Neurophysiology – lecture 24

April 12, 2011

1 Review:
1. We began a discussion of Chemical Synaptic Transmission
2. Of the rather long sequence of events that occurs during chemical synaptic transmission we shall only
discuss 1) the presynaptic currents leading to transmitter release and 2) the generation of postsynaptic
currents and potentials.
3. Most of our understanding of chemical synaptic transmission comes from study of the neuromuscular
junction (NMJ). This preparation is relatively easy to study because:
(a) There is but 1 axon forming a synapse on each muscle fiber.
(b) This axon is easy to stimulate and the muscle fiber is easy to record from.
4. Next we asked what couples the presynaptic A.P. to neurotransmitter release?
The available data suggest that a change in the Ca2+ concentration inside the terminal triggers the
release of neurotransmitter.
5. The First Test of this conclusion is to ADD Ca2+ and then measure the postsynaptic response to
determine if synaptic transmission had occurred.
(a) Ca2+ was added to the bath containing the nerve-muscle preparation and the nerve was stimulated.
As extracellular Ca2+ was raised above 0.5mM the postsynaptic response increased markedly.
(b) If extracellular Ca2+ near the test NMJ was increased from 0mM into the mM range by ionophoretic
application of Ca2+ , a presynaptic A.P. began to produce a postsynaptic response. (Figure 16D)
(c) If “caged” Ca2+ is suddenly liberated inside the presynaptic terminal of the squid giant axon
synapse by a UV flash, then a postsynaptic response is produced.
6. The Second Test of this conclusion is to SUBTRACT Ca2+ from the inside of the presynaptic terminal
to see if this removes the postsynaptic response which otherwise would be present.
(a) Mg2+ was added to the extracellular fluid around the NMJ to block the Ca2+ channels in the
presynaptic terminals and thereby prevent Ca2+ from entering the terminal during or after an
A.P. With Ca2+ channels blocked no post synaptic response was produced.
(b) BAPTA was injected into the presynaptic terminal of thegiant axon synapse of the squid. BAPTA
blocked neurotransmitter release by “chelating” the Ca2+ which entered the terminal during an
A.P.

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2 Concomitant Variation
A Third Test of this conclusion requires measuring both Ca2+ inside the terminal and the postsynaptic
response after an A.P. invades the terminal and then correlating these 2 measures. This approach is called
“Concomitant Variation”.
Intracellular Ca2+ can be measured with the aid of fluorescent dyes whose fluorescence increases after they
have bound Ca2+ . These dyes can be injected into the presynaptic terminal of the giant squid synapse.
As shown in Figure 16D when the preparation was not stimulated to produce an A.P. there was little
fluorescence present inside the terminal. But when an A.P. invaded the terminal there was a marked increase
in fluorescence detected in the presynaptic cell.
This fluorescence occurred in a very small area and was present for but a few milliseconds. These fluorescent
flashes are aptly called “sparks” and indicate that Ca2+ concentration is high in the presynaptic terminal
only in very localized spots and for a very brief time. These localized areas of high Ca2+ concentrations are
referred to as “microdomains”.
The correlation is that “sparks” lead to transmitter release (which cannot be measured) and that leads to
the observed postsynaptic response.
From the 3 lines of evidence cited above it is concluded that a rise in [Ca2+ ] in the presynaptic terminal
produces neurotransmitter release.

3 Microdomains
Why does the increase in intracellular Ca2+ during a depolarization appear as localized and short-lived
“sparks”?
1. Note that the [Ca2+ ] increase is very localized and lasts for only milliseconds since the Ca2+ diffuses
away rapidly into the cytoplasm. This pattern of Ca2+ influx suggests that each spark is produced by
the Ca2+ concentration increase occurring close to a Ca2+ - selective ion channel during an opening of
that channel.
2. These Ca2+ channels must be voltage dependent, since a depolarization of the terminal markedly
increases their probability of their being open.
3. In searching for the relatively small Ca2+ channel influxes (i.e., Ca2+ channel currents) the much
larger currents carried by Na+ ions through Na+ channels and by K+ ions carried through K+ chan-
nels must be eliminated. Therefore, the Na+ channels and currents are blocked by tetrodotoxin (ab-
breviated TTX) and the K+ channels and currents are blocked by tetraethyl ammonium (TEA)or
3-aminopyridine. (See Figure 17A.)
4. The Ca2+ channels which have been identified are characterized by several properties:
(a) 1) by their voltage dependence. There are 2 major varieties LVA - low voltage activated, and
HVA - high voltage activated.
The probability of a LVA channel opening begins to increase for depolarizations at and above -60
mV and increases to 1 as the transmembrane potential is further depolarized to -20 mV.
The probability of HVA channels opening does not begin to increase until depolarizations to -
20mV and above are reached and increases to 1 when the transmembrane potential reaches about
+20 mV.
(b) by their kinetics - fast or slow to activate.

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 (c) by whether they inactivate or not when the transmembrane potential is held in at a depolarized
level.
(d) by where they are found in the nervous system
(e) by their sensitivity to drugs or toxins derived from all sorts of animals - snakes, frogs, snails,
spiders, etc. and pharmaceutical companies.
5. Analysis of the residual currents left in dorsal root ganglion cells after block by TTX and TEA showed
there were 3 types of Ca2+ currents and channels called “T”, “N” and “L” were found in these cells.
Subsequently, two additional Ca2+ channel types labeled “P/Q” and “R” have been found in other
neural tissues. Where “L” stands for “long-lasting”, “T” stands for “transient” , “N” stands for
“neuronal”, “P” stands for “Purkinje” and “R” stands for “residual.”
6. Table of Calcium channel types:
Type Locus Voltage Dependence Kinetics Blocked by
L muscle, gland cells HVA Slow, no inactivation dihydropyridines (DHP)
T some neurons LVA Fast, inactivates Ni2+ (nonspecific)
N neurons at synapses HVA Slow, slow inactivation ω-conotoxin
P/Q Purkinje cells, synapses HVA Slow, slow inactivation ω-agatoxin
R neurons HVA intermediate tarantula toxin (SNX-486)
7. At the top of the table some of the properties of these channels are indicated. For the purposes of
studying synaptic transmission we need only be concerned with channel types N, P/Q and R, since
these are the channel types that are found in synaptic terminals and that are engaged in synaptic
transmission.
8. Of the information on this table the information which is relevant for synaptic transmission is the
fact that these 3 channel types all are HVA channels, they have fast channel gating (i.e., they open
with considerable delay when the synaptic terminal is depolarized and then close fairly rapidly), they
regulate neurotransmitter release, and there is a specific toxin which binds to and blocks each one of
them.
Thus, ω-conotoxin (ωCgTX), a toxin derived from marine cone snail venom, specifically blocks N
channels.
On the other hand, ω-agatoxin (ω-AgaIV), a toxin derived from funnel web spider venom, specifically
blocks P/Q channels.
And SNX-4861 , a synthetic compound based on the venom of tarantulas, specifically blocks R channels.
9. Figure 17B shows the time course of Ca2+ current which flows into neurons through each of these
channel types when an action potential of the form shown at the top of the figure occurs in the
synaptic terminal containing the channel type. Note 2 things in this figure:
(a) the channels do not begin to open and pass current until well (2.5 ms or more) after the onset of
the action potential and
(b) for both P/Q and N type channels the influx of Ca2+ into the terminal rises rapidly and then
falls rapidly as the action potential voltage falls.
10. At many types of synapses electrical stimulation of the presynaptic axons will produce a large post-
synaptic potential. Addition of ω-conotoxin will reduce the amplitude of this postsynaptic potential
indicating that there are N channels present in the synaptic terminal membrane. However, addition
of ω-agatoxin may also reduce the amplitude of the postsynaptic potential indicating that there are P
channels in the synaptic terminal membrane.
1I can find only limited documentation on SNX-486. It seems that SNX-482 is more common

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 Finally addition of both ω-conotoxin and ω-agatoxin often will more drastically reduce the postsynaptic
amplitude but not completely eliminate it, which suggests that there are R channels also present in
the synaptic terminal membrane.
11. It is common that there is more than one type of Ca2+ channel present in presynaptic terminals.

4 Ionic selectivity
1. Channels of all types are not perfectly ion selective. For instance, Na+ channels show the following
pattern of ion selectivity:
Na+ : Li+ : Ca2+ : K+
1 : 1.1 : 0.1 : 0.083
2. Ca2+ channels are relatively unselective. Ca2+ channels are more permeable to Ba2+ than to Ca2+
and also more permeable to Sr2+ .
3. However, if Ba2+ is used to replace Ca2+ in the extracellular medium outside an NMJ and the axon
to the junction is stimulated to produce an A.P., no postsynaptic response will be recorded. The
neurotransmitter release machinery does not respond to Ba2+ .
4. Thus, ion specificity in Ca2+ systems can be determined not only by the ion channel but also by the
enzymes or intracellular binding proteins that are activated by increases in intracellular Ca2+ .