mod 5 -soley valencia

Module 5 Soléy ValenciaCBNS 130L

Electrophysiology

Abstract

I made a 2 Cell model and observed the communication between them while varying

conductance to A, T, AHP, L, C, and M currents and manipulating GABAa, GABAb, NMDA, AMPA to

fine tune each action potential.

Introduction Much of the currents that I varied rely on calcium dependent potassium channels such as C, AHP, and T currents. Calcium dependent potassium channels have been further studied and have been linked with rhythmic motor movements sequenced into timing of muscle contractions that rely on communications between neurons. Scientists found that calcium dependent potassium channels may be linked to rhythmic movement while studying Drosophila. BK channels activate with an increase in extracellular calcium and are involved in fast repolarization and burst firing. Mutations in slo genes that require BK channels were identified in movement disorders. The slo gene was cloned in the Drosophila and it was observed that slo genes encode for calcium dependent potassium channels and found that they delay repolarization at neuromuscular junctions (McKiernan 2013).

Methods and Materials Through “My first Neuron” created my own experiment between 2 Cells by inserting

and removing mechanisms to create the intended parameters.

- Applied A-current first to the excitatory cell then to the inhibitory cell changed Cell 1 AMPA = 0.1 NMDA = 0.001 Amplitude = 2n and Cell 2 GABAa = 0.01 GABAb = 0.001 gkbar_iA = 0 then compared these results to one I had set with gkbar_iA = 0.000345. Then applied changes to the second cell; Cell 1 AMPA = 0.1 NMDA = 0.001 Amplitude = 3nA Cell 2 GABAa = 0.01 GABAb = 0.001 gkbar_iA = 0.001.

- Applied T-Current first to the excitatory cell then the inhibitory Cell 1 was set to GABAa = 0.4 GABAb = 0.1Cell 2: AMPA = 0.1 NMDA = 0.01 iT = 0.005 then these results were compared with a graph setting Cell 1: GABAa = 0.4 GABAb = 0.1 Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iT = 0.007 Base Current = 5nA and applied Initial voltage of -80mV with 1000ms.

- AHP current is applied while its conductance is varied when setting AHP first to the excitatory neuron then to the inhibitory neuron. Settings were set to Cell 1: GABAa = 0.4 GABAb = 0.1Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iAHP = 0.01 iT= 0.008 and a Base Current = 2nA [Ca++]out = 0.1mM while maintaing an initial voltage of -80mV. I then compared these results with settings Cell 1: GABAa = 0.2 GABAb = 0.1 gkbar_iAHP = 0.002 [Ca++]out = 0.1mM Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iT = 0.008 Base Current = 2nA.

- L and C current is applied while its conductance is varied by first applying it to an excitatory and then to the inhibitory neuron. The settings Cell 1: GABAa = 0.3 GABAb = 0.1 Cell 2: AMPA = 3 NMDA = 0.01 iL = 0.000276 iC = 0.00345 Base Current = 3nA and compared the results with a graph set to Cell 1: GABAa = 0.3 GABAb = 0.1 Base Current = 1nA iL= 0.003 iC= 0.004 Cell 2: AMPA = 3 NMDA = 0.01 Base Current = 3nA.

- M Current is applied first on the excitatory neuron then on the inhibitory neuron while the conductance is varied. I set Cell 1: GABAa = 1 GABAb = 0.1 Base Current = 1nA Cell 2: AMPA = 0.3 NMDA = 3 Base Current = 5nA iM = 0.001 while maintaining the initial voltage at -65mV. I compared these results with settings I set the graph in at Cell 1: GABAa = 1 GABAb = 0.1 Base Current = 1nA iM = 0.0009 Cell 2: AMPA = 0.1 NMDA = 3 Base Current = 3nA

Cell 1 (Excitatory) Cell 2 (Inhibitory)The graph on the left shows Cell 2 firing more frequently since the conductance of A-Current is set to 0. Cell 1 depolarizes Cell 2 which sends an IPSP back to Cell 1, this is why the amplitude of Cell 2 is smaller. The graph on the right, Increase GABAa to 0.1 to 2 didn’t inhibit Cell 2 even when it was successfully responding with IPSP to Cell 1 with a GABAa level of 0.01. This is because I introduced A-current at 0.00345 settings which inhibited Cell 2 from firing. To overcome iA inhibition on Cell 2

I increased Cell 1 Base current to 3nA.

Cell 1: AMPA = 0.1 NMDA = 0.001 Amplitude = 2nACell 2: GABAa = 0.01 GABAb = 0.001 gkbar_iA = 0

A-Current Applied to Cell 2

Cell 1: AMPA = 0.1 NMDA = 0.001 Amplitude = 3nA Cell 2: GABAa = 2 GABAb = 0.001 gkbar_iA = 0.000345

Cell 1 (Excitatory) Cell 2 (Inhibitory)The graph on the left; Cell 2 shows less inhibition on Cell 1 because I changed GABAa to 0.01

keeping everything else the same. The graph on the right; I lowered A-current conductance on Cell 2 which allowed it to send an IPSP to Cell 2. Cell 1 is able to still send an EPSP to Cell 2 after it

receives an IPSP because I added a base current injection of 3nA.


Cell 1: AMPA = 0.1 NMDA = 0.001 Amplitude = 3nACell 2: GABAa = 0.01 GABAb = 0.001 gkbar_iA = 0.00345

Cell 1: AMPA = 0.1 NMDA = 0.001 Amplitude = 3nACell 2: GABAa = 0.01 GABAb = 0.001 gkbar_iA = 0.001

Cell 1 (Excitatory) Cell 2 (Inhibitory)The graph on the left; with an increased gkbar_iA conductance from 0.00345 to 0.06 Cell 1 became too inhibited to reach threshold and have an action potential so that it could no send an IPSP to Cell 2, therefore, we see no Action potential from Cell 2. The graph on the right; A-current conductance is set to 0.01 and Cell 2 is sending an IPSP to Cell 1 so that Cell1 action potentials are smaller in

amplitude in spite of Cell 1 sending less EPSP to Cell 2 because of the increase IPSP sent on Cell1.


Cell 1: AMPA = 0.1 NMDA = 0.001 Amplitude = 3nA gkbar_iA= 0.06

Cell 2: GABAa = 0.1 GABAb = 0.001

Cell 1: AMPA = 0.1 NMDA = 0.001 Amplitude = 3nA gkbar_iA= 0.01


Cell 1 (Inhibitory) Cell 2 (Excitatory)Initial Voltage -80mV

The graph on the left; Cell 2 is activating faster than there are sodium channels available because sodium channels have not recovered by the time Cell 2 has recovered so that its action potential

amplitude decrease sending less EPSP to Cell 2. The graph on the right; T-current on Cell 1 activates due to the initial voltage set at -80mV so that there is an increase influx of calcium. I also increased

potassium outside the Cell 1 to 5mM leading to a further depolarization as potassium influxes. Cell 2 sent an EPSP that depolarized Cell 1 too much. It cause it to increase firing rate faster than sodium

channels available because sodium channels have nor recovered at that time so that there isn’t enough sodium for the next action potential. The decrease in action potentials stopped Cell 1 from

firing an EPSP to Cell 2 so that it was able to depolarize, but because I also added -20nA as base current, it cause T-current to activate and influx of calcium further drove the cell to fire more action

potentials


Cell 2: AMPA = 0.1 NMDA = 0.01 iT = 0.005

Cell 1: GABAa = 0.4 GABAb = 0.1 [k+]o = 5mM

Cell 2: AMPA = 0.1 NMDA = 0.01 iT = 0.005 Amplitude -20nA

T-Current Applied on 2 Cell Model

Cell 1 (inhibitory) Cell 2 (Excitatory)iT influx calcium into Cell 2, causing action potential at Cell 2 so that

Cell 2 fires EPSP to Cell 1. In turn, Cell , sends an IPSP to Cell 2 hyperpolarizing Cell 1, plus calcium gated potassium dependent and channels are activated causing further

hyperpolarization of cell 2.



Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iT = 0.007

1000 msInitial Voltage -80mV

Cell 1 (Inhibitory) Cell 2 (Excitatory)By adding 5nA of base current to the excitatory cell, it cause it to fire an stronger EPSP

to Cell 1 which sent an a strong IPSP back to Cell 2 but, since 5nA of current was applied to Cell 2, it prevented it from staying in a state of hyperpolarization so that it

was able to fire subsequent EPSP to Cell 1.



Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iT = 0.007 Base Current = 5nA


Cell 1 (Inhibitory) Cell 2 (Excitatory)In spite of adding more iAHP to Cell 2, managed to depolarize due to the addition of 2nA o base current. Because initial voltage was set to -80mV, iT current was able to

activate that allowed an influx of calcium which led to the opening of potassium channels that hyperpolarized the cell. iT and iAHP are both calcium dependent

potassium channels so both were working on inhibiting the cell right after reaching a depolarized state of 59mV.

AHP-Current Applied to Cell 2


Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iAHP = 0.01 gkbar_iT= 0.008 Base Current = 2nA [Ca++]out = 0.1mM


Cell 1 (Inhibitory) Cell 2 (Excitatory)I added 2nA of current to Cell 2 but little IPSP from Cell 1 was received from Cell 2, which lowered Cell 2 because of the base current and because Cell1 has 0.1 mM of

extracellular calcium so that there will be less calcium available inside the cell.

AHP-Current Applied to Cell 1

Cell 1: GABAa = 0.2 GABAb = 0.1 gkbar_iAHP = 0.002 [Ca++]out = 0.1mM

Cell 2: AMPA = 0.1 NMDA = 0.01 gkbar_iT = 0.008 Base Current = 2nA


Cell 1 (Inhibitory) Cell 2 (Excitatory)Cell 2 reaches threshold and displays action potentials because iL increases

calcium influx an iC is calcium dependent potassium channel. I also added 3nA of base current to Cell 2 so that it does not stay hyperpolarized for long after

receiving an IPSP from cell 2.

L & C-Current Applied to Cell 2


Cell 2: AMPA = 3 NMDA = 0.01 iL = 0.000276 iC = 0.00345 Base Current = 3nA


Cell 1 (Inhibitory) Cell 2 (Excitatory)Cell 1 is very depolarized because there is an influx of calcium due to iL and iC current

which are dependent in the influx of calcium. When calcium binds to potassium channels it hyperpolarizes the cell so that

Cell 1 stops sending IPSP to Cell 2 and Cell 2 can then fire back an EPSP to Cell 1.

L & C-Current Applied to Cell 1

Cell 1: GABAa = 0.3 GABAb = 0.1 Base Current = 1nA iL= 0.003 iC= 0.004

Cell 2: AMPA = 3 NMDA = 0.01 Base Current = 3nA


Cell 1 (Inhibitory) Cell 2 (Excitatory)I had to add 5nA of current to Cell 2 so that it could fire because otherwise iM would cause Cell 2 to become inhibited, and would have otherwise stopped it

from producing an EPSP to Cell 1.

M-Current Applied to Cell 2

Cell 1: GABAa = 1 GABAb = 0.1 Base Current = 1nA

Cell 2: AMPA = 0.3 NMDA = 3 Base Current = 5nA iM = 0.001


Cell 1 (Inhibitory) Cell 2 (Excitatory)iM current is inhibiting Cell 2 so that it sends a weaker IPSP to Cell 2. I added base

current to Cell 2 so that it wouldn’t stay hyperpolarized. Otherwise, we wouldn’t be able to see action potentials from Cell 2, which would make Cell 2 stop sending EPSP

to Cell 1 and communication between the cells would stop.

M-Current Applied to Cell 1

Cell 1: GABAa = 1 GABAb = 0.1 Base Current = 1nA iM = 0.0009

Cell 2: AMPA = 0.1 NMDA = 3 Base Current = 3nA


Discussion- The more A-Current Conductance that is added to Cell 2 the less excitable it becomes which in turn, sends less IPSP signals to Cell 1.

- When a neuron is overexcited the amplitude of its action potentials decreases because it is firing at a faster rate than that of the recovery of sodium channels, meaning that without sodium available threshold can’t be reached and no action potentials are seen.

- When the excitatory neuron is stimulated so that it produces a strong EPSP the inhibitory neuron will respond strongly and inhibit the excitatory neuron so that communication between the neurons becomes interrupted.

- L-Current is a persistent current that lets calcium inside the cell. Once calcium is inside the cell, it can then help modulate other channels such as, C and AHP currents. AHP is calcium dependent potassium current.

- When T-Current is equal to zero, we don’t have Ca++ spikes but, we still get depolarization even though it does not reach threshold so that we observe no bursts. Given that it is active at hyperpolarized state, the cell that was observed without iT current needed a positive base currents.

- C-Current is calcium dependent potassium channel. Once calcium is inside the cell it tries to reach calcium equilibrium which pushes the membrane potential close to threshold where sodium channels act as positive feedback and start firing.At this more depolarized membrane potential potassium channels are activated leading to the hyperpolarization of the cell.

- M-Current is activated when current shows depolarization for a long time which means that iM can modulate long term neuronal states.

Literature Cited

"McKiernan (2013) Effects of manipulating slowpoke calcium-dependent potassium channel expression on rhythmic locomotor activity in Drosophila larvae. PeerJ 1:e57." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 01 Sept. 2013.

mod 5 -soley valencia

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