Department of Biology, Queen's University, Kingston, Ontario K7L
3N6, Canada
 |
INTRODUCTION |
Synaptic facilitation, a
short-term strengthening of synaptic connections, plays an integral
role in the functional reorganization of synaptic information
processing and provides important physiological insight into
learning-related synaptic modulation (Byrne and Kandel 1996
; Davis and Murphey 1994
; Dittman et
al. 2000
; Fisher et al. 1997
; Zucker
1989
, 1999
). Stressful environmental conditions can influence
and detrimentally affect synaptic transmission through modulation of
ionic diffusion and flux within the active zone and structural
stability of proteins intrinsic to the functional integrity of the
synapse (Hu et al. 2000
; Le Corronc et al.
1999
; Robertson 1993
). Within a natural ecology,
stressful conditions come and go, and the prior history of an
organism's environment can alter how neural circuitry operates in the
long term (Barclay and Robertson 2000
;
Karunanithi et al. 1999
; Robertson et al. 1996
).
The locust provides an excellent in vivo model system for examining the
effect of a prior environmental stress on synaptic facilitation and,
more generally, the properties of synaptic transmission. An extended
exposure to elevated temperatures (45°C for 3 h) is a potent
environmental stress, inducing natural cellular stress responses
(Whyard et al. 1986
) and mimicking ecologically relevant conditions experienced by the organism (Cloudsley-Thompson
1975
; Uvarov 1966
). Here, we show that prior
exposure to a stressful condition acts to enhance short-term synaptic
facilitation and increase the limit for functional survival during
repeated exposure to the environmental stress. This enhancement in
plasticity is concurrent with a decrease in all other synaptic
parameters (excitatory junction potential latency, rise time, duration
and amplitude), regardless of the subsequent temperature stress.
 |
METHODS |
Experimental preparation
Mature locusts Locusta migratoria (at least 3 wk past
the imaginal molt) were obtained from a crowded colony (25-30°C;
16 h:8 h light:dark photoperiod) maintained at Queen's
University. The experimental setup was as previously described
(Barclay and Robertson 2000
). The axon of the slow
extensor tibia (SETi) motoneuron, originating in the metathoracic
ganglion and innervating the tibial extensor muscle in the locust
hindleg, was stimulated with bursts of just-suprathreshold square
voltage pulses (0.3 ms duration) at 40 Hz (burst frequency = 0.1 Hz; burst duration = 250 ms) using a suction electrode placed on
the severed end of nerve 3 within the thorax. The stimulation protocol
was chosen to ensure adequate facilitation reliably within the
physiological range of SETi firing frequency (Burrows
1996
). Intracellular recordings were made using glass
microelectrodes (filled with 2 mol/l potassium acetate; resistance,
60-80 M
). Prior environmental stress was experimentally induced by
exposing locusts to heat-shock (HS) conditions (45°C for 3 h).
These conditions are known to be stressful to locusts, rapidly inducing
up-regulation of heat shock proteins (Whyard et al.
1986
). Control locusts were kept in a similar environment for
the same period of time at room temperature (20-25°C). Experiments were performed within 1-3 h following the HS treatment.
Procedure and analysis
Excitatory junction potentials (EJPs) were recorded from
SETi-innervated muscle fibers at the proximal end of the hindleg, located just distally to the "fan" region of muscle fibers
(Hoyle 1978
). This cluster of fibers is innervated by
SETi and the common inhibitor (CI) motoneuron. However, in our
recordings, there was no evidence that the CI was recruited by varying
the stimulus strength. Recordings were made initially at room
temperature (20-25°C) and then continuously as the saline
temperature was gradually increased (approximately 5°/min) until
complete failure of synaptic transmission. Intracellular recordings at
their corresponding temperature were digitized and recorded onto VHS
videotape for analysis using Brainwave analysis software (Datawave
Technologies, Longmont, CO). In each animal, EJPs were analyzed for
amplitude (measured from baseline to the peak of the 1st event),
duration (event measured at half-maximal amplitude), rise time
(measured from the beginning to the event peak), and latency (measured
from the stimulus artifact to the event onset). An EJP facilitation index was calculated as the ratio of the amplitudes of the first and
last EJP within each stimulus train. For each parameter, the results
from one fiber per animal were averaged in 5°C bins such that each
animal contributed only one value per temperature bin. The dataset
consisted of nine control and seven HS animals. Significance (P < 0.05) between control and HS animals was assessed
with two-way ANOVA and unpaired t-test where applicable. All
values are reported as means ± SE.
 |
RESULTS |
EJPs in the locust hindleg extensor muscle were recorded
intracellularly as temperature was continually increased, eliciting consistent changes to the individual parameters of the EJP in both
control and previously stressed (HS) animals. As temperature increased,
there was a decrease in all temporal parameters (EJP latency, rise
time, and duration) and an increase in EJP amplitude (Fig.
1A). With an increase in
temperature, the extent of EJP facilitation also decreased (Fig.
1A). The temperature at which synaptic transmission failed
was increased by 3.9°C in animals exposed to prior HS stress (Fig.
1B). The improvement in upper temperature limit from
46.6 ± 1.5°C in controls to 50.5 ± 0.9°C in HS animals
was found to be significant (t-test, t = 2.25, d.f. = 15, P = 0.04). Resting membrane potential
was monitored throughout the experiment and was not significantly
different between control and HS animals.

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Fig. 1.
Synaptic transmission is altered in animals exposed to a prior
environmental stress, induced by exposure to heat shock (HS)
conditions. Excitatory junction potentials (EJPs) were evoked via
extracellular stimulation of the slow extensor tibia (SETi) motoneuron.
A: representative EJP traces recorded from hindleg extensor
muscle fibers of the locust at indicated temperatures. B:
the upper temperature limit for synaptic transmission was significantly
(*, P < 0.05) increased in HS animals. Reported values
are means ± SE.
|
|
Individual EJP parameters were altered in HS animals. Prior exposure to
HS stress caused a reduction in EJP latency (Fig. 2A), rise time (Fig.
2B), and duration (Fig. 2C). Using two-way ANOVA
assessment, this effect was found to be significant for both latency
(F = 23.45, d.f. = 1, P < 0.01) and
rise time (F = 5.82, d.f. = 1, P = 0.02). Although HS stress did not have a significant effect on EJP
duration (F = 3.514, d.f. = 1, P = 0.07), the observable trend was similar to that for latency and rise time. An increase in temperature decreased all temporal parameters (2-way ANOVA, P < 0.01); however, there were no
significant differences in the effect of temperature between control
and HS animals (2-way ANOVA, P > 0.80).

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Fig. 2.
Preexposure to an environmental stress (HS) decreases temporal
parameters of EJPs. A: EJP latency was significantly
reduced in HS preparations in comparison to controls. B:
EJP rise time was significantly reduced in HS preparations.
C: there was a nonsignificant trend for a reduction in
EJP duration in HS preparations. Values are means ± SE.
|
|
Prior HS stress also affected the amplitude of the EJP (Fig.
3A), which was reduced by 63%
at room temperature (20-25°C). This effect of HS was found to be
significant (2-way ANOVA, F = 33.67, d.f. = 1, P < 0.01). Temperature also had a significant effect
(2-way ANOVA, F = 2.58, d.f. = 6, P = 0.03), increasing EJP amplitude with an increase in temperature. The
effect of temperature on EJP amplitude was not found to be different
between control and HS animals (2-way ANOVA, F = 0.79, d.f. = 6, P = 0.58). This dampening effect of HS on EJP
amplitude was evident for each response to the stimulus train (data not
shown). Although prior HS stress reduced EJP amplitude and all temporal
parameters, it increased EJP facilitation (Fig. 3B). In HS
animals, there was significantly increased synaptic facilitation by a
factor of 1.85 at room temperature (2-way ANOVA, F = 21.57, d.f. = 1, P < 0.01). An increase in temperature significantly reduced the amount of facilitation (2-way ANOVA, F = 3.14, d.f. = 6, P < 0.01);
however, temperature did not affect control and HS animals differently
(2-way ANOVA, F = 0.44, d.f. = 6, P = 0.69).

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Fig. 3.
Short-term synaptic plasticity is enhanced by prior exposure to
environmental stress (HS). A: EJP amplitude was
significantly reduced in HS preparations compared with controls.
B: short-term EJP facilitation was significantly
increased in HS preparations. Values are means ± SE.
|
|
 |
DISCUSSION |
Increases in temperature can have deleterious effects on synaptic
transmission, which is of considerable importance to poikilothermic animals. Whereas prior exposure to heat stress is known to reduce the
thermosensitivity of synaptic transmission (Barclay and
Robertson 2000
; Dawson-Scully and Robertson
1998
; Karunanithi et al. 1999
), in this paper,
we have demonstrated that following stressful environmental conditions,
neuromuscular transmission at the locust SETi synapse is altered
(temporal parameters and EJP amplitude are reduced) but that the
thermosensitivity of transmission is unaffected. It is thus becoming
increasingly apparent that exposure to prior HS can exert strikingly
different effects at different synapses. Previous work has shown that
relative EJP (Barclay and Robertson 2000
;
Dawson-Scully and Robertson 1998
) and excitatory
junctional current (EJC) (Karunanithi et al.
1999
) amplitudes decrease with increasing temperature and that
this decrease is mitigated by prior HS. The opposite result occurs at
the SETi synapse; EJP amplitude increases with temperature, which is
similar to that seen for mEJC amplitudes in Drosophila
(Karunanithi et al. 1999
). Furthermore the reduction of
absolute EJP amplitudes at the SETi synapse in HS animals is not
evident at other synapses. This reduction in EJP amplitude could be due
to a stress-induced alteration to postsynaptic input resistance or
resting membrane potential. While both control and HS animals had
similar membrane potentials, a difference in input resistance following
HS cannot be ruled out. However, at Drosophila neuromuscular
junctions, HS had no significant effect on either membrane potential or
input resistance (S. Karunanithi and J. W. Barclay, unpublished
observations). The reductions in EJP rise time and duration at the SETi
synapse in HS animals are also in contrast with previous work
describing no such effect in Drosophila (Karunanithi
et al. 1999
) and at the FETi synapse in the locust hindleg
(Barclay and Robertson 2000
). The effects of HS on
latency are even more varied, ranging from no effect whatsoever
(Barclay and Robertson 2000
; Karunanithi et al.
1999
) to an increase in latency (Dawson-Scully and
Robertson 1998
) and finally to our reported decrease in latency
at the SETi synapse. Although our measure for latency does not
differentiate between axonal conduction velocity or synaptic delay,
previous work in the locust has indicated that HS has the opposite
effect, slowing both conduction velocity (Gray and Robertson
1998
) and synaptic delay (Dawson-Scully and Robertson
1998
). The varied effects of heat stress at different synapses
may indicate multiple pathways for HS alteration to synaptic
performance or a single mechanism acting on different cellular targets
depending on the synapse.
The ability of a synapse to regulate the relative strength of
individual connections is absolutely critical for modulation of neural
circuitry. During periods of stress, synaptic plasticity could become
even more consequential by rapidly and appropriately modifying the
postsynaptic message as environmental conditions change. It is
therefore interesting that the preexposure of the animal to stressful
conditions also increased the short-term plasticity of the
neuromuscular synapse regardless of the subsequent temperature stress.
Enhancement of synaptic facilitation most likely indicates an increase
in presynaptic residual calcium levels during repetitive synaptic
transmission (Dittman et al. 2000
; Kamiya and
Zucker 1994
). Short-term facilitation could be achieved by
axonal spike broadening increasing calcium influx, such as is seen in
serotonin-induced short-term facilitation in the locust (Parker
1995
) and in Aplysia (Byrne and Kandel
1996
). It is unknown whether the effects seen here are achieved
via a stress-activated release of neuromodulators, such as serotonin,
that are known to enhance facilitation via cAMP pathways (Chen
and Regehr 1997
; Sugita et al. 1997
). However, previous studies have linked a prior exposure to environmental stress
with alterations to potassium channel dynamics and spike broadening
(Nicol et al. 1997
; Ramirez et al. 1999
;
Saad and Hahn 1992
). Alternatively, the enhancement of
synaptic facilitation could occur through spike broadening-independent
effects on the synaptic machinery for transmitter release. This could
be achieved via an upregulation in heat shock protein synthesis
(Parsell and Lindquist 1993
), protecting proteins
integral to short-term facilitation, or by enhancing the persistent
action of residual calcium within the presynaptic nerve terminal. It
also cannot be ruled out that the effects of HS to alter the plasticity
of the SETi synapse could be the result of chronic activity of the
intact SETi motor axon activated during the heat stress. For example,
similar changes to synaptic function (depression of EJP amplitude,
potentiated EJP amplitude at higher frequencies) were found following
long-term tonic stimulation of crayfish muscle fibers (Lnenicka
and Atwood 1985
; Mercier and Atwood 1989
). An
increase in neural activity during the 3-h HS period could induce a
substantial modification to the performance of the synapse. Although
much remains to be determined, it is an important and novel result that
the short-term ability of a synapse to reorganize its connective
strength is enhanced following a period of environmental stress.
We thank Dr. R. D. Andrew (Queen's University) for critical
comments on an earlier draft of the manuscript.
This work was supported by a grant from the Natural Sciences and
Engineering Council of Canada to R. M. Robertson.
Address for reprint requests: J. W. Barclay (E-mail:
barclayj{at}biology.queensu.ca).