1Department of Organismal Biology and Anatomy, The University of Chicago, Chicago, Illinois 60637; and 2Department of Biology, Queens University, Kingston, Ontario K7L 3N6, Canada
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ABSTRACT |
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Ramirez, J. M.,
F. P. Elsen, and
R. M. Robertson.
Long-term effects of prior heat shock on neuronal potassium currents
recorded in a novel insect ganglion slice preparation. Brief
exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and
behavior of poikilotherms. However, little is known about heat shock
effects on neuronal properties. To investigate how heat shock affects
neuronal properties we developed an insect ganglion slice from locusts.
The functional integrity of neuronal circuits in slices was
demonstrated by recordings from rhythmically active respiratory neurons
and by the ability to induce rhythmic population activity with
octopamine. Under these "functional" in vitro conditions we
recorded outward potassium currents from neurons of the ventral midline
of the A1 metathoracic neuromere. In control neurons, voltage steps to
40 mV from a holding potential of 60 mV evoked in control neurons
potassium currents with a peak current of 10.0 ± 2.5 nA and a
large steady state current of 8.5 ± 2.6 nA, which was still
activated from a holding potential of
40 mV. After heat shock most of
the outward current inactivated rapidly (peak amplitude: 8.4 ± 2.4 nA; steady state: 3.6 ± 2.0 nA). This current was inactivated
at a holding potential of
40 mV. The response to temperature changes
was also significantly different. After changing the temperature from
38 to 42°C the amplitude of the peak and steady-state current was
significantly lower in neurons obtained from heat-shocked animals than
those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to
investigate ion currents in insect ganglion slices.
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INTRODUCTION |
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Brief exposure to high sublethal temperatures
(heat shock) has lasting adaptive sequelae for cells and tissues in
organisms across all taxa. This is most relevant for
poikilotherms,which can be adapted by heat shock to extreme
temperatures in their natural environments. There is increasing
awareness that for these organisms heat hock has far-reaching
consequences for their behavior and population dynamics (Coleman
et al. 1995; Feder 1996
; Feder and Krebs
1997
; Gehring and Wehner 1995
; Hofmann
and Somero 1996
; Norris et al. 1995
;
Ulmasov et al. 1992
). These adaptive effects are
primarily mediated by various heat shock or stress proteins. The
molecular biology and anatomic distribution of these proteins were well
characterized in a variety of animal models (Buchner 1996
; Craig et al. 1993
; Lindquist and
Craig 1988
; Mayer and Brown 1994
). However,
surprisingly little is known as to what extent heat shock affects
neural function, particularly the mechanisms that underlie electrical
signaling in nervous tissue.
We examined the effect of heat shock on neuronal potassium currents in
locusts. These animals are ideal to study heat shock effects on
neuronal function for the following reasons. First, in their natural
environments, locusts are routinely exposed to ambient temperatures
>40°C (Uvarov 1966), and during vigorous activity,
such as flight, the temperature in the thorax can be as much as 10°C
above ambient (Weis-Fogh 1956
). Second, it was demonstrated that locusts exhibit a robust heat shock response, and
exposure to 43°C for 3 h induces the production of heat shock proteins (Baldaia et al. 1987
; Whyard et al.
1986
). Third, it was demonstrated that heat treatment alters
neuronal operation in locusts in ways that can be interpreted as being
ecologically adaptive (Robertson et al. 1996
). For
example, it has been shown that heat pretreatment permits tethered
flight at higher thoracic temperatures than are normally permissive,
and this is a consequence of an extended temperature range of operation
in the neuronal circuitry controlling the wingbeat (Robertson et
al. 1996
). Most interestingly the temperature sensitivity of
flight motor rhythms is markedly reduced after nonlethal heat shock.
Furthermore, heat shock affects the temperature sensitivities of
amplitude potential amplitude and conduction velocity in this system
(Gray and Robertson 1998
). We tested the idea that heat
shock induces lasting modifications of neuronal properties.
To examine the effect of heat shock on ion channel properties we developed a slice preparation obtained from insect nervous tissue. This approach to studying neuronal properties in insects provides an exciting possibility to study adaptive changes in channel properties in neurons that are still embedded in their normal histological environment. We show here that heat shock treatment has a long-term effect on whole cell potassium current recorded from the somata of neurons visualized in a functional slice preparation of the metathoracic ganglion of the locust.
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METHODS |
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Heat shock
Adult locusts (Schistocerca americana) were kept in a crowded colony at the University of Chicago. Five days before performing the experiments 50 locusts were isolated from the colony and maintained in the laboratory in cages at room temperature (~24°C). All locusts were placed into a ventilated plastic container (1-L capacity) for 3 h. Heat shocked locusts were exposed to 45°C, whereas control locusts were kept at room temperature for the same time period. None of the locusts died as a result of the heat treatment. Heat shocked locusts were maintained for 6-24 h at room temperature before dissection to allow them to recover from the treatment. No difference was noted between the results of animals that recovered for different periods of time.
Slice preparation
Locust slices were prepared by modifying the mammalian slice
preparation technique (Ramirez et al. 1996). Locusts
were decapitated, and the metathoracic ganglia were removed in saline
(containing, in mM, 128 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose
equilibrated with continuous bubbling of carbogen [95%
O2-5% CO2] to pH 7.4). The dorsal surface of
an isolated ganglion was glued onto an agar block with cyanoacrylate
glue. This was secured in a vibratome with the rostral end of the
ganglion up, and thin slices (100 µm) were sectioned serially from
rostral to caudal to expose the rostral portion of the metathoracic
ganglion. In most experiments described in this study we used the next
slice of 400-600 µm from this rostral boundary extending into the
rostral border of the first abdominal neuromere. In only two cases we used a slice obtained from the posterior portion of the metathoracic ganglion and the first fused abdominal neuromere. The slices were immediately transferred into a recording chamber and held in place with
an overlying nylon mesh on a titanium frame. The preparation was
further stabilized because the overlying mesh covered not only the
slice itself but also the polymerized glue that remained partly
attached to the slice. The preparation was submerged under a stream of
saline (31°C; flow rate 16 ml/min) and stabilized for 10 min before
recording neural activity.
Recording technique and analysis
Extracellular recordings were obtained with a suction electrode
(low resistance glass pipettes, 0.3 M) and were integrated electronically with a leaky RC circuit (set at a time constant of
20-30 ms). Whole cell patch recordings were obtained with unpolished electrodes that were manufactured from filamented borosilicate glass
(Clark GC150F) and had a resistance of 1-2 M
when filled with a
solution containing (in mM) 140 KCl, 1 CaCl2, 10 ethylene glycol-bis-
-aminoethyl
ether-N,N,N',N'-tetraacetic
acid, 2 MgCl2, 4 Na2ATP, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (pH 7.2). Locust neurons on the ventral midline of the A1
neuromere of the metathoracic ganglion were recorded with the visual
patch-clamp technique with a Zeiss upright microscope in conjunction
with infrared Normarski optics. The electrodes with an internal
pressure of 30-50 mmHg were positioned onto a soma of a locust neuron
with a three-dimensional (3D) piezomicromanipulator (Burleigh). For seal formation, positive pressure in the pipette was released, and
gigaseals were formed by slight negative pressure. The membrane was
ruptured to achieve whole cell configuration by applying negative pressure pulses. Membrane currents were recorded with an Axopatch 1D
amplifier and analyzed with the pClamp 6.0 software in conjunction with
a Digidata 1200 interface (Axon Instruments, Foster City, CA). Current
response traces were recorded with either off- or on-line leak
subtraction, eliminating linear leak current. Serial resistance was
always 80% compensated, and the input resistance of all neurons was
between 180 and 800 M
. All quantitative data are given in mean ± SE, if not indicated otherwise. Statistical significance was
assessed with the Students t-test, and significance was
assumed for P < 0.05. We recorded from 20 neurons in
16 preparations and only quantitatively analyzed those recordings that
had an initial seal resistance 2.5 G
. All substances used in this
study were obtained from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Functional ganglion slice preparation
Previous investigations in Manduca sexta (Oland
et al. 1996) already indicated that it is possible to preserve
the anatomic architecture of an insect ganglion in a slice preparation.
We confirmed this finding in our study with 400- to 600-µm transverse slices obtained from the metathoracic ganglion. Characteristic fiber
tracts and intraganglionic tracheal supply of the metathoracic ganglion
were preserved in the slice preparation (Fig.
1). In addition, the slices were
sufficiently thick to preserve not only the 3D arrangement of cell
bodies, neuropile, and tracheal supply but also the functional
integrity of neuronal circuits (Fig. 2). In slice preparations containing the anterior portion of the
metathoracic ganglion, superfusion of the biogenic amine octopamine
induced rhythmic neuronal activity. To induce the rhythmicity we used a
concentration of 10
3 M as was previously described
(Ramirez and Pearson 1991a
). Suction electrode
recordings from the ascending connectives reveal that octopamine
activated not just single neurons but a population of neurons (Fig. 2,
top panel). Previous studies demonstrated that octopamine
can release flight rhythms in locust preparations (Ramirez and
Pearson 1991a
,b
; Sombati and Hoyle 1984
;
Stevenson and Kutsch 1987
). Thus it is conceivable that
this octopamine-induced rhythm reflects residual rhythmic activity in
flight circuitry (see also Wolf et al. 1988
). In slices that contained
the posterior portion of the metathoracic ganglion and the first fused
abdominal ganglion, it was possible to record intracellularly from
spontaneously active respiratory rhythmic neurons (Fig. 2, bottom
panel). The characteristic activation patterns of these neurons
were previously described (Ramirez and Pearson 1989
),
suggesting that this respiratory neuron was inhibited during
inspiration and activated during expiration. The finding that neuronal
network activity was preserved indicates that neuronal discharge
properties remained largely unaffected in the slice preparation. Thus
any heat-shock induced alterations in ion channel properties evident
under these in vitro conditions may resemble those in intact animals.
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Heat-shock induced alteration in potassium currents under control conditions
To characterize ion channel properties, unstained living locust
neurons were visualized with infrared Nomarski optics (Figs. 1 and
3A) and routinely voltage
clamped with the whole cell patch configuration (Fig. 3, B
and C). All recordings were obtained from cell bodies
located in the ventral midline of the A1 neuromere (Fig. 3A,
see also bottom right square in Fig. 1). Hyperpolarizing and
depolarizing test potentials (duration: 80 ms), incrementing in 10 mV
steps, from 80 to 40 mV from a holding potential of
60 mV evoked a
series of outward currents (Fig. 3, B and C). These currents were identified as K+ currents because they
were blocked by tetraethylammonium chloride (TEA 30 mM) and CsCl (110 mM) (not shown). Figure 3 shows the outward current responses to
similar voltage steps in a neuron obtained from a control animal (Fig.
3B) and a neuron obtained from a locust that was exposed to
a heat shock 24 h before the experiment (Fig. 3C). In
control neurons the evoked K+ outward current consisted of
a small rapidly inactivating component and a large slowly inactivating
component. The average current amplitude evoked by a depolarizing
voltage step from
60 to 40 mV was initially 10.0 ± 2.5 nA
(peak, n = 5) and after 80 ms 8.5 ± 2.6 nA
(steady state, n = 5). Thus after 80 ms only 15.5% of the outward current was inactivated (Fig. 3B, top
panel). In contrast, in heat-shocked neurons most of the
K+ current inactivated rapidly (Fig. 3C,
top panel). The average peak current amplitude evoked by a
voltage step from
60 to 40 mV was initially 8.4 ± 2.4 nA (peak,
n = 5) and after 80 ms was 3.6 ± 2.0 nA (steady
state, n = 5). Thus 57% of the outward current was
inactivated after 80 ms. The peak outward current was not significantly
different in control and heat-shocked neurons (Fig. 3D,
left panel). However, because in heat-shocked neurons most of the K+ current inactivated there was a significant
difference in the steady-state component of current in control and
heat-shocked neurons (Fig. 3D, right panel).
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At a holding potential of 40 mV most of the current in control
animals was still activated when stepping to more depolarized membrane
potentials (Fig. 3B, bottom panel). In contrast
in all neurons obtained from heat-shocked locusts, most of the outward current was inactivated at a holding potential of
40 mV (Fig. 3C, bottom panel), suggesting that outward
currents in heat-shocked animals were dominated by an A-type current.
Heat-shock induced alteration in the response to temperature changes
The response to a temperature increase was assessed with a holding
potential of 60 mV and the same hyperpolarizing and depolarizing step
protocol as described in the previous section. In control animals, the
amplitude of the voltage-dependent K+ current decreased
after a temperature step from 31 to 35°C and increased again after a
subsequent step to 41°C (Fig.
4A). During each temperature
step, the same temperature was maintained for 3 min before obtaining a
measurement to ensure that the slice equilibrated to the new
temperature. The temperature-induced decrease and subsequent increase
in the current amplitude are demonstrated by the current-voltage
relationship measured at the peak current (Fig. 4B). In the
heat-shocked animal the amplitude of the K+ current
continued to decrease in response to temperature steps from 31 to
35°C and to 41°C (Fig. 4, C and D). The
current remained reduced for
10-20 min after returning to the
starting temperature.
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The temperature effect on the K+current amplitude evoked by
a voltage step from 60 to 40 mV was quantitatively evaluated for five
control and five heat-shocked preparations (Fig.
5). When stepping from 31°C to a
temperature of 34°C the peak amplitude (Fig. 5A) was not
significantly different between control and heat-shocked animals. When
stepping to a temperature of 38 and 42°C, the peak amplitude (Fig.
5A) as well as the steady-state amplitude (Fig.
5B) were significantly lower in heat-shocked than in control
animals. The outward current after heat shock was presumably reduced
because of a shift in the inactivation properties. We assessed the
inactivation properties of the K+ current by applying
holding potentials in 10-mV steps from
90 to
10 mV. These holding
potentials were maintained for 2 s before stepping to the same
test potential of 40 mV. As demonstrated in Fig. 5C the
K+ current was fully inactivated at a temperature of 36 and
41°C when holding a heat-shocked neuron at
60 mV.
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Neither the control nor the heat-shocked animals recovered fully from heat exposure. The current amplitude, obtained 10 min after returning to the control temperature of 31°C, was variable and not significantly different between heat-shocked and control preparations (Fig. 5, A and B), although it is notable that the variability is greatly increased in control compared with heat-shocked preparations.
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DISCUSSION |
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We have shown that heat-shock pretreatment can lead to
long-lasting modifications in K+ outward currents of
neuronal somata. Under control conditions voltage-dependent outward
currents consisted of a large component of a slowly inactivating
current. After heat shock, outward currents were characterized by a
large component of a rapidly inactivating current. This alteration
corresponds with a different response to temperature changes, which may
be due to a shift in inactivation properties of the rapidly
inactivating component. Our finding suggests that, under control
conditions, K+ flows primarily through K+
channels with delayed-rectifier characteristics. This currents inactivate only very slowly. After heat shock, voltage steps evoke only
very little delayed-rectifier current and instead evoke an inactivating
K+ conductance with A-type characteristics. The
inactivation properties of this A-type current were altered at higher
temperatures because at a holding potential of 70 mV most of the
current was fully inactivated at a temperature of 36 and 41°C. This
explains why at higher temperatures K+ outward currents
were very reduced in heat-shocked animals.
Despite the dramatic alteration in potassium conductances the locusts
that were heat shocked showed no evidence of neural or behavioral
impairment and were alert, walking, and jumping, apparently normally,
before dissection. Perhaps these alterations in potassium conductances
become behaviorally significant only at higher temperatures. The heat
shock treatment as used in this study induced thermotolerance that
permits survival of intact animals at temperatures well in excess of
41°C. This increased thermotolerance was similar to thermotolerance
shown after the same treatment of a different species of locust
(Robertson et al. 1996). Thus the effect on
K+ conductance as described in this study was evident in
slices from thermally protected animals that showed no signs of damage. Whether this alteration in K+ conductances might contribute
to an increased heat tolerance is another interesting issue that needs
further investigation. It is interesting to note, however, that the
protective effects on motor pattern generation can be at least
partially explained by alterations in the temperature sensitivity of
synaptic parameters (Dawson-Scully and Robertson 1998
);
the amplitude of postsynaptic potentials reduces less with increases in
temperature for heat-shocked animals compared with controls. In
parallel with this, intracellularly recorded action potentials are
longer in duration and tend to lack an afterhyperpolarization (Wu and
Robertson, unpublished observations). With the caveat that these
observations were of the inexcitable membranes of cell somata, it is
nevertheless a testable hypothesis that the effects on potassium
conductances described here result in action potentials with increased
durations and protected synaptic transmission thus protecting flight
motor patterns. It remains to be determined whether the properties of potassium conductances in excitable membranes are similar to the properties of those described here. In addition to any possible effect
on neural signaling the alterations of potassium conductances could
have more general beneficial consequences for the health of the animal
at stressful temperatures.
A change in voltage-gated K+ currents was previously
described in various vertebrate and invertebrate neuronal systems. Most commonly such changes were associated with a developmental change. For
example only a noninactivating delayed rectifier current was observed
in rat embryonic sympathetic neurons; a fast transient current appeared
later during postnatal development (Nerbonne et al.
1986). A differential development of a fast-transient and noninactivating delayed rectifier current was also described for tadpole spinal neurons (Ribera and Spitzer 1992
). This
developmental change was correlated with the temporal regulation of
different potassium channel genes (Gurantz et al. 1996
).
In Drosophila flight muscles a fast transient K+
current appears during development before a delayed rectifier K+ current (Salkoff and Wyman 1981
). This
change may be due to a change in calcium sensitivity (Salkoff
1983
).
A temperature dependency in the expression of voltage-gated
K+ currents was previously described in insects. In
Drosophila the relationship between a delayed sustained and
a rapidly inactivating current depends on the developmental temperature
(Chopra and Singh 1994). A more rapidly occurring
alteration in voltage-gated K+ conductances was
demonstrated in locust photoreceptors. These neurons express a delayed
rectifier during the day and an inactivating K+ conductance
during the night (Weckström and Laughlin 1995
). These authors suggest that the inactivating conductance is
metabolically less demanding, which might be advantageous during the
night when the increased dynamic range and frequency response imparted
by the sustained current would be unnecessary. In fact, our finding can
similarly be interpreted, and the switch we observed in K+
conductance could be a part of a coordinated physiological response to
heat shock, which results in a reduced K+ outward current.
This could protect the locust from a detrimental increase of
extracellular K+ during high temperature exposure. Clearly,
this is only one possible interpretation of our finding, and further
investigations will be necessary to examine the physiological
consequences of the observed alteration in K+ conductances.
The mechanism that caused the modification of K+
conductances remains to be determined. This alteration could be due to
a direct or indirect effect of heat shock proteins on potassium
channels. If this is the case we would expect that HSP70 antibodies
might block the observed channel modification by reducing levels of available HSP70. Some support for this hypothesis is provided by
previous studies that have described effects of exogenous application of HSP70 on neuronal calcium flux (Smith 1995) and on
calcium-dependent potassium channels in a human promonocyte culture
(Negulyaev et al. 1996
). Alternatively, the described
alterations could also be due to a heat shock-induced release of
neuromodulators, which then alters the expression of different types of
potassium currents. For locust photoreceptors the diurnal switch in
potassium currents was attributed to the release of serotonin
(Cuttle et al. 1995
). This possibility could be examined
with selective antagonists for serotonin. By using the ganglion slice
preparation that we describe here, it is possible not only to test
these working hypotheses but also to examine how heat shock can
condition and protect the operation of neuronal circuitry underlying
motor pattern generation in this model system. We predict that the
effects of heat shock on neuronal properties we describe are not
restricted to locusts but may be also evident in other invertebrates
and vertebrates.
Although there are many interesting issues raised by our findings that
remain unresolved, we clearly demonstrated that heat shock has
long-term effects on neuronal properties. This important aspect of heat
shock so far received only very little attention in the literature. The
effect we describe is likely to have important consequences for the
signaling function and/or the metabolic well-being of neurons in their
extracellular environment. Another important outcome of this study is
that we demonstrated that the sliced ganglion approach has great
utility for the study of the cellular properties of neurons in
functional circuits in the locust. We anticipate that such an approach
could easily be modified for use with smaller insects (e.g.,
Drosophila) and other invertebrates (Ermentrout et
al. 1998). This would avoid the current necessity to perform
detailed cellular investigations in cell cultures containing dissociated and functionally isolated neurons.
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ACKNOWLEDGMENTS |
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This work was in part supported by an award to the University of Chicago's Division of Biological Sciences under the Research Resources Program for Medical Schools of the Howard Hughes Medical Institute to J. M. Ramirez and National Sciences and Engineering Research Council of Canada to R. M. Robertson.
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FOOTNOTES |
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Address for reprint requests: J.-M. Ramirez, University of Chicago, OBA, 1027 East 57th St., Chicago, IL 60637.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 April 1998; accepted in final form 14 October 1998.
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REFERENCES |
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