Selective recruitment of the reserve pool of vesicles by serontonin in motor nerve terminals

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Selective recruitment of the reserve pool of
vesicles by serontonin in motor nerve
terminals S. Logsdon, A. Johnstone, and R. L.
Cooper Dept. of Biology, Univ of Kentucky,
Lexington, KY 40506-0025
Results
Introduction
The number of vesicles used for neurotransmitter
release is limited, so vesicles must be retrieved
from the NMJ to be reused for additional
neurotransmitter release. Vesicles are readily
available for recurrent fusion through several
different mechanisms. There is evidence
suggesting that when a vesicle recycles after
release it might be able to follow two different
routes for recycling a rapid loop, or a slower
one which reprocess the vesicle within an
endoplasmic reticulum (Klingauf et al., 1998
Kuromi and Kidokoro, 2002 Richards et al., 2000
Stevens and Williams, 2000). This might produce
two slightly different pools of reserve vesicles.
Results in the field of synaptic transmission
predict that electrical stimulation of a nerve
terminal can selectively utilize a rapid
recycling population of vesicles separately from
vesicles that are kept in reserve. Recent
findings of vesicular kinetics and recycling
processes also support the notion that it might
be possible for recycling in the presynaptic
membrane to occur in different pathways. It is
possible that multiple paths might be regulated
independently by second messengers, such as those
activated by 5-HT. However, direct electrical
stimulation of nerve terminals could use a
different intracellular path than is used for
5-HT. In the crayfish motor nerve terminals, one
route might be differentially influenced by 5-HT
via secondary messenger activation, as compared
to another route that might be more tightly
regulated by electrical activity patterns of the
terminal. Such a differential regulation is
reasonable since incubation of the NMJ with 5-HT
in the absence of persistent electrical activity
results in more vesicles being released with a
single or within a train of stimuli. The
neuromodulator serotonin (5-HT), which is
endogenous to the hemolymph of crustaceans,
greatly enhances transmitter release (Dudel,
1965a) presynaptically at the neuromuscular
junction by increasing the probability of
vesicular fusion (Southard et al., 2000). 5-HT,
at a high concentration (1 µM), also causes
spontaneous fusion of vesicles from the
presynaptic membrane by a mechanism that has not
been previously described. We have noted that in
the presence of dl-threo-beta-benzyloxyaspartate
(DL-TBOA, 10µM), the glutamate uptake blocker,
with repeated stimulation the synaptic responses
(i.e., EPSPs) attenuate in amplitude. The evoked
EPSPs depress with TBOA since the rapidly
recycling vesicles are depleted of glutamate.
Thus, the pool of evoked vesicles for release
might not be depleted, but they are recycling
with little or no glutamate present. However,
when 5-HT is added in the presence of TBOA, a new
pool, also referred to as a reserve pool, of
vesicles are recruited which have glutamate
already packaged in them. We suggest that the
population of readily recycling vesicles during
electrical activation of the nerve terminal is
distinctly separate from reserve
vesicles. Studies about mechanisms that regulate
the use of presynaptic vesicles in motor nerve
terminals at chemical synapses provide insight
into the mechanisms of vesicle recycling and the
kinetics within neurons. These studies are very
relevant to humans as well as general principals
in biology since chemical communication among
nerves and from nerves to muscles work by a
similar means in all living animals.
Figure 1 A schematic of the opener muscle in
the crayfish walking leg. Representative EPSP
responses to a train of twenty stimulation pulses
given at 40 Hz and 60Hz before and during
exposure to 5-HT (100nM). The amplitude of the
EPSPs is measured from the trough preceding the
EPSP of interest to peak response, as shown for
the twentieth pulse during saline exposure. Note
that the EPSP amplitudes are greater throughout
the entire stimulus train when exposed to 5-HT in
comparison to saline.
Figure 5 A schematic representation of the
vesicle pathways within the presynaptic motor
nerve terminal. (A) In the absence of electrical
stimulation (i.e., Inactive, A) of the nerve
terminal few vesicles will spontaneously be
released at slow rate thus, a slow recycling
path (path 1 to 2) will be utilized. Upon
electrical stimulation (i.e., Active, B) the
high-output synapse (Sy1), the one with two
active zones as shown with dark hemispheres
sitting on the inner face of the synapse, will be
recruited before the low-output synapse (Sy2)
with only one active zone (B). Vesicles maybe
recruited from the reserve vesicle pool (RV, path
1) to the readily releasable vesicle pool (RRV)
for docking and fusion with the presynaptic
membrane as well during electrical stimulation.
The vesicles may then recycle through a slow
process and intermediate endosomal (ES) stage
(path 2), as well as a different path (path 3)
that is relatively rapid in recharging the
vesicles with transmitter before the vesicle ends
up back at the RRV pool. When the nerve terminal
is not electrical stimulated but is exposed to
5-HT (i.e., 5-HT Inactive, C), at a low
concentration (100nM), there is a priming of the
synapses by promoting path 1 (C). The activation
of the second messenger IP3 can recruit vesicles
from RV as well as enhance priming and docking of
vesicles at the synapses within the RRV by
phosphorylating synaptically relevant proteins.
In this case, both the high-output (Sy1) and
low-output (Sy2) synapses will be influenced.
When combining electrical activity and exposure
to 5-HT (i.e., 5-HT Active, D) a marked
enhancement of transmission will occur which will
activate path 1 and path 3 for rapid recycling.
Figure 2 The mean amplitude of EPSP during the
10th pulse in a 20 pulse train for 10 consecutive
trials was used to show the effects over time due
to the increase in stimulation frequency and/or
exposure of 5-HT (100nM). The effect of
increasing the stimulation frequency from 40 to
60 Hz is relatively rapid. The means and standard
error of the mean is shown for each 50 seconds.
Upon completion of the 60Hz stimulation in
saline, the preparation was allowed to recover
for 5 minutes. The responses were tested to
insure that baseline conditions resumed for 40Hz
stimulation prior to exchanging the bathing media
with 5-HT solution. The bathing media was
exchanged three times with one containing 5-HT.
The preparation was then left to soak for 5
minutes before data collection in the 40 Hz
stimulation paradigm with 5-HT exposure. The
break in the time axis illustrates this recovery
and incubation time before adding the 5-HT
containing bath.
Summary
1.The neuromodulator, 5-HT, at a high
concentration (1 µM) causes spontaneous fusion of
vesicles from the presynaptic membrane by a
mechanism that has not been previously described.
  2. Dl-threo-beta-benzyloxyaspartate (DL-TBOA,
10µM), the glutamate uptake blocker, causes
synaptic responses to be attenuated in amplitude.
Thus, the pool of evoked vesicles for release are
recycling with little or no glutamate
present.   3. 5-HT, added in the presence of
TBOA, recruits a reserve pool of vesicles which
have glutamate already packaged in them.   4.
The electrically excitable pool of vesicles and
the 5-HT modulated vesicle pool are divisible
within the presynaptic nerve terminal.
Figure 3 The percent change in the 10th (A) and
20th (B) EPSP amplitudes for each preparation
induced by 5-HT during the 40Hz and 60Hz
stimulation indicated that a smaller change
occurred for the 60Hz paradigm. In addition, the
preparations which showed the largest change at
40Hz also showed the largest change at 60Hz
during the 5-HT exposure for the 20th event.
 
Methods
Animals Mid-sized crayfish (Procambarus clarkii),
measuring 8 - 10 cm in body length and weighing
20 to 36 grams, were obtained from Atchafalaya
Biological Supply Co. (Raceland, LA). Animals
were housed in an aquatic facility within the
laboratory in individual tanks, and were fed fish
food pellets every three days. Only male
crayfish in their intermolt stage were used.
Dissection Physiology In brief, the
experimental paradigm is to record intracellular
EPSPs from muscle fibers during stimulation of
the motor nerve in the presence of saline and
then saline containing TBOA. As the EPSP
responses get smaller and unable to be discerned,
a saline containing 5-HT is used. The second
phase of experiments consist of using a similar
experimental paradigm but to record quantal
responses by use of a focal macropatch electrode
placed over a defined region of the motor nerve
terminal. Excitatory Postsynaptic Potentials
(EPSPs) in crayfish EPSPs at the crayfish NMJ
were recorded by intracellular electrodes, with
30-60 MO resistance microelectrodes, filled with
3 M KCl. Responses were recorded with a standard
intracellular electrode amplifier (AxoClamp 2A,
Axon Instruments). Electrical signals were
recorded onto VHS tape and on-line to a Power Mac
9500 via a MacLab/4s interface. EPSPs were
recorded at 10 kHz. All events were appropriately
scaled to known values measured on an
oscilloscope. The opener muscle preparations were
stimulated to induce a short-term facilitation
(STF) by giving a 40 Hz train of ten pulses at
intervals of 5 or 10 seconds.
Figure 4 The presence of TBOA resulted in a run
down of evoked transmission. Here the 10th EPSP
amplitude in measured over time. After a short
while the EPSP amplitude is not detectable, but
after adding 5-HT (1um) the evoked EPSP responses
are revived to larger amplitudes than control
levels.
References
Southard RC, Haggard J, Crider ME, Whiteheart
SW, and Cooper RL. 2000. Influence of serotonin
on the kinetics of vesicular release. Brain
Research 87116-28. Sparks G and Cooper RL.
2004. 5-HT offsets homeostasis of synaptic
transmission during short-term facilitation. (In
Press- J. of Applied Physiology). Tabor J and
Cooper RL. 2002. Physiologically identified 5-HT2
-like receptors at the crayfish neuromuscular
junction. Brain Research 93291-98.
Funding was provided by NSF grant IBN-0131459
(RLC) and a G. Ribble Fellowship for
undergraduate studies in the Department of
Biology at the University of Kentucky (SL).
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