BIPN 105 Sciatic Nerve and NMJ

What did we observe in the sciatic nerve lab?

Axon function, we cut cell bodies and terminals to just have axons

Membrane potential (V_m)

• sodium potassium pumps set up and maintain V_m

  • maintain more potassium inside, low sodium inside

• Determined by the Goldman Equation

Goldman Equation

potassium permeability is much higher than sodium due to significantly more leak channels being available

at 37 degrees C

Equilibrium Potential

• use nernst equation

• E_K=-90mV

• E_Na=+61mV

• E_Cl=-70mV

• when the chemical potential is equal to the electrical potential

Action Potential

an all or none response to depolarization

Action potential Phases

• are generated by voltage-gated ion channels activity dependent on the V_m

• Depolarization, Repolarization, Hyperpolarization

Depolarization

Triggered by sodium channel activation as a result of depolarization, the pore activates creating a pathway for sodium influx. The electrochemical gradient is driven by the very positive equilibrium potential of sodium. This facilitates sodium entry further depolarizing the membrane. This triggers a cascade of sodium channel activation until all channels are engaged

Repolarization

The time dependent inactivation gate of the sodium channels closes blocking the pore and potassium channels activate opening their pore as a result of depolarization, allowing the potassium to exit resulting in a rapid repolarization since the equilibrium potential of potassium is highly negative

Hyperpolarization

when you have excess potassium permeability and excess potassium current leading to the membrane potential to go past Vrest.

Return to V_rest

Potassium channels deactivate as a result of repolarization and sodium channels remove their inactivation gate as a result of repolarization resulting in the deactivation of sodium channels. Then the membrane potential returns to rest as a result of leak channels being the only open channels

Refractory Period

how long a neuron must wait before it can fire a second AP

Absolute Refractory

No matter the strength of a second stimulus, there is no resulting AP due to the sodium channels being inactivated

relative refractory

when a larger second stimulus could yield a second action potential due to the hyperpolarization phase and high permeability of potassium

Lab assumption for refractory

The stimulus is large enough that any neuron in RRP will fire a second action potential and if the neuron does not fire a second action potential, it is in its absolute refractory period.

Conduction velocity

how fast an action potential propagates down an axon

What increases conduction velocity?

• larger diameter

• greater myelination

• fiber types

myelination

a lipid bilayer wrapped around the axon that acts as insulation so passively spreading depolarization doesn’t drop below V_T

Saltatory Conduction

AP jumps from one node of ranvier to another and only regenerates AP at the nodes resulting in much faster AP spread

Fiber types

A fibers - large myelinated, 20-100 m/s

C fibers- small unmyelinated- 1-2 m/s

Compound Nerves

bundles of different types of axons

CAP

• summation of individual APs; this is what is measured from the sciatic nerve by extracellular recording

• not all or none

• max CAP is when all neuron types have been recruited

Stimulus

• using an electrical stimulator

• enough positive ions accumulate to reach V_T starting the AP at the negative

  • AP goes both ways

• at the positive stimulating electrode there is hyperpolarization,

Extracellular Recording

Recording electrodes only measure the voltage outside the neuron near the electrode- DOESNT measure membrane potential

• intracellluar recording would be for a single neuron, extracellular for the nerve

• Record the positive and then subtract the outside voltage of the negative recording electrode, if the AP is too far it will not be recorded by either axon

• the max diff in - and + gives a large positive deflection

• negative monopolar- - recording is subtracted from the ground

Sciatic Nerve

mainly A fibers, record with - first by convention to yield a positive deflection, using an AC couple

Conduction velocity in the Lab

position E- Position D/ latency at E- latency at D

refractory period lab setting

double pulse with decreasing interpulse interval , maintain delay and duration

CAP2/CAP1 decreases= less neurons recruited and reaching Vt, IN ARP

Stimulus polarity results explained

with the negative stimulus, the positive stimuluating electrode serves as the negative and the negative stimulating electrode serves as the positive therefore there is hyperpolarization that the AP must pass through resulting in a lowered stimulus potential and thus less neurons reaching threshold and a lower cap

this also increases the distance from the stimulating electrode resulting in an increased latency

Strength-Duration

threshold depends on amplitudes and duration of the stimulus.

• decreased duration requires a greater stimulus amplitude to yield a criterion sized CAP

Rheobase

the minimum voltage to elicit a criterion sized CAP at infinitely long durations

chronaxie

a measure of nerve excitability, smaller chronaxie means the nerve is more excitable . Duration of the stimulus needed when amplitude is twice the rheobase

Neuromuscular Junction Anatomy

1. vesicles

   1. 300,000/ terminal

   2. 10,000 ACh/ Vesicle

2. synaptic cleft

   1. 30 nm wide

   2. filled with basal lamina

3. receptors

   1. n_mAChR

      1. respond to ACh- cholinergic

      2. sensitive to nicotine- nicotinic

      3. on skeletal muscle

      4. Vrest is close to Ek

         1. Fk= very small

         2. FNa= very large

            1. therefore, despite being a mixed ion channel, mainly sodium enters for depolarization

n_mAChR

• ligand dependent ion channel

  • requires 2 ACh to activate

  • nonspecific mixed ion channel

Nerve AP→ MAP

1. Aα motorneuron→AP→ Presynaptic Terminal→ activates P/Q Ca⁺⁺ Channels→Ca⁺⁺ influx

2. vesicle exocytosis (~300)→ACh release→ ACh diffuses across the cleft→binds to n_mAChR

3. n_mAChR activation→ postsynaptic depolarization→ EPP is graded (increase ACh increases EPP)

4. MAP?

   1. small ACh?→ EPP too small to reach V_T, no MAP

   2. enough ACh→EPP> V_T→ get MAP!

1 NMJ per muscle, in the middle of the muscle cell

What must EPP do for a MAP

the EPP starts in the center of the muscle cell and must activate the sodium channels for the MAP

Termination

1. stop exocytosis

   1. ca++ diffuses away from the vesicles

   2. [Ca++]in—ATPase Pumps—>[Ca++] out

2. ACh disassociates from n_mAChR→deactivates→ EPP dissipates

3. ACh removed due to the fact that as long as it is in the cleft it will activate receptors

   1. diffuses away and enzymatic breakdown in cleft ( ACh—AChE→ choline+acetate

      1. ACh is removed in <1 ms

Recycling

1. ACh broken down into Choline by AChE

2. Choline uptake into the presynaptic terminal by the choline- Na+ cotransporter

   1. the Na+ concentration gradient from the Na+-K+ pumps allows this

3. choline +AcCoA—ChAT(choline acetyltransferase)—>ACh+CoA→ put in vesicles by VAChT

Fatigue

• after high frequency stimulation

• store 300,000 vesicles/terminal, release 300 vesicles/ AP

  • deplete ACh/ vesicles resulting in fatigue

• Recovery requires the formation of new ACh/ vesicles which may take seconds to minutes

Synaptic Delay

• delay between presynaptic depolarization and postsynaptic depolarization

• around 0.5 ms due to

  • release of NT ~0.3ms

    • longest step

    • if you slow this step, you increase synaptic delay

  • diffusion across the cleft ~0.05ms

    • fastest step

  • n_mAChR activation ~0.15 ms

• IN LAB, the delay is from presynaptic depolarization to MAP recording, not the beginning of the EPP. This adds the time that sodium enters the cell, time the EPP needs to reach threshold, the time to fire an action potential and the time for the AP to spread throughout the muscle membrane to be detected by recording

Synaptic Delay calculation in Lab

• MAP latency- conduction time

  • conduction time: distance from - stimulating electrode and negative muscle recording electrode divided by the CV of the nerve

  • MAP latency- onset of the MAP- onset of the stimulus

Facilitation

• may occur if multiple stimuli are used

  • does NOT change the AP of the motorneuron, these are all or nothing

• with a wide interval , there is no facilitation

• with a short interval

  • residual intracellular Ca++ near presynaptic terminal membrane increases P/Q channel activation, sensitive to calcium channels so they activate better increasing calcium influx

    • if the stimulus occurs very quickly exocytosis doesn’t get rid of all the calcium from the previous

  • increased Ca++ influx caused increase ACh release and therefore a greater EPP every time due to more nicotinic receptor activation

Nerve vs SKM comparison

• V_rest

  • Aα motorneuron: -70 mV

  • SKM- -90mV

• V_threshold

  • Aα motorneuron: -50 mV

  • SKM- -50mV

• ARP

  • Aα motorneuron: 1 ms

  • SKM: 3-4 ms

• CV

  • Aα motorneuron: 20-100 m/s

  • SKM: 1 m/s, no myelination

Other NT/ Receptors

• Glutamate

  • glutaminergic receptors

  • CNS excitatory

• GABA

  • GABAnergic

  • CNS inhibitory

• Norepinephrine

  • adrenergic

  • sympathetic→postganglionic neuron (released)→tissue CNS receives

ACh

• cholinergic

  • can be blocked with atropine

  • nicotinic

    • NMJ (n_mAChR)

    • ANS+ CNS n_NAChR

• muscarinic

  • parasympathetic: postganglionic neuron releases it onto CNS tissue

Myasthenia Gravis (Symptoms, Cause, Diagnosis, Treatment)

• Symptoms

  • muscle weakness/ fatigue with repetitive use

  • starts in small cranial muscles

    • extraoccular- double vision when reading for a while

    • throat- swallowing problems after a while

  • progresses to larger muscles

    • arms, legs, breathing?

• Cause

  • autoimmune attack on n_MAChR

• Diagnosis

  • have the patient do repetitive contractions until fatigue starts

    • squeeze a ball, hold arms above head, etc

  • inject edrophonium ( short acting AChE inhibitor)

    • 30 seconds→ decrease fatigue

    • 10 minutes→ fatigue returns

• Treatment

  • long acting AChE inhibitor

  • anti-immune therapy

Drugs applied in lab to?

middle of the muscle

For fatigue and recovery

we used the grass stimulator with no delay at repetitive stimulation for 4 minutes

Why is stimulus artifact smaller in the MAP than the CAP

• we used two grounds

• there is a longer distance between stimulating and recording electrodes resulting in passive spread dissipating

• the artifact hits both recording electrodes at the same time and thus is subtracted from the recording- MAP is bipolar

Normal synaptic delay range in lab

1-3 ms

why would a CV be too low?

• nerve is damaged

• CAP didn’t record right, they are too far apart