
Modulation of Internal Calcium and Synaptic 
Function by Neurotransmitters 
Steven A. Siegelbaum, Ph.D. — Associate Investigator 
Dr. Siegelbaum is also Associate Professor of Pharmacology in the Center for Neurobiology and Behavior, 
Columbia University College of Physicians and Surgeons. He received his A.B. degree in biochemical 
sciences from Harvard College and his Ph.D. degree in pharmacology from Yale University, studying the 
role of calcium in cardiac electrical activity. He then spent two years as a postdoctoral fellow in the labo- 
ratory of David Colquhoun at University College, London, and one year with Philippe Ascher at the Ecole 
Normale Superieure in Paris, studying the nicotinic acetylcholine receptor ion channel, before joining the 
faculty of Columbia University. He has received the Herbert J. Kayden Award in Biomedical Science of the 
New York Academy of Sciences. 
THE ability of neurons to regulate and alter the 
strength of their synaptic connections is 
thought to play a crucial role during learning and 
development. Changes in synaptic function can 
be brief, lasting seconds to minutes, or pro- 
longed, lasting days to months. One important 
mechanism by which neurons regulate synaptic 
transmission is by regulating the amount of neuro- 
transmitter released from the presynaptic cell. 
Since this release is triggered by the influx of cal- 
cium ions into the presynaptic terminal, it has 
been widely proposed that modulation of release 
results from the modulation of presynaptic cal- 
cium levels. However, there is little direct evi- 
dence to support this hypothesis. 
For the past several years we have focused our 
attention on the up- and down-modulation of neu- 
rotransmitter release from the terminals of Aply- 
sia sensory neurons in response to the mod- 
ulatory transmitter serotonin (5-HT) and the 
neuropeptide FMRFamide, respectively. Electro- 
physiological recording techniques have shown 
that 5-HT increases action potential duration by 
closing a particular potassium channel (the S 
channel) and increases an inward calcium 
current that is sensitive to block by dihydropyri- 
dine drugs (the L-type calcium channel) . In con- 
trast, FMRFamide increases the opening of the S 
potassium channel, decreasing action potential 
duration, and decreases current flow through a 
class of calcium channels not blocked by dihy- 
dropyridines (the N-type channels) . 
In a recent study, Brian Edmonds, Nicolas Dale, 
Marc Klein, and Eric Kandel (HHMI, Columbia 
University) have shown that only calcium that 
enters through the N-type calcium channels is 
able to trigger transmitter release. This conclu- 
sion was based on the finding that application of 
dihydropyridines to block the L-type calcium 
channels has little effect on transmitter release. 
Thus presynaptic facilitation with 5-HT is 
thought to result from an indirect increase in cal- 
cium influx via the N channels into the sensory 
neuron presynaptic terminals due to the increase 
in action potential duration (which allows the 
N-type calcium channels to remain open lon- 
ger). Presynaptic inhibition with FMRFamide is 
thought to result from a direct decrease of cal- 
cium influx via the N-type calcium channels. 
Do 5-HT and FMRFamide modulate calcium in- 
flux during an action potential in the presynaptic 
terminals of sensory neurons? To what extent is 
such modulation due to changes in calcium in- 
flux via N-type as opposed to L-type calcium 
channels? Why doesn't calcium entry via the L- 
type channels contribute to transmitter release? 
To address such questions we have used the 
fluorescent dye fura-2, developed by Roger Tsien 
(HHMI, University of California, San Diego), 
which binds calcium and reports intracellular cal- 
cium concentration, to study the effects of 5-HT 
and FMRFamide on intracellular calcium levels in 
response to action potential stimuli. In a previous 
study we showed that the action potential- 
induced calcium transient was increased by 5-HT 
and decreased by FMRFamide in sensory cell bod- 
ies, growth cones, and neurites. Over the past 
year we have investigated whether such modula- 
tory changes occur at presynaptic terminals, by 
coculturing sensory neurons with postsynaptic 
motor neurons. Pseudocolor images of intracel- 
lular calcium levels were obtained at presumed 
presynaptic sensory neuron terminals at regions 
of contact between sensory and motor neurons. 
The intracellular calcium transient induced by 
action potentials was markedly enhanced by 5- 
HT. These results show that presynaptic calcium 
transients are modulated at defined regions of 
contact between sensory and motor neurons. 
Why is it that calcium influx via the L-type 
channels does not contribute to transmitter re- 
lease? One possibility is that during an action po- 
tential, the L-type channels contribute relatively 
little to total calcium influx. Alternatively, these 
channels may carry substantial amounts of cal- 
cium into the cell, but the channels may be local- 
ized in regions of the cell far from the site of 
synaptic contacts. 
407 
