, ∼15–20% of the virions were double-labeled.

Virus-Cell Fusion

Strategies and procedures similar to those previously described were used to monitor fusion of pseudovirions to target cells (Markosyan et al., 2005). Pseudoviruses containing the desired fusion protein, DiD within the envelope, and a GFP-tagged MLV Gag core were adhered to polylysine-coated cover slips by a 2-h spinoculation at 4°C. After washing off unbound virus, target cells were added and allowed to adhere on top of the pseudovirions at 15°C for ∼30 min. Typically, each cell was in contact with ∼5–10 virions that were labeled by both DiD and GFP. This arrangement immobilizes the pseudovirions and places them within a single plane of focus, allowing unambiguous identification of individual virions and their fusion. A coverslip was rested upon heat-adsorbing (i.e., IR adsorbing) glass that formed the bottom of the experimental chamber. The chamber was placed on the stage of the microscope. The solution within the experimental chamber was maintained at 12°C by Peltier control (20/20 Technology, Wilmington, NC). It consisted of (in mM) 150 N-methylglucamine aspartate, 5 MgCl2, 2 Cs-HEPES (pH 7.2), and 1 n-propyl gallate in order to reduce photobleaching. Patch pipettes were filled (in mM) with 155 Cs glutamate, 5 MgCl2, 5 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, and 10 Cs HEPES (pH 7.4). A 30-s pulse of a pH 5.7 solution (also maintained at ∼12°C) was delivered, through one channel of the four-channel pipette, immediately above the cells. By this means, the neutral pH solution was transiently displaced by an acidic one. Fusion was triggered by illuminating the cells with an IR laser. As described (Melikyan et al., 2000a), the laser illuminated an ∼300-μm-diameter region of the heat-adsorbing glass and thereby raised the temperature of the solution surrounding the cells of interest. Through computer-controlled feedback, temperature was raised to 37°C within 2 s, as measured by an immersed miniature thermistor. We define time = 0 as the moment the temperature reached 37°C and refer to this time as the moment of fusion activation.

Virus-cell fusion was visualized with a laser scanning confocal Fluoview 300 microscope (Olympus America, Melville, NY), using an UPlanApo 60×/1.20 NA water-immersion objective. Gag-GFP and DiD were excited simultaneously with a 488-nm argon and a 632-nm HeNe laser, respectively. Images were collected at 10 s/frame and later were analyzed offline. All cell-bound fluorescent particles were visually inspected, and virions that were labeled by both fluorescent dyes were analyzed. Virions that fused (i.e., released DiD and GFP), only hemifused (i.e., released only DiD), or did not respond to a manipulation were separated into groups for analyses. For pseudovirus containing the fusion protein of SFV, we used HEK 293T cells (rather than HAb2 cells as in cell–cell fusion) as target for virus containing the fusion protein of VEEV and VSV, Huh-7.5 cells were used as target. Lower extents of cell–cell fusion and pseudovirus-cell fusion occurred for VEEV E. Expression levels of this fusion protein may be lower than those for SFV E1/E2 and VSV G.

Creating the Cold-arrested Stage

The fusion intermediate of CAS (cold arrested stage) was created by locally applying a pH 5.7 solution around cells (bound to virions) for 30 s at 12°C, followed by a reneutralization to pH 7.4, also at 12°C. After creating CAS, conditions were altered at varied times, as indicated. The alterations were raising of temperature to 37°C to induce fusion, adding CPZ, and changing the polarity of the holding potential across the target cell membrane. CPZ was locally applied to the cells of interest through a channel of the pipette.

Statistical Analysis

Histograms of DiD spread under different conditions (see Figure 5) were compared using a χ 2 -analysis. For each of the 12 histograms (three viruses, four histograms per virus, each displaying hemifusion or fusion for +40 or −40 mV), the first 15 time frames contained almost all the events and these frames were used for comparisons. To compare two different histograms, a 2 × 15 table (two conditions, 15 time frames) was constructed and the χ 2 value was calculated. A Holm-Bonferroni test was applied (to avoid biases that would otherwise be introduced because multiple pairs of histograms were compared) to obtain the statistical significance, p, at which the null hypothesis could be rejected. The null hypothesis was always taken as statistical equivalence of two histograms and was only rejected if p < 0.05.

Time courses for DiD spread from individual virions at +40 and −40 mV were compared by using the mean fluorescence profiles of individual virions. Each profile was generated by first aligning, for each virus and condition, the zero time points, defined as the last frame before commencement of the decrease in fluorescence. For each type of virion that hemifused, the aligned fluorescence intensities of individual virions were averaged over the first 18 frames to yield the fluorescence profile. For SFV E1/E2 and VSV G, the first 50 virions, as arranged chronologically, that hemifused for each voltage were used to generate the profile. The data set was smaller for VEEV E at positive potentials, and here all 44 hemifused virions were used for the analysis. To compare a +40 and −40 mV fluorescence profile, a t test was applied to pairs of points at each time, employing a Holm-Bonferroni procedure to provide a multicomparison correction. The 0.05% confidence interval for the mean difference between the time profiles was calculated. All statistical analyses were performed with MatLab 7.0.1 (MathWorks, Natick, MA).


Membrane Potential across the Cell Membrane: 3 Types | Biology

Membrane potential is classified as: 1. Resting Membrane Potential 2. Action Potential 3. Graded Potential.

All cells in animal body tissue are electrically polarized, in other words they maintain a voltage difference across the plasma membrane known as membrane potential. The cell membrane acts as a barrier that prevents intracellular fluid from mixing with the extracellular fluid. Therefore, the electrical potential difference results from a complex interplay between protein structures embedded in the membrane called ion pumps and ion channels.

Type # 1. Genesis of Resting Membrane Potential:

The membrane potential across the cell membrane when the cell is at rest is called resting membrane potential (RMP). We always express RMP by comparing ICF potential to ECF potential keeping ECF potential to be zero. For example: RMP for large nerve fibre is –90 mV. That is the potential inside the fiber is 90 millivolts more negative than the ECF potential. The nerve cell at this state is said to be in polarized state.

RMP in a cell is generated due to two reasons:

i. Contribution of Simple Diffusion to the Genesis of RMP:

Simple diffusion through protein channels like sodium and potassium channels, which allows movement down the concentration gradient, is influenced by factors such as size, charge on surface of protein, hydration of the ion, etc.

Biophysical basis for membrane potential is caused by simple diffusion alone.

The Gibbs-Donnan effect (also known as the Donnan effect, Donnan law, Donnan equilibrium, or Gibbs-Donnan equilibrium) is a name for the behavior of charged particles near a semipermeable membrane which sometimes fail to distribute evenly across the two sides of the membrane. The usual cause is the presence of a different charged substance that is unable to pass through the membrane and thus creates an uneven electrical charge.

In the body, it is the Gibbs-Donnan effect of intracellular negatively charged protein forms the basis of the negative resting membrane potential. If it was not for the electrogenic activity of Na/K ATPases the resting membrane potential would be even more negative. This forms the basis for equilibrium potential of ions.

b. Equilibrium Potential and Nernst Equation:

Particular ion will flow across a membrane from the higher concentration to the lower concentration (down a concentration gradient), causing a current. However, this creates a voltage difference across the membrane that opposes the movement of ions. When this voltage reaches the equilibrium value, the two balances (concentration gradient and the voltage) and the flow of ion stops.

The voltage at which the flow of the ion stops is called the equilibrium potential of that ion. The equilibrium potential for any ion can be calculated by an equation called Nernst equation. Equilibrium potential for potassium is –94 mV equilibrium potential for sodium is +61 mV.

Equation for calculating the equilibrium potential for potassium is as follows:

Eeq K + = RT In [K + ]o/zF [K + ]i

i. Eeq K + is the equilibrium potential for potassium in volts

iii. T is the absolute temperature

iv. Z is the number of elementary charge of the ion

vi. [K + ] o is ECF concentration of potassium

vii. [K + ] i is ICF concentration of potassium

viii. RT is a constant and the value is calculated as 61 and formula can be written as-

c. Goldman-Hodgkin-Katz Equation:

If membrane is permeable to only one ion, the membrane potential is the equilibrium potential of that ion. But in reality, animal cell is permeable to many ions. So the membrane potential has to be calculated taking equilibrium potential of all ions into consideration.

Hence, membrane potential depends on:

i. Polarity of electric charge of each ion

ii. Permeability of membrane

iii. Concentration of ion in ICF and ECF.

The major ions involved in generating membrane potential are sodium, potassium and chloride. Membrane potential can be calculated from Goldman-Hodgkin-Katz equation ―

Where P is permeability of the cell membrane to the ion. Due to the negative charge of chloride ion, the concentration of chloride in ECF is written in the numerator.

At rest the cell membrane is 100 times more permeable to potassium diffusion than sodium because the hydrated form of potassium is smaller in size compared to that of hydrated form of sodium ion. This is reason why RMP will be close to equilibrium potential of potassium.

ii. Contribution of Sodium Potassium Pump for the Genesis of RMP:

It is an electrogenic pump which pumps three Na + to ECF and two K + to ICF against concentration gradient leaving a net deficient of one positive ion on the inside, which causes a negative voltage inside the cell membrane.

Let us calculate the RMP of the large nerve fiber:

i. The resting membrane potential of large nerve fiber, if potassium alone is considered as permeable is –94 mV which is calculated from Nernst equation.

ii. The RMP if sodium alone is considered will be +61 mV.

iii. But actually RMP is due to both sodium and potassium which can be calculated from Goldman equation to be –86 mV, which is nearest to equilibrium potential of potassium.

iv. But still we have –4 mV left to get –90 mV as RMP in large nerve fiber. What it is due to? It is due to the sodium potassium pump which leaves negativity inside the cell contributing to –4 mV by pumping an extra positive charge outside the cell.

v. So totally –86 mV and –4 mV negativity inside the cell is due to simple diffusion and active transport respectively which contributes to –90 mV RMP in large nerve fiber.

For calculating RMP in a muscle, calcium ion should also be considered for calculating Goldman equation (Fig. 2.18).

Type # 2. Action Potential (AP):

An action potential is a short lasting event in which the electrical membrane potential of cell rapidly rises and fall after sufficient strength of a stimulus is applied (during action). This occurs in excitable cell like neu­rons and muscle cell. In neurons, they play a central role in cell to cell communication (Fig. 2.19). In muscle cell, AP is the first step in chain of events leading to contraction.

The stages of action potential:

I. Resting (Polarized) Stage:

This is the membrane potential before the stimulus, i.e. RMP and the membrane is said to be polarized.

II. Depolarization Stage:

After the sufficient stimulus to an excitable cell, there occurs a change in voltage which opens the voltage gated sodium channel, (Sodium permeability is more than potassium during action in contrast to what it is at rest) allow­ing tremendous flow of positively charged sodium ions through voltage gated sodium channels by simple diffusion to the interior of cell.

The membrane potential which was negative compared with outside, now rapidly shifts to positive side due to flow of positive charge sodium ion. This shift from negative to positive potential is called as depolariza­tion (i.e. polarized to depolarized state).

III. Repolarization Stage:

The gated channel is open only 1/10000th of a second. Within a few 10000th of a second the sodium channels begin to close. But the voltage for opening potassium channel is attained, and therefore, voltage gated potassium channel opens. Since K + is more inside, the potassium flows tremendously to the outside of the cell (Fig. 2.20). This rapid diffusion of K + which is also a positive charge to outside of the cell, re-establishes the negative RMP. This stage is called repolarization stage. After a fraction of second the gated potassium channel closes.

The ionic basis with an example of action potential in a large nerve fiber is explained as:

RMP nerve fibre is –90 mV (resting stage) → Sufficient strength of a stimulus is applied → Opening of voltage-gated sodium channel → Sodium flows slowly from outside to inside through these channels thereby slowly decreasing the negativity inside the cell to –50 mV to –70 mV (threshold potential) → Once firing potential or threshold potential is reached, more voltage gated sodium channels is recruited which causes rapid inflow of sodium (sodium permeability increases by 500 to 5000 times) → The voltage inside the cell shoots up to +35 mV. This is depolarization stage → Inactivation of sodium gate at +35 mV occurs, but this is voltage required for opening the potassium gates → Potassium pours from inside to outside through these channel because ICF potassium is more than ECF concentration of potassium. There is rapid fall of potential. This event brings back the potential towards negativity. This is repolarization stage (sharp rise and fall of potential is called spike potential) → At the end there is slow fall of potential towards RMP called after depolarization → Potassium channel is slow to close, so there is extra outflow of the potassium ion to the ECF → More negativity than RMP is created called after hyperpolarization → Here comes Na + K + pump which is re-establish RMP

There will be some extra flow of K + ions outside the cell because they are very slow to close. This causes after hyperpolarization. The gates will not reopen until the membrane potential returns to the original RMP. This is possible only with the help of Na + K + pump which helps to re-establish the RMP.

Propagation of Action Potential:

An action potential elicited at any one point on an excitable membrane usually excites the adjacent portion of the membrane, resulting in propagation of action potential over the membrane. The local change in potential is carried inwards to several millimeter of adjacent membrane in both the directions, which slowly opens more Na + channel. This newly depolarized area in the same manner propagates the action potential. This transmission of depolarization process is called an impulse.

Saltatory Conduction:

The need for fast transmission of electrical signals in nervous system results in myelination of neuronal axons. Myelin sheath around axons is separated by intervals known as nodes of Ranvier. In saltatory conduction, an action potential at one node of Ranvier caused inward current that depolarize the membrane at the next node, provoking a new action potential, the AP hops from node to node because myelin offers resistance in internodal intervals.

The significance of this is that:

i. The conduction of AP is faster.

ii. The energy is conserved, which otherwise requires lot of energy if it travels through the entire neuron.

Properties of Action Potential:

It states that a threshold or sub-thre­shold stimulus is capable of producing action potential will produce the maximum possible amplitude of action potential or will not produce an action potential if the stimulus is sub-threshold. In other words, large strength do not create large action potential, therefore, action potential are said to be all-or-none.

It is the period during which excitability of excitable tissue to second stimulus is decreased.

i. Absolute Refractory Period (ARP):

It is the period during which even a second strongest stimulus cannot produce an action potential. The period extends from firing level to one-third of repolarization stage of first action potential. This is responsible for unidirectional conduction of action potential in axon.

Reason ― Inactivation of sodium gates.

ii. Relative Refractory Period (RRP):

It is the period during which a stronger than normal stimulus can produce action potential. It extends from one-third of repolarisation stage to after depolari­zation.

Reason ― The excitability is increased but threshold is decreased, so you need stronger stimulus.

III. Strength-Duration Curve:

This is a curve plotted to show the relationship of action potential with strength of a stimulus and duration of stimulus. A stimulus must be of adequate intensity and dura­tion to evoke a response. If it is too short, even a strong pulse will not be effective. A long pulse below certain strength will evoke only a local non- propagated response.

It is the minimum threshold strength which can pro­duce an action potential.

It is duration for which twice the rheobase strength has to be applied to produce an action potential. It is the measure of excitability of the tissue. Chronaxie is inversely proportional to excitability.

Variations in Action Potential in Other Tissues:

I. Plateau in Action Potential:

For example, in cardiac muscle. This type action potential happens in cardiac muscle.

b. Depolarization Phase:

Due to rapid sodium influx through voltage-gated sodium channel which is same as nerve action potential, but it is followed by plateau phase.

The membrane is held at high voltage for a few milliseconds prior to being repolarized.

This is due to two reasons:

(i) Voltage-gated calcium sodium channel, which is slow to open causing slow inflow of calcium and sodium,

(ii) Voltage-gated potassium channel are slow to open which causes slow outflow of potassium, and therefore, potential remains in positivity for some time. This delays the return of membrane potential to resting level.

d. Repolarization Phase:

Due to rapid outflow of potassium through K + channels and closing of slow calcium sodium channel which returns the action potential to resting level.

II. Pacemaker Potential:

For example ― cardiac pace­maker and smooth muscle (Fig. 2.25).

The cardiac pacemaker cells of the sinoatrial node in the heart provide a good example. They have self-induced rhythmical action potential without any stimulus. This is due to spontaneous excit­ability.

This is attributed to two reasons:

a. The resting membrane potential of pacemaker cells is between -55 mV to -60 mV. It is very close to the threshold potential which makes the cells to depolarize easily.

b. The resting sinoatrial nodal cells have sodium leaking channel called funny channels that are already open to sodium. Without any stimulus, the sodium leak to the inside of the pacemaker cell causes a slow rising RMP between heart beats, this slow rising membrane potential when reaches -40 mV attains threshold for firing due to rapid entry of sodium and calcium ions at -40 mV. After repolarization the cycle continues. Thus the inherent leaking of sodium ion causes self-excitation.

Type # 3. Graded Potential:

It is the localized change (depolarization or hyper-polarization) in the potential difference across a cell surface membrane. Strength of the graded potential varies with the intensity of the stimulus and causes local flows of current which decrease with distance from the stimulus point (Fig. 2.26).

Graded potentials are given different names according to their function (Fig. 2.27). The membrane potential at any point in a cell’s membrane is determined by the ion concentration differences between the intracellular and extracellular areas and by the permeability of the membrane to each type of ion.

The ion concentrations do not normally change very quickly (with the exception of calcium, where the baseline intracellular concentration is so low that even a small inflow may increase it by orders of magnitude), but the permeability can change in a fraction of a millisecond, as a result of activation of ligand-gated or voltage-gated ion channels.

The change in membrane potential can be large or small, depending on how many ion channels are activated and what type they are. Changes of this type are referred to as graded potentials, in contrast to action potentials, which have a fixed amplitude and time course. Graded membrane potentials are particularly important in neurons, where they are produced by synapses ― a temporary rise or fall in membrane potential produced by activation of a synapse is called a postsynaptic potential.

Comparison between Graded Potentials and Action Potentials:

1. Origin ― Arise mainly in dendrites and cell bodies

2. Types of channels ― Chemical, Mechanical or light

3. Conduction ― Not propagated, localized, thus permit communication over few mm

4. Amplitude ― Depends on strength of stimulus varies from less than 1 mV to more than 50 mV

5. Duration ― Longer, ranging from msec to several minutes

6. Polarity ― May be hyperpolarizing, depolarizing

1. Origin ― Arise at trigger zones and propagate along axon

2. Types of channels ― Voltage-gated ion channels

3. Conduction ― Propagated, thus permit communication over long distance

4. Amplitude ― All-or-none, typically 100mV

5. Duration ― Shorter, ranging from 0.5-2 msec

6. Polarity ― Always consist of depolarizing phase followed by repolarizing phase and then return to resting membrane potential


When the electrons don't cancel out

Note, however, that if we are combining two half reactions to obtain a third half reaction, the values are not additive, since this third half-reaction is not accompanied by another half reaction that causes the charges to cancel. Free energies are always additive, so we combine them, and use &Delta = &ndashnFE° to find the cell potential.

Calculate for the electrode Fe 3 + /Fe(s) from the standard potential of the couples Fe 3 + /Fe 2 + and Fe 2 + /Fe(s)

Tabulate the values and calculate the &Deltas as follows:

(i) Fe 3 + + e &ndash &rarr Fe 2 + 1 = .771 v , &Delta1 = &ndash.771 F
(ii) Fe 2 + + 2 e &ndash &rarr Fe(s) 2= &ndash.440 v , &Delta2 = +.880 F
(iii) Fe 3 + + 3 e &ndash &rarr Fe(s) 3 = ? , &Delta3 = +.109 F


What are negative and positive after potentials? - Biology

Negative (-) electrode positioned on Cz.

Measure with the paper ruler the distance between de nasion and inion of the subject and the circumference of the head as indicated in the diagram above. Then affix the negative electrode in the midline just at the half way point of the distance between de nasion and inion (Cz).

Positive (+) electrode positioned on the ipsilateral mastoid process

ERPs are classified according to the nature of the stimulus: visual, somato-sensory, and auditory they can also be classified according to the latency at which their components occur after stimulus presentation: short latency (<100msec) and long latency (>100msec) potentials.

The shorter latency components are generated during the sensory stimulus processing stages (exogenous components). The longer latency components represent the cortical processing stages, which are less determined by the physical features of the stimulus (endogenous components).

The classification into early or late components ERPs is useful in practical terms, however it is more theoretical than realistic since ERPs generation is a continuous process.

In this session the stimulus used to evoke the responses is auditory, so the responses will be auditory related potentials.

Early auditory related potentials include five positive waves that occur during the first 10 msec after stimulus presentation and are labeled from I to V according to their order of appearance. They are very stable in shape, amplitude and latency in subjects with no hearing impairment. It is well proven that these components are generated as a result of the activation of brain stem nuclei of the auditory pathway during auditory stimuli information processing. Due to their stereotyped behaviour, even during sleep and unconsciousness states, these potentials have been very helpful as an objective functional evaluation of auditory system in newborns and psychological deafness.

Long latency potentials are referred to those components that appear after 100 msec of stimulus presentation and are thought to represent cortical information processing. They are affected by level of attention, stimulus significance, task relevance and stimulus processing requirements.

We are going to record P100 (first positive [P] component appearing 100 ms after the stimulus) using auditory stimuli, although this component can also be evoked visually the most important factor is that the stimulus must be unpredictable in time.

Variables analyzed from ERPs:

Absolute latency: is the time interval between stimulus presentation and the point of maximal value (peak) of a defined component. It is expressed in milliseconds and represents the time taken by the stimulus information to generate the component.

Relative latency (inter-peak latency) : is the time interval between two components and measures the conduction of the impulse between two generators.

Amplitude: vertical distance measured from the trough to the maximal peak (negative or positive). It expresses information about the size of the neuron population and its activation synchrony during the component generation.

Ask the subject to close their eyes. Put the headphones on the subject. Set the volume button of the sound amplifier to zero. This way, the subject will not perceive any auditory stimulus delivery. Make sure that the subject does not hear anything!


Lecture 22: Neurons, Action Potential, and Optogenetics

Professor Martin begins his lecture on electrical signaling by talking about neurons, followed by action potentials, synapses, and optogenetics.

Instructor: Adam Martin

Lecture 1: Welcome Introdu.

Lecture 2: Chemical Bonding.

Lecture 3: Structures of Am.

Lecture 4: Enzymes and Meta.

Lecture 5: Carbohydrates an.

Lecture 9: Chromatin Remode.

Lecture 11:Cells, The Simpl.

Lecture 16: Recombinant DNA.

Lecture 17: Genomes and DNA.

Lecture 18: SNPs and Human .

Lecture 19: Cell Traffickin.

Lecture 20: Cell Signaling .

Lecture 21: Cell Signaling .

Lecture 22: Neurons, Action.

Lecture 23: Cell Cycle and .

Lecture 24: Stem Cells, Apo.

Lecture 27: Visualizing Lif.

Lecture 28: Visualizing Lif.

Lecture 29: Cell Imaging Te.

Lecture 32: Infectious Dise.

Lecture 33: Bacteria and An.

Lecture 34: Viruses and Ant.

Lecture 35: Reproductive Cl.

ADAM MARTIN: All right, let's get started. So I'm starting with this video here. What's happening here is there's this mouse, and you see there's like this fiber optic cable going into its brain. And the mouse is asleep right now. And now the researchers are shining light into its brain, a specific region of the brain, to activate specific neurons in order to test whether they function in arousal.

And here, you see the mouse is going to wake up. There it goes. It's awake now. So for today's lecture, we're going to work towards understanding how this experiment works. And we're going to talk about how neurons function and how researchers are able to control that function in order to modify behavior-- in this case, the arousal of this mouse.

OK, so this is going to involve a particular type of cell in our body, which is the neuron. And neurons are highly specialized cells that have a function to transmit information from one part of the body to another. And so neurons are highly polarized cells, which you can see here. On the left of this neuron, you see this arbor of protrusions, which are called dendrites. And then on this side of the cell body, you see a single extension, which is an axon, and then the terminus of the axon over here.

And this nerve cell transmits information in a single direction. It will transmit information from this side to this side. And these neurons are able to communicate with each other. And they communicate at the ends of the neuron, which are known as synapses, which I'll come back to and talk about later on in the lecture. So neurons could be making synapses on this side and also making synapses on this side with other neurons.

So to start to unpack the function of this neuron-- and I should highlight that this flow of information can occur over very long distances, right? Your sciatic nerve extends from the base of your spine all the way down into your foot, OK? So that axon is one meter in length. So that's an extremely long distance to transmit information along a single cell.

And so we're going to go from thinking about how signals are transmitted in single cells, and this will evolve electrical signaling. Then we'll talk about synapses and how synapses function to communicate between neurons. And this is going to involve also sort of understanding how certain antidepressants, like Prozac, work. And then we'll end by talking about how researchers did this experiment to wake up the mouse.

And it all starts with something that I told you about at the beginning of the semester, which is that the plasma membrane separates distinct compartments the outside of the cell from the cytoplasm. And there are distinct ion concentrations on either side of this boundary. So we're starting now talking about a single neuron cell. And we're going to talk about a type of signal known as an action potential. Oh, that's right.

So we're going to talk about an action potential. And what an action potential is, is it's an electrical signal that travels the length of the neuron. So this action potential, I'll abbreviate this AP. So when I refer to AP, I'm not referring to advanced placement, but action potential, OK? So this is an electrical signal that travels the length of the axon and the neuron.

And so in order to have an electrical signal propagate, we need some sort of electrical property that the cell has that enables this. And so I showed you or I told you earlier in the semester how sodium ions are concentrated on the outside of the cell and potassium ions are concentrated on the inside. You see here's the sodium gradient here, potassium gradient here. And now I'm going to tell you how it is that this happens, because this is thermodynamically not favored, right?

These ions would prefer, by diffusion, to be equal concentrations on both sides of this plasma membrane, which means that the cell to shift this from equilibrium has to expend energy to set up this situation. And so in the plasma membrane of the cell, there is a protein. It's an integral membrane protein and sits inside the plasma membrane. So this here is the plasma membrane.

And this integral membrane protein is called a sodium potassium ATPase. So it's going to have a subunit that hydrolyzes ATP to ADP. And the protein uses the energy of ATP hydrolysis to pump sodium ions up their concentration gradient. So the sodium ions are going out of the cell. And this is going against the flow that sodium would normally like to take, which would be going downstream.

And it pumps potassium ions into the cytoplasm such that there's a higher concentration of potassium ions in the cytoplasm, OK? So these neurons expend a huge-- a quarter of their ATP is used by pumping ions like this, such that there is gradients of ions across the plasma membrane.

Now, if one sodium ion was pumped out for every potassium ion pumped in, there'd be no charge difference between the exterior and the cytoplasm. But what happens in the plasma membrane is that in addition to the sodium potassium ATPase, there are other channels that are present. There are sodium channels. These are mostly closed, but there are some potassium channels that are leaky. And they're basically leaking potassium from the cytoplasm out into the exoplasm, OK?

And if you have positive charges going out the cell, then the inside of the membrane is going to have a net negative charge. And the outside of the membrane is going to have a net positive charge. And this charge across the membrane, where you have positive on the outside and minus on the inside-- I should label this exterior, and this is cytoplasm.

This voltage difference is known as a membrane potential. So this is a membrane potential. And it's an electrical potential across the membrane. If you're an electrical engineer, you can think of the plasma membrane as a capacitor, OK? So this plasma membrane is holding this charge difference across it. And so there's a voltage across the membrane. And in a resting state, the cell's resting potential is negative 70 millivolts.

So if the cell is not getting stimulated by something like a neurotransmitter, the resting potential is negative 70 millivolts, where the inside is negative and the outside is positive, OK? So now I just want to define some terms that are going to be useful for us when we talk about action potentials. So when there's this negative inside potential, a negative inside potential is referred to as polarized. So it's polarized because there's a polarity across this membrane, where one side is positive and the other side is negative, OK?

So polarized refers to if there's a negative inside potential. So the resting state of the side is there's a polarized-- it's polarized. However, the cell can lose this polarity and not have a charge differential, or it can flip and be positive on the inside. And when that happens, if there's either zero or positive inside potential, this is referred to as depolarized.

Anyone have an idea as to how the cell would flip the potential? What would have to happen in the plasma membrane to flip this potential and depolarize the cell? Yes, Stephen?

AUDIENCE: You could open the ion channels.

ADAM MARTIN: So Stephen suggested opening ion channels. Which ion channels would you open?

AUDIENCE: The sodium channels.

ADAM MARTIN: Yeah. So Stephen suggested if you open these, it's going to depolarize the cell. Because remember, sodium is high on the outside, out here. And so if you open these channels, positive ions are going to flow in. And that's going to make this less negative and this less positive, OK?

So this is the situation here, where these sodium channels open, and the sodium channels-- or the sodium ions rushing in is going to create a depolarization, where you now flip the potential. And there's a greater positive charge on the inside of the plasma membrane. Everyone see how? Because the sodium ions are going to just go downstream. They're higher concentration out here. So just by opening these channels, the cell doesn't have to do any work to do this. Sodium is just going to flow down its gradient into the cytoplasm.

So what an action potential is, is it's a transient depolarization of the nerve cell. So the Action Potential, or AP, is a transient depolarization of the neuron, which means it doesn't just depolarize and stay depolarized, but it depolarizes and then restores itself back to the resting polarity. And so what you see when you measure the voltage across the plasma membrane in a neuron, you see that it can spike and depolarize, but then it's rapidly restored to its resting state, OK? So it's a transient process.

When we think about the neuron at higher resolution, what you're going to see is not only is it transient, but it's also a traveling wave that propagates along the entire length of the cell. So this is also a traveling wave. And one thing that you can notice about these neurons, or the action potentials here, is that they all depolarize to the same extent. So they all depolarize to this positive 50 millivolts.

And so this illustrates a key property of neurons, in that the level of activity of a neuron is not determined by the size of this action potential. This action potential is an all-or-nothing event. It either happens or it doesn't. And when it happens, it depolarizes to the same level. So the action potential is all or nothing. You can think of it as a binary signal.

And therefore, the way that neurons encode sort of the magnitude of activation is not through the level of depolarization of a single action potential, but it is able to distinguish between different frequencies of action potentials that are propagating along the neuron. So signal strength, in this case, is related to the frequency of action potentials firing.

So now we're going to unpack how it is a nerve cell fires an action potential and how it propagates along the entire cell length, right? In the case of the sciatic nerve, this has to happen across an entire meter, OK? That's a very long distance to propagate this change in electrical signal, at least for a cell. And so we're going to talk about the mechanism. And I'm going to start at the beginning, when this action potential initiates.

So we'll start at the initiation of the action potential. So how is it that this nerve cell is told to start depolarizing at the dendrites? Because there's going to be another neuron here, which is going to communicate to this neuron over here to tell it to start depolarizing. It does this at the location known as the synapse, which is basically sort of the connection between the two neurons.

And the way this process is initiated is similar to the type of signaling that you saw in the past few lectures, where you have a ligand and a receptor, OK? In this case, the ligand is going to be what's known as a neurotransmitter, which is a small molecule. And I'll show you some later on. And the receptor is going to be a receptor that binds to this ligand.

But in this case, rather than being something like a G protein coupled receptor or a receptor tyrosine kinase, the receptor is going to be an ion channel, OK? So the receptor is going to be an ion channel. And so you see one example in the slide up here, where here's a receptor. And these receptors are what are known as ligand-gated ion channels. In this case, it's a sodium channel.

So it's going to be-- whether or not it's open depends on the presence of the ligand. So if we take a neurotransmitter like serotonin, if it's not bound to the receptor, the receptor is closed. But if serotonin binds to the receptor, it opens up the channel, which can selectively let in a type of ion-- in this case, sodium. In this case, this is an activating channel, because letting in sodium is going to depolarize the cell, OK?

So this ligand receptor binding uses a ligand-gated-- there's a ligand-gated sodium channel. And it's this ligand-gated sodium channel which starts the depolarization. So that's how you sort of knock over the first domino, right? But then there has to be some mechanism to propagate this along the length of a very long cell.

And so I'll tell you this involves a different type of sort of signaling mechanism from what you're used to thinking about, because this involves a different type of an ion channel. And it's called a voltage gated. And I'll abbreviate voltage gated just VG. And in this case, it will be a sodium channel.

So what's a voltage-gated sodium channel? This is a voltage-gated sodium channel here. And you can see, in the resting state of the cell, this channel is closed. And it's closed because of this red rod structure that's positively charged. That's a positively charged alpha helix that is a part of this protein and is embedded in the membrane. But this alpha helix is positioned down towards the cytoplasm, because it's positively charged. And the cytosolic face of the plasma membrane is negatively charged, OK?

And the confirmation of this protein then depends on the charge across this membrane. Because when there is depolarization, that shifts the position of this alpha helix, such that now it shifts up towards the exterior face of the plasma membrane. And that opens the channel, which lets sodium ions rush in, OK? Again, sodium ions here, they're always rushing downstream. They're concentration gradient.

So in this case, whether or not this channel is open or closed depends not on the presence of a ligand, but on the membrane potential across the plasma membrane. So these voltage-gated sodium channels, they're opened by depolarization.

And then the question becomes, if you open these channels at the very end of the neuron, how do you get it such that this electrical signal moves unidirectionally along the neuron? So what leads to unidirectionality? Who's been to a sporting event lately? OK, good. You guys know the wave?

So we're going to do the wave. Once you to stand up, you're going to be tired, and you're going to have to sit down for a while. I'm going to be a ligand-- I'm a ligand-gated sodium channel, so I'm going to start things off, OK? You ready? All right, here we go. OK, that's basically an action potential.

So the way that this was unidirectional is once you stood up and did the wave, you then sat down, and you stopped doing anything. And so these voltage-gated sodium channels have a similar property. If we look at the next step in this, the sodium channel is opened by depolarization. And you see there's this ball of chain segment of the protein. You see that yellow ball?

Once the sodium channel opens, after about a millisecond, that ball sticks in the channel pore and blocks it, OK? So these sodium channels open to let in sodium ions, but then they're immediately inactivated after about a millisecond, OK? And so that enables unidirectionality. So this is what I'll call voltage-gated sodium channel inactivation.

And how this promotes a traveling wave of depolarization is that if we consider an action potential moving along this axon from left to right and if the sodium channels in the green zone are currently open, it came from the left, which means that all the sodium channels to the left of this green zone are going to be inactivated. So because they're inactivated here, there won't be further depolarization going to the left, but depolarization will have to move to the right. And you basically get this traveling wave. And it goes one direction, because if it came from somewhere, which it always does, then where it just was coming from, all those sodium channels, the voltage-gated sodium channels are going to be closed.

So this allows it to move in a single direction along the neuron. Also, once the action potential gets to the end of the neuron, it doesn't reflect back the other way in the neuron. This can only go one direction. So this provides unidirectionality. So it's this inactive or refractory period of the voltage-gated sodium channel which prevents the action potential from moving backwards.

Now, if you look at these action potentials in the cell, you see that they happen, but you don't just depolarize and stay depolarized. The cell body depolarizes and then repolarizes very rapidly. So there's an oscillation. So there has to be some way to terminate the action potential. So there's a termination or repolarization of the cell.

So there has to be a way for this nerve cell to rapidly restore membrane potential. And I want you to think for just a couple of seconds about what type of channel might you open to re-establish this polarity. What ion do you need to flow from where to where in order to get a net negative charge on the inside? Udo?

AUDIENCE: You need to move the sodium ions from the inside to the outside.

ADAM MARTIN: OK, you could pump the sodium ions out, and that's totally accurate. So that's going to require moving sodium ions up a concentration gradient, which is going to take energy and is going to be slow. So is there another option we could take advantage of here to repolarize? Rachel?

AUDIENCE: Move the potassium ions.

ADAM MARTIN: So Rachel has suggested to moving the potassium ions to the outside, which is how this is done. So remember, potassium is high in the cytoplasmic, low on the exoplasm. And therefore, if you have a voltage-gated potassium channel, that's going to cause a rush of positive ions out of the cell. And that will be able to restore the net negative potential on the inside of the cell.

So this termination or repolarization is the result of the opening of voltage gated, in this case, not sodium channels, but potassium channels. When do you think these have to open relative to the sodium channel? Should they open right with the sodium channel? Carmen's shaking her head no. Do you want to explain your logic?

AUDIENCE: Well, I mean, they both carry the same charge, so they wind up getting out at the same time [INAUDIBLE].

ADAM MARTIN: Exactly. So what Carmen said is if they open simultaneously, you have sodium flowing in. You have potassium flowing out. And that's not going to necessarily change the charge. So when would these have to open relative to sodium channels? Yeah, Carmen?

AUDIENCE: When it reaches that potential [INAUDIBLE].

ADAM MARTIN: So after it's depolarized, yeah. So this has to be delayed relative to the sodium channels, OK? So this has to be delayed relative to the voltage-gated sodium channels. Because if you're thinking about this traveling wave of depolarization, the depolarization is going to be high where the sodium channels are only entering. And then following that, you would have potassium ions getting pumped out and basically repolarizing the cell.

Everyone see how you sort of get depolarization with sodium rushing in, and then after that, you repolarize with the potassium getting pumped out, right? So here, you have a spike, and you complete the cycle. It can even get hyperpolarized, where it gets even more negative than it normally does. And then it eventually gets back to this resting potential of around negative 60 or negative 70 millivolts.

OK, so this has to happen fast. And I want to tell you about one process or property of neurons and another helpful cell that enables this to go extremely fast. And that is that there are these glial cells in your body and your brain that wrap around the axons of the neurons and basically function like electrical tape for neurons, OK? So they are these-- there's electrical insulation around the axons of these neurons. And this is provided by another specialized cell type called a glial cell. So this is by a glial cell.

And here are two examples of glial cells. There are oligodendrocytes-- and you can see how the cell is extending processes that wrap around the axons of these two neurons. Here's a Schwann cell over here, which again, wraps around the axon. And these cells basically form what's called a myelin sheath. So they form a myelin sheath around the axons. And that insulates the plasma membrane of the axon such that-- so here is an axon.

You have glial cells that are wrapped around, and it sort of forms like beads on a string. And so there are these gaps between the myelin sheath that are known as the nodes of Ranvier. So there are these nodes of Ranvier, which are gaps in the myelin sheath. And these nodes perform an important function for the neuron, because where the axon is wrapped, the membrane is electrically insulated.

And so the sodium ions-- or the sodium channels and potassium channels, the voltage gated ones, localize to these nodes. And when the action potential is traveling along the axon, because these regions where the myelin sheath is are electrically insulated, the axon potential doesn't just move continuously, but jumps from node to node, such that you are just opening the sodium channels at these nodes. And that allows the action potential to travel about 100-fold faster along the axon. And that's what allows your neurons to transmit these electrical signals from the base of your spine to your foot so rapidly.

So you get an increase in speed because the action potential is jumping from node to node. And one important reason to bring this up is because there is an important human disease that affects the electrical insulation in the myelin sheath here, and that's multiple sclerosis.

So we're going to unpack multiple sclerosis in a couple lectures. This is an autoimmune disorder. And so we're going to talk about immunity later in the semester, and we'll talk about how that happens. But for now, I just want to point out that multiple sclerosis happens when the immune system attacks this myelin sheath.

So in multiple sclerosis, the myelin sheath is damaged. And if you damage this electrical insulation, you greatly slow down these action potentials, and that has a significant impact on nerve impulses in the brain and throughout the entire body. And that's why multiple sclerosis is such a devastating disease.

All right, I'm going to start moving now to consider more than one neuron. So until now, we've just talked about how an electrical signal is sent along the length of one cell. And now we're going to start thinking about multiple neurons and how they connect and how neurons integrate information from multiple other neurons to decide whether or not to send an action potential.

And so if we consider this connection right here, there's a synapse right here. Here's a cell that's sending information and a cell that is receiving that information. When we're considering a synapse-- so if we consider a synapse, there's a cell that is sending the signal, which is called the presynapse. This is the sender cell. And there's a postsynaptic cell.

But you can have more than one neuron sending a signal to a neuron at a given time, right? So here, you have one neuron that's sending a signal at this synapse, but you might have another neuron sending a signal to a synapse on this part of the cell. And you could have another signal coming in here. And so this neuron will then have to decide whether or not to fire an action potential down its axon.

And the way that the neuron decides this is to integrate the signals. So there's a signal integration process. And what's important for signal integration in a neuron is whether or not the cell body-- whether the voltage increases above a certain threshold potential. So if the cell body doesn't increase-- if the voltage doesn't increase above this potential, there will be no action potential fired. But if the voltage increases above the threshold potential, then it fires the action potential and signals to a downstream neuron or muscle or another cell.

So here, it is the threshold potential in the cell body that determines whether or not an action potential is sent down the axon. And there are different types of signals that nerve cells can send. So there are different types of signals. Signals can be excitatory, meaning it will tend to depolarize the neuron. So there are excitatory signals, which result in depolarization.

For example, with serotonin, that opens the sodium channel, and that results in depolarization, so that's an excitatory signal. But there are other types of signals that bind to different types of receptors that are inhibitory. What might be a type of receptor that would inhibit this process of sending an action potential? What might an inhibitory receptor be to lower the chance that this action potential will be fired?

What if I told you it's an ion channel? What ion would you expect it might pass? Udo?

ADAM MARTIN: Potassium. Udo is exactly right, right? If it passes potassium, then it's going to make the inside more negative. And that's what's known as hyperpolarization. So receptors that result in hyperpolarization would have an inhibitory effect on this process.

And remember, if you're hyperpolarizing, then you could cause this to actually go down and get even farther away from this threshold potential, right? And if you have an activating signal and an inhibitory signal, they might cancel out, because one will depolarize and the other will hyperpolarize. So it's in this way a neuron is able to integrate signals coming from different neurons. And that influences whether or not it will send the signal to a downstream cell.

OK, so now we're focusing on what is the communication between one neuron and another. And this revolves around this thing that's called the synapse, which is basically the gap between the axon terminal of one neuron and the dendrites of a postsynaptic neuron. And so the way that multiple neurons communicate with each other are through a type of signal known as a neurotransmitter. And this is what initiates the signal.

So there's a signal initiation process at the synapse. Initiation. And this involves the presynaptic neuron secreting a neurotransmitter. So the signal, in this case, signals between neurons are called neurotransmitters. And as you see on the slide, these are examples of neurotransmitters. They're often derived from amino acids, and so they're small molecules. They're not the proteins that you often see with receptor tyrosine kinase ligands. This is a different class of signal.

So one example is serotonin. And if you look up at those, we'll find serotonin here. There it is. Here, you can see it's a derivative of tryptophan. So it's a small molecule, and it's able to bind to a receptor on the postsynaptic cell and induce depolarization.

And so neurons are-- the way that they communicate is-- neurons are a case of where luck favors the prepared. Neurons are totally prepared to send signals to each other. They have everything ready to go when they get word from upstream, and they're ready to send signals to the next cell. And that's because if we look at the synapse prior to an action potential, everything is ready to go. The cell has neurotransmitter, and it's packaged in these vesicles, and it's tethered to the plasma membrane, ready to be released.

So prior to the action potential, there are vesicles filled with neurotransmitter that are docked at the plasma membrane. I abbreviate plasma membrane PM, just so I don't have to write it out, OK? So these contain neurotransmitter, right? But you see in this docked vesicle, the neurotransmitter is in red, and it can't get out if that vesicle does not fuse with the plasma membrane. So these contain neurotransmitter.

But at this point, the vesicles haven't fused. But the vesicle's not fused. When should they fuse? In this system of neuron signaling to each other, when should the vesicle fuse with the plasma membrane? What should trigger the fusion process? Yes, Miles?

AUDIENCE: So after [INAUDIBLE] axon when it's time for the [INAUDIBLE] that's when the vesicles fuse.

ADAM MARTIN: Yeah, so Miles is exactly right. If we consider my diagram here, there's an action potential traveling along this axon. When it gets to the axon terminus, that should be the signal for these vesicles to fuse to the plasma membrane and to release neurotransmitter. So it's the arrival of the action potential, right?

So remember, in this case, serotonin is going to be in blue. If serotonin is inside my vesicle here, it's going to need to exocytose. And now the serotonin is going to be outside the cell, ready to bind to the receptor.

All right, so as Miles pointed out, you have an action potential. The fusion should be triggered by the action potential. In order to fuse, there needs to be some signal inside the cytoplasm to tell the vesicles to fuse. That signal is increased calcium ion concentration. And then when calcium concentration increases in the cytoplasm, that triggers the fusion of these vesicles. And when you get fusion, that's exocytosis, and the serotonin is now on the outside of the cell, where it can travel across the synaptic cleft and bind to a receptor on the postsynaptic neuron.

So this fusion is when neurotransmitter is released. Neurotransmitter is released here. And the way that this increasing calcium has to happen, when the action potential arrives at the axon terminus. So when it arrives in the axon terminus, there's depolarization of that part of the cell. And so there's a special type of protein called a voltage-gated calcium channel.

All these channels are very selective for different ions. So a voltage-gated sodium channel isn't letting in all of the ions outside the cell. It's selective to sodium. And this case, this voltage-gated calcium channel is just going to let in calcium. And then there's a mechanism that links calcium entry to vesicle fusion. And that's going to be shown here.

What you see on this docked synaptic vesicle is this calcium-binding protein called synaptotagmin that's present on the vesicle. And so when calcium goes into the cytoplasm, that protein binds to calcium, and it activates the fusion machinery such that the plasma membrane of the vesicle fuses-- or the membrane of the vesicle fuses with the plasma membrane of the cell, thus releasing the neurotransmitter into the synaptic cleft.

So this is what starts the signal. Now, you probably know that these neurons are not active or on all the time. So something has to terminate the signal, usually quite rapidly. So now I want to talk about that. So like all signaling pathways, signaling is useless if you can just turn it on. You have to be able to toggle it on and off in order for biological systems to function properly, right? And that's the case with neurons.

If you just turn on a neuron and you don't have a way to turn it back off again, then that's pretty useless. And so we have to have a way to turn off the signal. And if we consider the synapse, this is the presynaptic neuron here. I'm going to draw a postsynaptic neuron here. And neurotransmitter is released by the presynaptic neuron to the postsynaptic neuron here. Neurotransmitter is released into the synaptic cleft.

So the sort of extracellular region between these two neurons is called the synaptic cleft. So now the cell just dumped a whole boatload of neurotransmitter into the synaptic cleft, right? How is it going to turn this off? What does it have to do? Yeah, Stephen?

AUDIENCE: It could absorb the-- take back in the [INAUDIBLE].

ADAM MARTIN: Stephen's exactly right. What Stephen suggested is, is there a way for the presynaptic neuron to reabsorb this neurotransmitter and, thus, recycle it? So it could either reabsorb it or degrade the neurotransmitter. Different process for different neurotransmitters. For serotonin, there are channels that are present in the plasma membrane, and these mediate reuptake of the serotonin.

So you have channels that are basically-- after the neurotransmitter is released, it sucks the neurotransmitter back into the presynaptic cell such that it can then reuse the neurotransmitter later on. And so this process of reuptake highlights a very important process that's been utilized by drug companies to create antidepressants. So antidepressants like Prozac and Zoloft affect this reuptake process. And what that does is it keeps the neurotransmitter in the synaptic cleft for longer, such that it enhances the signaling.

And so the idea behind these drugs is that if you are suffering depression from a lack of serotonin, then you can rescue that by preventing the rapid reuptake of the neurotransmitter into the cell after the synapse is stimulated and the neurotransmitter is released. And so Prozac, Zoloft, these are a class of drugs that are known as selective serotonin reuptake inhibitors.

It's kind of a mouthful. This is abbreviated SSRIs. But the way they function is to leave the neurotransmitter in the synaptic cleft for longer so that you enhance signaling, even if you have low levels of the neurotransmitter to begin with.

I also want to point out that if we look at this diagram here, the synaptic vesicle fuses, and then this releases the neurotransmitter. But all the machinery on this vesicle is recycled by endocytosis such that it can be reused again, OK? So cells are really good at recycling stuff. If this is sort of the membrane, you endocytose and then you can use it again later on, OK?

And so there's recycling not only of the neurotransmitter, but also all of the machinery on the synaptic vesicles that are responsible for the fusion event. All right, now I want to end by just telling you how this experiment works, where we're able to activate specific neurons in a brain and that leads to the animal sort of waking up. So in a normal neuron-- so this is the last part, optogenetics. And I'm going to go through this very fast.

But normally, you need a neurotransmitter to induce depolarization. But what optogenetics is, is an approach to control the activity of a cell with light, OK? So in this case, we're going to have light inducing depolarization. And the way this is done is there's a protein discovered from photosynthetic algae that's responsive to light, and it is a sodium channel. And this protein is called channelrhodopsin, specifically ChR2. And this is a light-sensitive protein where light induces sodium channel opening.

So that's going to depolarize the cell. And what you can do is if you have a gene that you know is specifically expressed in a certain type of neuron, you can take the promoter and enhancer region of that gene and hook it up to this single component, channelrhodopsin, that open reading frame, using recombinant DNA technology. And if that's expressed specifically in the neurons that you're trying to test, you can then shine a light into the brain of the organism and activate, specifically, this type of neuron. And that allows you to test the function of the neuron in the behavior of an organism.

So, in this case, this mouse, the light is shined into its brain, and they're testing a specific type of neuron that is involved in arousal of the mouse, and it wakes up. Oh, it's not playing. So here, this is the brain activity on the top, and the muscle activity on the bottom. So you're going to see light. There's the light. You see it? Light going into the brain.

They induce light at that frequency for a while. And then they're going to wait and see when the mouse wakes up. And it's going to wake up right now. There it goes. It woke up. You see now its muscle activity is going, OK?

So you can test the function of specific nerve cells using this approach, and it's because you have a light-sensitive sodium channel. So I'm done for today. Have a great weekend. I will see you on Monday.


Negative Staining Procedure

  1. Take a clean, grease-free and dry glass slide.
  2. Put a minimal drop of nigrosin towards one end of the glass slide via a dropper.
  3. Then, take the inoculum from the culture plates or slant culture via a sterilized inoculating loop and mix it with a drop of nigrosin.
  4. After that, take another clean, grease-free and dry glass slide and place the one end of it towards the centre.
  5. Then, tilt the glass slide over the stain containing the test organism by making an acute angle.
  6. Slightly draw the tilted slide until it touches the drop of the culture organism, and drag it across the edge of the glass slide to make an even, broad and thin bacterial smear.
  7. Allow the glass slide to air dry (do not heat fix).
  8. At last, put a drop of oil immersion and observe the glass slide under the microscope for the appearance of a colourless bacterial cell with a grey background.


Advantages

  • Through negative staining, clear unstained cells are easily observable against the black coloured stained background.
  • A negative staining method does not involve the heat-fixing of the specimen. As a result, the cell will not deform by the heat.
  • It can also stain heat-sensitive microorganisms like Spirochetes, Yeasts etc.
  • The negative staining technique also permits examining a transparent capsule around the cell wall of various microorganisms like Cryptococcus neoformans.
  • It is quite an easy and rapid method that makes the use of a single acidic stain only.

Disadvantages

  • Negative staining does not provide much information about the cell rather than the cell size, shape and arrangement.
  • By using this technique, we cannot examine a particular strain or a type of organism.

Conclusion

Therefore, we can conclude that a negative staining technique is a simple method to examine the microorganism using a single acidic stain. Negative staining results in an unstained or clear specimen with a dark coloured background.

Repulsion occurs between the negatively charged stain and the specimen, after which we could observe cells of different shapes and sizes as unstained outlines against a stained background.


Why some people test positive for malaria after successful anti-malarial treatment

Treating patients with malaria first requires a confirmed diagnosis. It has recently come to light that not all diagnosis tests are equal, and some tests, including rapid diagnostic tests (RDTs), can potentially show false positive results in certain patient populations. The following study explores the accuracy of different malaria RDTs that recognize specific malaria antigens.

The human malaria parasite has developed partial resistance to every drug used to treat it, particularly the first-line treatment, ACT (artemisinin combination therapy).

In order to curb the further spread of ACT resistance, in 2010 the World Health Organization recommended that all suspected malaria cases receive a parasite-based diagnosis (rather than presumptive treatment based on external symptoms such as a fever) before issuing anti-malarial medication.

This was challenging for most local clinics because a parasite-based diagnosis relies primarily on observing blood samples under a microscope a time consuming process that uses expensive equipment and training, which is neither readily available nor cost-effective for a small-scale health clinic.

However, rapid diagnostic tests (RDTs) have been developed that are quick, easy and cheap to use. These tests resemble pregnancy tests, with an indicator band appearing on the display when a malaria-infected blood sample is present. The RDT works by detecting antigens (an umbrella term for proteins that indicate an infection) produced by the malaria parasite, and induce a chemical reaction which causes the indicator band to fluoresce.

Between 2010 and 2015, sales of RDTs from manufacturers worldwide tripled from 90 to 270 million, and in 2015 RDTs constituted 74% of diagnostic testing used for suspected malaria cases. By reducing the number of anti-malarial medications given without first confirming the presence of the parasite, the rate of overtreatment declined, and the probability of developing anti-malarial resistance reduces.

Understanding the duration of antigen persistence is critical for correctly interpreting RDTs from recently-treated individuals

After a successful anti-malarial treatment, the malaria parasites clear from the bloodstream and the treated individual begins to feel better within a couple of days.

At this point, an issue with the use of RDTs for diagnosis purposes arises. The antigens produced by the recently-cleared malaria parasites persist in the blood after treatment for a period of time, and this duration of antigen persistence has been widely reported to be highly variable. Therefore, subsequent RDT tests can still appear positive if a recently-treated individual is tested, despite the fact that they are no longer infected with malaria.

Additional problems can occur if the individual falls ill with a fever for a second time if the test returns positive, the clinician may assume that they have been re-infected with malaria, but the possibility exists that the individual has been infected with a non-malarial fever-causing illness (for example, dengue virus or tuberculosis) but the antigens from the previous malaria infection still persist in large enough quantities to return a positive RDT.

Understanding the duration of antigen persistence is critical for correctly interpreting RDTs from recently-treated individuals, and reduces the probability of clinicians mismanaging non-malarial fevers contributing to ACT resistance.

In a recent publication in Malaria Journal by the Malaria Atlas Project at Oxford University, the authors systematically collated studies documenting the persistence of RDT positivity after treatment, and applied a Bayesian survival model to the dataset to measure the amount of time RDTs remained positive after treatment.

The authors found that approximately half of RDTs remained positive for more than a week after treatment, and a small fraction remained positive for more than three weeks after treatment.

RDTs come in a variety of forms, typically testing for either or both types of malaria antigens: histidine-rich protein II (HRP2) and Plasmodium lactate dehydrogenase (pLDH) enzyme. This study found that between these two types of RDTs, HRP2-detecting RDTs showed persistent positivity for much longer than pLDH-detecting RDTs (or combination RDTs that detect both HRP2 and pLDH simultaneously) after successful anti-malarial treatment.

In addition to these findings, the study explored collated studies on the age ranges of the sampled patients, and found children experienced longer durations of persistent positivity than adult patients. This is most likely due to pediatric malaria infections frequently being associated with a higher blood parasite density, and adults in high malaria exposure areas typically develop immune responses to malaria that keeps their blood parasite density low.

The results from this study suggest that clinicians should treat positive RDT results from recently-treated patients with caution, particularly if the patient is young and the clinician only has access to RDTs that detect HRP2.

Fortunately, with malaria prevalence continuing to decrease in most areas of the world, reinfection after successful treatment is becoming less and less likely for human populations. RDTs have contributed in no small part to this development, but their correct usage is instrumental in continuing this trend. Over-diagnosis of malaria leads to over-prescription of anti-malarial drugs and a systematic mismanagement of non-malarial fevers globally. On an individual level, it is of utmost importance to provide the best diagnosis possible for a patient in order to achieve an optimal health outcome.


Core

The Action Potential

The action potential describes the phenomenon by which excitable cells create an electrical signal via the movement of ions across the membrane. The key features of an action potential are:

  • It relies on ionicgradientsPre-existing ionic gradients are required for the movement of ions across the membrane. Changing the membrane’s permeability to different ions (i.e. opening and closing ion channels) allows the cell’s membrane potential to be changed.
  • It is predictableinnature – Although the shape of the action potential can vary between excitable cell types, in a particular cell type (e.g. a neurone) the action potential should be the same every time.
  • It is ‘all or nothing’ – For an action potential to be generated, the voltage across the membrane must reach a thresholdlevel any lower than this threshold and no action potential will be fired.
  • It is propagated without loss of amplitude – The strength of the action potential is maintained along the length of the axon as the local spread of depolarisation triggers new action potentials to be generated.

The action potential relies on the movement of Na + and K + ions. Recall that Na + influx causes depolarisation, whereas K + efflux causes hyperpolarisation.

The stages of the action potential are as follows:

  1. Initial stimulus – This is the initialdepolarisation that triggers the action potential it is generally due to the movement of Na + , either due to the activation of receptors or the local spread of depolarisation from an adjacent action potential.
  2. Depolarisation – If the initial depolarisation reaches the thresholdlevel, around -55 mV, voltage-gated Na + channels (VGSCs) open which results in rapid depolarisation.
  3. Repolarisation – After the membrane is fully depolarised, the membrane becomes more negative again as VGSCs become inactivated and voltage-gated K+ channels open.
  4. Hyperpolarisation – Often the cell ‘overshoots’ the repolarisationphase due to the movement of K + , resulting in a brief period of hyperpolarisation before returning back to the resting membrane potential.

Diagram - Graph showing the stages of the neuronal action potential

SimpleMed original by Joshua Bray

Voltage-Gated Na + Channels and the Refractory Period

The voltage-gated Na + channel is different from other ion channels in that apart from being open or closed, it also has an ‘inactivatedstate. VGSCs become inactivated in response to depolarisation and in this state Na + ions cannot pass through the channel. From the inactivated state, the channel must first ‘recover’ into the closed state before it can be open again. This provides the basis for the refractory period.


Diagram - Voltage-gated Na + channels in the closed, open and inactivated states

SimpleMed original by Joshua Bray

The refractory period describes the period of time in which the cell cannot generate an action potential. There are 2 terms that you should be familiar with:

  • Absolute Refractory Period – Within this period, the cell cannotgenerate an action potential whatsoever, as all Na + channels are in the inactivated state.
  • Relative Refractory Period – In this period, the cell can generate another action potential, although it is harder to do so. This is because the VGSCs are beginningtorecover, although some are still inactive. The relative refractory period ends when all Na + channels have recovered.

Local anaesthetics, such as lidocaine, work by blocking Na + channels in small afferent neurones responsible for pain. By blocking these channels, it prevents depolarisation so an action potential cannot be generated. It is often referred to as a ‘use-dependentblock, meaning that the drug has a preference for blocking Na + channels which are in the open or inactivated state.

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Propagation of the Action Potential

It is necessary for the action potential to travel, or be propagated, along the entire length of the axon (which can be up to 1 meter long!). To do this, there is a local spread of current which triggers new action potentials to be generated along the length of the axon.

Local current theory dictates that depolarisation at a given point in the membrane causes a spread of depolarisation in the surrounding area of membrane. This is because an influx of Na + ions repels other positive ions in the cell, resulting in local depolarisation. However, the further this current spreads, the weaker it gets.

The length constant (λ) is the distance travelled before the membrane potential falls to 37% of its original value (i.e. the higher the length constant, the further the spread of charge without loss of amplitude). The length constant is determined by 2 factors:

  1. Membrane Capacitance (Cm) – This is the abilityto storecharge. The higher the capacitance, the slower the change in membrane potential (lower length constant).
  2. Membrane Resistance (Rm) – This is determined by the numberofopenionchannels. The fewer channels open, the higher the resistance and the further a change in voltage will spread along the axon (higher length constant).

Nervous transmission is made quicker by myelination of nerve fibres. Axons are wrapped in a fatty substance called myelin, but small portions of axon are left unmyelinated and these are termed Nodes of Ranvier. It is very important to note that a myelinated axon does not have any ion channels in the membrane, channels are only present at the Nodes of Ranvier.

Image - An electron micrograph image showing a transverse section of the myelin sheath

My = Myelin sheath, Mt = Mitochondrion, Ax = Axonal cytoplasm, N = Schwann cell nucleus

Creative commons source by Roadnottaken [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

The myelin sheath acts as an electrical insulator, causing the local current to spread to the next node before the next action potential is generated. This is described as saltatory conduction, because the action potential appears to ‘jumpfrom node to node. This is much faster than if action potentials had to be continuously generated along the entire length of axon.

Myelination improves conduction by increasing the length constant. This is achieved by:

  • Decreased membrane capacitance (Cm)
  • Increased membrane resistance (Rm)

Diagram - Model of a myelinated nerve axon demonstrating saltatory conduction

SimpleMed original by Joshua Bray

Demyelination diseases, such as multiple sclerosis (MS) and Charcot-Marie-Tooth disease, result from breakdown of the myelin sheath. They are characterised by impaired nervous transmission. Areas of damaged myelin sheath (plaques) have low resistance and high capacitance, so the local current fails to spread to the next node so the next action potential cannot be generated.

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The Neuromuscular Junction

The neuromuscular junction is the synapse between a neurone and a skeletal muscle cell. As with all synapses, it involves the release of a neurotransmitter which diffuses across the synaptic cleft and activates receptors on the post-synaptic membrane – at the neuromuscular junction, this neurotransmitter is acetylcholine (ACh).

The process of synaptic transmission at the neuromuscular junction is as follows:

  1. The actionpotentialarrives at the synaptic terminal and the localdepolarisation causes voltage-gated Ca 2+ channels to open, resulting in Ca 2+ influx.
  2. Ca 2+ binds to synaptostagmin on the pre-synaptic membrane, which brings vesicles containing ACh to the membrane and forms a snarecomplex. The ACh is consequently released via exocytosis.
  3. ACh diffuses across the synaptic cleft and binds to nicotinicAChreceptors (nAChRs) on the sarcolemma (this area is termed the ‘motor end plate’). Activation of these receptors results in Na + influx and consequent depolarisation of the endplate. ACh is degraded by an enzyme called acetylcholinesterase (AChE) and its constituents are taken up by the neurone for recycling.
  4. Local spread of depolarisation across the sarcolemma causes VGSCs to open resulting in action potential generation. The action potential initiates contraction of the skeletal muscle fibre, a process which is mediated by a rise in intracellular Ca 2+ (see article on Membrane Transport).

Diagram - Synaptic transmission at the neuromuscular junction

SimpleMed original by Joshua Bray

Drugs which block action at the neuromuscular junction can be used as muscle relaxants in conjunction with general anaesthesia. They are particularly useful for relaxing the vocal cords to facilitate endotracheal intubation.

Neuromuscular blockers can work via 2 different mechanisms:

  1. Non-depolarising blockers (e.g. rocuronium bromide) – DirectlyblocknAChRs at the motor end plate, thereby preventing depolarisation.
  2. Depolarising blockers (e.g. succinylcholine) – These drugs activatenAChRs, however the sustaineddepolarisation causes the surrounding Na + channels to become inactivated so an action potential cannot be generated.

Myasthenia gravis is an autoimmune disease where autoantibodies target and destroy nAChRs at the neuromuscular junction, so it is harder for the end plate potential to reach the threshold for action potential generation. This disease is characterised by fatigable muscle weakness and is treated using acetylcholinesterase inhibitors such as pyridostigmine.


Watch the video: Math Antics - Negative Numbers (May 2022).