physio lec 1/2/97-----start-----
E.Neil Moore, DVM, PhD

Handout: NEUROPHYSIOLOGY LECTURES, 14 pp

Dr. Spears == course coordinator. He reminds us to go over the handouts 
we got in our boxes. This course will have 3 labs, multiple instructors, 
4 exams. See handout for details.

adams-kerr group A
knighton-end of alphabet group B

[hillary: you are group A!!]

Note that Dr. Moore is using a laptop for his presentation, so he can't 
be all bad.

Dr. Moore's exam questions will come from the handout, which will take 
the place of note service. There are illustrations in the handout on the 
last three pages.  Dr. Moore won't be correcting note service this 
quarter, it seems. He recommends Guyton's Physiology textbook as a good 
book. Dr. Moore remarks that he will not be LECTURING directly from the 
handout but that we will be RESPONSIBLE for the handout.

EXAM INFO: "my exams are all the same, i just change the answers"
a series of true/false statements: select the one false statement from 
these 4

Lecture overview: stimulus->receptor->nerve->synapse CNS->muscle

We will be skipping a lot of the CNS entirely but by the end of these 
lectures we should understand how a stimulus alters a receptor, and how 
the receptor gives rise to a potential which is sent in "digital format" 
along nerve to CNS and is modulated again causing action potential and 
resulting in muscle action.

Why does one have potentials existing across an excitable membrane? Why 
are there electrogradients? how do these gradients come about and give 
rise to action potentials?

Excitation is Electrical

if you take two nerve/muscle preps from a frog, and two types of metal eg 
copper and zinc, and place them on a damaged area of the muscle (crushed) 
nothing happens, but if you touch one of them to the nerve and the other 
to the crushed area, it will cause a contraction. also showed that static 
charge can cause muscle contraction, or those lightning jars they used to 
have. it's been known a long time that electrical events cause muscle 
contraction.

some guy took frog heart and a nerve/muscle prep. when nerve placed on 
heart, it would cause muscle to contract with each heartbeat - it 
conducted the impulse to the muscle. if the nerve was crushed, it no 
longer conducted the impulse, and the muscle remained relaxed.

Lippmann capillary electrometer - records nerve action potentials
what this does, it's an extracellular recording - an electrode is 
attached to the muscle or nerve and another into the electrometer acid 
tube, and the meniscus will move up and down with electrical events. in 
this way in the 1800s they were able to record action potentials. there 
were other older methods using string galvanometer etc. so it's been 
known for many years that electrical events are associated w/excitation.

Bernstein in 1902 suggested that there is a concentration difference 
across the cells between intra and extracellular levels of potassium. So 
there is an ionic basis for nerve potentials. this acts like a battery. 
bernstein thought the nerve potential was due to this "potassium battery" 
existing. what he said is that there is this gradient of potassium 
concentration, so if you make the membrane impermeable and then permeable 
it's like flipping a switch on or off. so he suggested that the impulse 
is generated by changes in membrane permeability to potassium ion. 

we know that there are ion differentials - K+ is high intracellular and 
low extracellular, Cl- high extracellular and low intracelllar, Na+ high 
extracellular and low intracellular, and lots of impermeable anion inside 
locked inside the cell - important in establishing initial concentration 
gradients and maintaining them. KNOW the concentrations. these gradients 
are in all excitable tissues and form the basis of excitability.

How did these gradients come about? Donnan Equilibrium: ions equilibrate

given a semipermeable membrane, if one adds KCl to one side of the 
membrane, it will pass through back and forth between the membrane, until 
the concentration equilibrates such that there is electroneutrality - 
equal concentrations on both sides of the membrane. We know that there 
are a lot of other ions involved besides K+ and Cl=. The DOUBLE DONNAN 
equilibrium:

there are these impermeable anions which can't go across the membrane 
because they are too large. So if you add them to the situation described 
above, the K+ and Cl- on one side are no longer in 
electroneutrality/equilibrium w/the K+ Cl+ and A- on the other side. So 
you will get a different osmolarity across the membrane. On one side 
there is KCl in solution, and the other side has the A- as well, so the 
osmotic pressure is different, because there is a different number of 
ions, because you need more K+ to balance the A- on that side of the 
membrane, so water will go to that side as well, to balance the osmotic 
pressure. So you get a "non-osmotic" equilibrium. this is bad in 
biological systems: the cells will burst. So, if you now add NaCl to the 
system, to the side with the A-, ions start to redistribute across the 
membrane. Na+ and Cl- move over to balance out the K+ and A- on the other 
side of the membrane.

Now, the thing is we end up with lots of K+ inside the cell, anion in the 
cell, and bound Ca++ in the cell so very little ionized Ca++ in the cell. 
Lots of Ca++ outside the cell, Cl- and Na+ high levels outside the cell. 
Ca++is good signal molecule because it's bound within the cell so it 
won't equilibrate within the cell freely as does potassium, sodium, etc.

squid nerve action potential:

back in 1936 it was seen that the axon was large, very big, millimeter in 
diameter. it could be dissected out and used for experimental puporses. 
You could actually thread a silver wire through the axon. Young suggested 
the use of the squid in 1936 and it has proven quite useful. earthworms 
also have large axons, and are cheaper than squid. lobsters also have 
large axons but are quite expensive. but, you might eat the lobster after 
dissecting out the axon... :)

Curtis and Cole in 1939 did cool exp't with squid before WWII. one of the 
neat things they did was they took a wheatstone bridge 
(R1=R2=R3=Rnerve===>no measured voltage across the cross connect) and 
showed that if you excited the nerve, the resistance would fall 
considerably so the bridge was no longer in equilibrium. this showed that 
the membrane's resistance was decreasing, meaning the conductance was 
increasing. so you can think of conductance as permeability - and as 
permeability increases, resistance will fall. Curtis and Cole also showed 
that there are conductance changes occuring in this way and they showed 
capacitance changes preceeding the action potential as well. so we knew 
we could find these differences, before WWII. 

Alan Hodgkins came over in 1938 or 39 and worked on this in the US but 
then was sent into radar research during the war. Then ANdrew Huxley - a 
british mathematician - worked with him, and after the war andrew huxley 
worked with him and they started working on physiology, and some other 
guy worked with them and during WWII the cathode ray oscilloscope was 
made. SO after wwII with this oscilloscope they didn't need to use old 
ways of looking at APs. They used the oscilloscope. An oscilloscope is 
interesting, it has plates within the tube and there is a potential 
across the plate, and it will make the fluorescent beam move up and down. 
so if you put an action potential across it at the amplifier and another 
potential at the time base unit you can make it sweep across. 

now, with digital computers, you don't really need an oscilloscope.
we won't be tested on how an oscilloscope works, he says, but it is 
interesting to know, he says.

Bernstein 1902 potassium. 
excitation seemingly develops secondary to concentration gradient of 
potassium across the cell membrane. now, tests showed the potential 
didn't go from zero to -80, but actually entered a positive state. This 
indicated that when no potassium permeability present, it's not zero, but 
positive, so there must be other ions involved. if only K+ involved, 
would be zero when impermeable, or -80 when permeable.

Microelectrodes: one micron tip diameter: ling and gerard 1949. these 
microelectrodes could record the transmembrane potential between the 
inside and outside of the cell. when the electrode is placed through the 
membrane the membrane seals up around it and you can record the gradient. 
You fill the electrode with 3 M KCl - so no potassium leaks out of it. if 
you used, say, 6 M KCl, some would leak into the cell. Now, 
microelectrodes aren't usedmuch, something has replaced them but I 
coudn't hear what it was.

----break----

This professor is a trip. He's playing intermission music from his 
laptop, which by the way has been making silly noises at random intervals 
all through this lecture.

Back to recording of transmembrane potentials....
using a microelectrode with the appropriate electrolyte solution, you can 
record the transmembrane gradient. as previously mentioned, when one does 
an intracellular recording, instead of going up to zero and back down, 
one finds a POSITIVE +35 mV potential. How does this positive potential 
come about? That interested people. Hodgkins and huxley worked it out 
mostly.

The hypothesis they proposesd was that sodium was responsible for the 
positive AP.

K+ has a 90 mV potential across the membrane.
the Nernst equation  : in order for a concentration gradient to persist 
there must be a simultaneous electrical gradient and they must resolve to 
zero. 

driving force= conc.grad + elec.grad = zero

if you have a potential than you can predict the gradient between the 
outside and inside. that's what you need to remember. If you change the 
gradient, the potential will also change. this is important.

if you have a concentration gradient, you MUST have an elec gradient and 
that elec gradient can be predicted with the Nernst equation. A high conc 
gradient results in a high potential, low gradient --> low potential. 

in the dog, [K+] in serum 3.5-4 or so. W/renal dz, [K+] increases to 6 or 
more. Therefore potentials change. Diuretics or GI problems can cause 
potassium losses, lowering membrane potentials.

If you have a ratio of 1:30 and you double the serum concentration you 
change the ratio to 1:15 and you have greatly changed the potential. When 
you gain or lose potassium you see EKG changes and clinical problems.

nernst eq basically says that the elctrical gradient = a constant times 
the extracellular concentration of K+ divided by intracellular 
concentration of K+

If you INCREASE the extracellular potassium level, it is similar to 
decreasing the weight pulling on a spring - the difference between the 
levels is not as much, the potential is decreased (spring tension 
decreases). so a lower concentration gradient requires a LOWER electrical 
gradient to maintain it.

if you DECREASE extracellular K+, it's like INCREASING the wt on a 
spring. it INCREASES the potential. It makes the concentration gradient 
BIGGER.

***********In order to have no net driving force, the concentration 
gradient must be equalized by an opposing electrical 
gradient.******************

how do we get a positive potential? well, if the sodium is present in 
high levels extracellularly, so you change the ion direction, then the 
potential is going to change from negative to positive. the concentration 
gradient wants to let sodium go into the cell, so there must be an 
electrical gradient present to allow the conc.gradient to persist. so the 
electrical potential will be POSITIVE.

[that laptop of his just started LAUGHING tee hee]

Goldman constant field equation:
looks like the nernst equation, corrects for the presence of Na and Cl. 
you use the relative permeabilities of the membrane to the various ions. 
the permeabilites and the gradients are different for each ion.

Baker, hodgkins and shaw: toothpaste experiment

axoplasm squeezed out of axon, microelectrode inserted and cell perfused 
with ion solution and potentials recorded. this allowed them to discover 
the relative permeabilities for various ions from inside to out and 
outside to in. this validated that goldman equation quite nicely.

Ion sensitive electrodes: you can use special K+ sensitive electrodes to 
sense only potassium current. other electrodes are extant which sense 
only other ions. so you can do very specific work. now, cardiac cells are 
too small to do this with, you can't squeeze the cytoplasm out.

LAG POTENTIAL: diffusion potential...

why do we see these + and - potentials, really? this concept of lag 
potential may help to see which way the gradient will actually go. 
consider that the membrane is highly permeable to potassium and potassium 
goes OUT into ECF, leaving a net negativity on the inside, because the 
Cl- can't follow rapidly, because Cl- travels AGAINST The concentration 
gradient to leave the cell to balance out the leaving K+. So the inside 
of the cell will be NEGATIVE.

VOLTAGE CLAMP TECHNIQUE
gave a lot of money to hodgkin and huxley in 1963 - they won the nobel 
prize.
what it does is allow one to pass current so you can set the membrane 
potential. you record what the potential is, you set the potential you 
want, and you pass current into the cell so you get the potential you 
want.in this way you can record how much current is required to maintain 
a specific potential. the inward current is sodium current, and outward 
is potassium. 

realize action potentials are very FAST. when you cause an action 
potential it happens in msecs. the voltage clamp recordings allowed you 
to see what currents would cause them. in this way, they discovered that 
there is an initial influx f sodium - so an increase in permeability to 
sodium is an early action potential event. 

so, tetrodotoxin was used to close the sodium channels. later tracings 
showed no sodium current at all. TEA, tetraethyl ammonia, blocks voltage 
gated K+ channels, when this was added, no K+ current showed on the 
tracings. 

there are a lot of channels in the squid axon - there is a potassium leak 
channel, there are sodium and potassium voltage gated channels. the 
potassium leak channels account for resting potential. depolarization is 
secondary to opening of sodium channels, and repolarization occurs when 
voltage gated potassium channels open.

sodium channel also allows hydrated lithium in, but hydrated potassium is 
too big to get in. 

VOLTAGE GATED CHANNELS:
when closed, covered by gate, membrane is polarized, potential exists
when open, there is an aqueous pore, membrane is DEPOLARIZED.
the gate holds an ion selective filter to determine what kind of ions get 
through. ions go through quickly, no carriers are involved, no ATP needed 
to get ions through.

slide of human cardiac sodium channel: very complex looking!
some guy named brian matthews became rich by discovering something about 
cathode ray tubes? now he's going off on a tangent on new job opps for 
vets because PhDs are all so molecule oriented. anyway, someone cloned 
the human cardiac sodium channel; it's made of tons of proteins that have 
been sequenced. there is some QT mutation that occurs but he didn't say 
the significance...oh, QT segment prolongation - genetic - (this syndrome 
is correlated with sudden death)- these patients have 3 AAs out of 
sequence in their cardiac sodium channels. Dr moore is also discussing 
the transgenic mouse work of Ralph Brinster who has transfected thin mice 
with the "fat" gene and gotten them to express it and become fat mice. he 
made a lot of money - won two prizes this year, and now he has a tax 
problem. i don't know why he's going off like this - now he's talking 
about the salary of that urologist who makes over a mill...I'm sure he 
has a point but I'm not sure exactly what....sorry.

Ralph is a potential nobel prizewinner according to Dr.Moore. 

ANyway. the plasma membrane is very THIN. 5 nm thick. voltage gradient 
100,000 V/cm WOW. proteins are very sensitive to voltage, so this really 
is quite significant even though the potentials are very small, they are 
RELATIVELY quite large. so the ion channels have a lipid layer, and 
aqueous pore, a gate, a voltage sensor,and selectivity filter. 

FUGU: japanese puffer fish: tetrodotoxin, ttd, ttx; is a voltage gated 
sodium channel blocker. the toxin is found in the ovaries and liver of 
the fish in high concentrations. "fat man" toxin (?) it sits on top of 
the channel and blocks it. if you eat a subfatal amount you get a kind of 
loopy feeling or something that people like, when the muscle tissue just 
has a few of the channels blocked but not all of them. you can use the 
toxin to count the channels by tagging the toxin, because it will mark 
the channels, and can also use it to see the gating current.


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