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The Journal of Neuroscience, March 15, 1999, 19(6):1932-1939
Temperature-Dependent Modulation of Excitatory Transmission in
Hippocampal Slices Is Mediated by Extracellular Adenosine
Susan A.
Masino1, 2 and
Thomas V.
Dunwiddie1, 2, 3
1 Neuroscience Program and 2 Department of
Pharmacology, University of Colorado Health Sciences Center, Denver,
Colorado 80262, and 3 Veterans Affairs Medical Research
Service, Denver, Colorado 80220
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ABSTRACT |
Although extracellular adenosine concentrations in brain are
increased markedly by a variety of stimuli such as hypoxia and ischemia, it has been difficult to demonstrate large increases in
adenosine with stimuli that do not result in pathological tissue damage. The present studies demonstrate that increasing the temperature at which rat hippocampal brain slices are maintained (typically from
32.5 to 38.5°C) markedly inhibits excitatory synaptic transmission. This effect was reversible on cooling, readily repeatable, and was
blocked by A1 receptor antagonists and by adenosine
deaminase, suggesting that it was mediated by increased activation of
presynaptic adenosine A1 receptors by endogenous adenosine.
This increase in adenosinergic inhibition was not a response to
hyperthermia per se, because it could be elicited by temperatures that
remained entirely within the hypothermic range (e.g., from 32.5 to
35.5°C). The increased activity at A1 receptors appeared
to be attributable to the direct release of adenosine via nucleoside
transporters; the release of adenine nucleotides, linked to either the
activation of NMDA receptors or the increased efflux of cAMP, appeared
not to be involved. These results suggest that changes in brain
temperature can alter the regulation of extracellular adenosine in rat
brain slices and that increased adenosine release may be an important regulatory mechanism for countering increased excitability consequent to increased brain temperature.
Key words:
adenosine; hippocampus; temperature; hyperthermia; A1 receptors; CA1
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INTRODUCTION |
Low concentrations of adenosine
normally are found in the extracellular space of brain, concentrations
that are sufficient to occupy, at least partially, the relatively
high-affinity A1 and A2A receptors even under
basal conditions [A1 (Dunwiddie and Hoffer, 1980 ; Bruns et
al., 1986; Dunwiddie and Diao, 1994); A2A (Cunha et al.,
1996; Svenningsson et al., 1997)]. Activation of A1
receptors exerts a tonic influence on electrophysiological activity in
regions such as the hippocampus (Dunwiddie, 1985), olfactory cortex
(Motley and Collins, 1983), and striatum (Flagmeyer et al., 1997),
where adenosine acts primarily on presynaptic glutamatergic receptors
to inhibit excitatory synaptic transmission. However, a variety of
situations can increase extracellular adenosine levels dramatically and
virtually abolish excitatory synaptic transmission. Most of the
conditions known to promote such large increases in extracellular
adenosine are those in which the supply of oxygen and energy substrates
is not adequate to meet the energy demands of the brain, such as
hypoxia (Rubio et al., 1975; Fowler, 1993; Zhu and Krnjevi ,
1993), ischemia (Berne et al., 1974), metabolic inhibition (Zhu and
Krnjevi, 1997), or electrical hyperactivity (Rubio et al., 1975;
Lloyd et al., 1993). All of these conditions ultimately can become
pathological and result in significant cell loss. Although these
stressful conditions that increase extracellular adenosine are
physiologically relevant, they are also relatively unusual. However,
smaller changes in brain metabolic balance or other variables occur on
a regular basis, and it is possible that more modest physiological
changes transiently regulate adenosine levels and neuronal excitability
in a more subtle manner.
A common aspect of many stimuli that release adenosine from brain
slices is a reduction in the ability of cells to synthesize ATP (e.g.,
by removing glucose or oxygen from the superfusion buffer) (Fowler,
1993; Zhu and Krnjevi, 1993, 1997). Another manipulation that
might alter metabolic balance and be physiologically stressful is
changing the temperature. In general, increases in temperature
correlate with an increased metabolic rate, whereas decreases in
temperature result in reductions in the metabolic rate; hypothermia is
a well known mechanism of cerebroprotection, both in intact animals
(Barone et al., 1997) and in brain slices (Tanimoto and Okada, 1987).
In vivo, small fluctuations in temperature occur throughout
the day (Refinetti and Menaker, 1992), and larger changes can be
initiated by physical exercise, heat stress, trauma, or illness
(Anderson et al., 1983; Kluger, 1991; Rothwell, 1994). In addition, the
function of the CNS is known to be particularly sensitive to
increased temperature (Anderson et al., 1983). Temperature is usually a
well controlled experimental variable in in vitro studies
and an important physiological one, but its direct effects have not
been characterized extensively in brain slices, particularly insofar as
its relationship with endogenous adenosine. To this end we have
characterized the effects of moderate, transient temperature increases
on evoked CA1 field potentials in the hippocampal slice and examined
the extent to which these effects can be attributed to changes in
endogenous adenosine.
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MATERIALS AND METHODS |
Slice preparation. Transverse hippocampal slices were
obtained from 6- to 8-week-old Sprague Dawley rats by using standard procedures (Dunwiddie and Lynch, 1978; Dunwiddie and Hoffer, 1980). Briefly, after decapitation into ice-cold artificial CSF (aCSF), 400 µm slices were made on a Sorvall TC-2 tissue chopper and
incubated either at room temperature (~23°C) or at 32.5°C. The
aCSF used for dissection, incubation, and submerged, perfused
recordings (2.0 ml/min) contained (in mM): NaCl 126.0, KCl
3.0, MgSO4 1.5, D-glucose 11, CaCl2
2.4, NaH2PO4 1.2, and NaHCO3 25.9 that was bubbled continuously with a
95%O2/5%CO2 mixture. Slices were
incubated undisturbed for 90 min before electrophysiological recording.
Electrophysiological recording. Slices were placed on a
nylon net in the recording chamber and superfused continuously (2.0 ml/min) with aCSF bubbled with
95%O2/5%CO2. Extracellular field EPSPs (fEPSPs) were recorded from the CA1 region of the stratum radiatum by using glass micropipettes (10-15 M ) filled with 3 M NaCl. A twisted bipolar stimulating electrode was placed
to stimulate the Schaffer collaterals in stratum radiatum every 30 sec.
Stimulation intensity was adjusted such that the fEPSP was between 0.5 and 1.2 mV. Data were recorded via an AC amplifier and digitized and
stored in the computer for later analysis.
Temperature manipulation. After a 5-10 min stable baseline
was established, the temperature of the superfusion medium was changed
slowly (~1.0°C/min; see Fig. 1A) from the
baseline recording temperature to either 32.5°C (slices starting from
a room temperature baseline) or 38.5°C (slices starting from a
32.5°C baseline). The temperature was regulated with a thermostatic
controller to within 0.5°C, and the chamber temperature was measured
with a small thermistor (YSI 511, Yellow Springs Instruments, Yellow Springs, OH) placed directly in the recording chamber along with the
tissue slice. In the majority of experiments the incubation and
baseline fEPSP measurements were made at 32.5°C; the higher temperature (38.5°C) was achieved, was maintained for 5-7 min, and
gradually was decreased back to baseline at a rate similar to that of
the previous increase. Unless noted otherwise, this initial test
protocol was used to characterize the temperature sensitivity of each
slice, and this was compared with the temperature sensitivity of the
same slice after a physiological or pharmacological manipulation. In a
subset of experiments the higher temperature was maintained for >40
and up to 65 min.
Drug application. A baseline fEPSP was reestablished after
the initial period of increased temperature to evaluate the degree of
recovery, after which any drugs that were applied were superfused for a
minimum of 12 min before the temperature again was increased. Thus, the
effect of the temperature changes on the fEPSP was compared before
(control) and after drug treatment within each hippocampal slice.
Analysis. Statistical analyses included linear regression
analysis and Student's two-tailed paired t tests.
Chemicals. Adenosine deaminase (type VI),
DL-2-amino-5-phosphonopentanoic acid (APV),
 , -methyleneadenosine 5'-diphosphate (AOPCP), dipyridamole,
guanosine 5'-monophosphate (GMP), probenecid, theophylline, and all
aCSF constituents were obtained from Sigma (St. Louis, MO);
8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), dizocilpine maleate
(MK-801), and 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (Ro 20-1724) were obtained from Research Biochemicals (Natick, MA). FPL
67156 was a gift from Fisons Pharmaceuticals (Leicestershire, UK).
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RESULTS |
Temperature increases inhibit excitatory synaptic responses
In initial experiments we observed that increasing the temperature
of hippocampal slices from the usual recording temperature of
32.5 38.5°C resulted in a profound depression in the amplitude of
the fEPSP recorded in CA1. This decrease was sustained for the duration
of the increased temperature and was completely reversible when the
temperature was returned to 32.5°C (Fig.
1A). When the same
slice was challenged with a second increase in temperature, the second
response was typically of the same magnitude as the initial response
(Fig. 1B). In six slices tested in this manner there
was no significant difference between the amount of inhibition produced
by the initial versus a subsequent temperature increase [72.9 ± 6.6% vs 73.8 ± 7.8%; not significant (n.s.); paired
t test]. Thus, in subsequent experiments each slice was
used as its own control to examine the effects of experimental
manipulations on the fEPSP response. There was considerable variability
in the amount of inhibition exhibited by individual slices with this protocol, with the degree of inhibition ranging from as much as 90% to
no change in the fEPSP. A sustained decrease in the fEPSP was observed
in 80% (58 of 72) of hippocampal slices tested with the
32.538.5°C paradigm; the remainder showed no change, a slight increase in the fEPSP, a transient decrease and spontaneous recovery, or a fluctuating response to the higher temperature. The 20% of slices
that did not exhibit a characteristic decrease in the fEPSP in response
to the temperature increase were not included in any additional
characterization.
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Figure 1.
The effect of temperature on extracellular field
potential amplitude. A, Changes in fEPSP amplitude
(top) during and after an increase in the chamber
temperature from 32.538.5°C (bottom). Temperature
changes were recorded with a small thermistor placed directly in the
recording chamber along with the tissue slice. The peak amplitude of
the fEPSP was inversely proportional to the temperature and showed full
recovery to the baseline fEPSP amplitude when the temperature was
returned to the initial recording temperature. B, The
decrease in fEPSP amplitude in response to the temperature increase is
repeatable in an individual slice. In this and all subsequent figures
the baseline recording temperature (32.5°C) is indicated by the
solid line, and the transition to 38.5°C, as
illustrated in A, is indicated by the open
triangles. The same temperature change illustrated in
A was executed twice in this slice, and a similar degree
of synaptic inhibition was observed both times.
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Figure 2A shows the
average response of 58 slices that responded to a transient temperature
increase from 32.538.5°C, as illustrated in Figure 1. The fEPSP
amplitude was decreased by 60.5 ± 3.5% on warming and fully
recovered to 100.7 ± 4.6% of the initial baseline. There was no
hysteresis in the temperature response, in that the relationship
between the change in the fEPSP amplitude and temperature was nearly
identical regardless of whether the temperature was being increased or
decreased (Fig. 2B). The inhibition also did not
appear to be dependent on the rate of change of the temperature, in
that a similar degree of inhibition was observed either after a
relatively slow change in temperature (T1/2 ~5 min, as illustrated in Fig.
1A) or when the temperature was increased more
rapidly (T1/2 < 2 min; data not shown). In addition, the synaptic inhibition did not occur at a specific threshold temperature, in that it could be elicited by increases from
32.538.5°C as well as by increases from ~2332°C. In six of
six slices that were tested with the ~2332°C protocol, the EPSP
initially increased on increasing the temperature (average increase,
58.0 ± 7.5%). Subsequent to this increase, three of six slices
showed a decrease in the EPSP (average decrease measured from increased
baseline, 46.0 ± 14.7%), similar to that seen with the
32.538.5°C protocol, which also recovered when the temperature was
decreased. Examples of synaptic responses recorded from individual slices before, during, and after the temperature increase from 32.538.5°C are illustrated in Figure
3. In addition to the inhibition of the
fEPSP (seen in Fig. 3C, trace b, D,
trace e), there was also a decreased latency to the
peak of the synaptic response, as would be expected because of faster
conduction velocity as well as faster kinetics of the synaptic response
at the higher temperature.
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Figure 2.
Effect of the 32.538.5°C protocol on fEPSP
amplitude. A, Shown is the time course of the mean
response of 58 slices to the increase in temperature; each
point is the mean ± SEM for 80% of the slices
tested with this protocol. The additional 20% not included in the
average did not show the characteristic sustained fEPSP decrease in
response to the increased temperature (the fEPSP increased slightly,
exhibited no change, or oscillated in amplitude during the increased
temperature). On average, the fEPSP amplitude was reduced to 39.5 ± 3.5% of the control value by the temperature increase and recovered
fully when the temperature change was reversed. B, Shown
are the same data, but with the fEPSP plotted as a function of
temperature instead of time. Responses elicited during the period when
temperatures were increasing are indicated by a plus and
during the decreasing phase by an open diamond. The
relationship between temperature and response amplitude was nearly
identical during both the temperature increase and decrease.
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Figure 3.
Responses of individual slices to changes in
temperature. A, fEPSP amplitude changes in response to
the standard temperature increase (32.538.5°C; the time course is
illustrated in Fig. 1A). Although the temperature
manipulation was identical, these two slices illustrate some of the
variability in the latency, the degree of inhibition, and rate of fEPSP
decrease in response to the temperature increase. B,
Synaptic depression induced by an extended 32.538.5°C protocol. In
this slice the temperature increase was maintained for 45 min; the
inhibition of the fEPSP was maintained for the duration of the
increased temperature and recovered when the recording temperature was
returned to 32.5°C. C, D, Examples of
synaptic responses recorded from the hippocampal slices shown in
A and B; individual records correspond to
responses evoked at the times indicated by the letters in
A (a-c) and B
(d-f). The superimposed traces highlight the
decreased latency to the peak of the fEPSP in traces b
and e (peak fEPSP in baseline recordings
a and d are indicated by a dashed
vertical line), indicative of the faster kinetics of the
response at the increased temperature.
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Because there did not appear to be an absolute temperature threshold
for the effect, we determined if a relative threshold temperature
increase was needed to produce significant inhibition. The change in
temperature required to inhibit the fEPSP by 20% was determined for a
population of slices incubated and recorded at 32.5°C and gradually
raised to 38.5°C (Fig. 4). There was no clear-cut threshold for this effect; 12 of 58 slices showed a 20%
inhibition with a <2°C change, but the thresholds ranged from <2°C to >5°C in others. There were three slices that exhibited a
decrease, but that did not reach the 20% criterion with a 6°C increase tested. Although it was not investigated extensively, it also
was noted that slices prepared from the same animal tended to respond
more similarly than did slices from different animals, suggesting that
minor differences in slice preparation, age, or other differences
between individual animals may confer increased or decreased
sensitivity to temperature changes.
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Figure 4.
Temperature change needed to produce a 20%
inhibition in the fEPSP with the 32.538.5°C protocol. This
cumulative histogram illustrates the temperature change required to
produce a 20% decrease in fEPSP amplitude. Although a significant
number of slices (12 of 58) exhibited this level of inhibition with a
<2°C increase in the recording temperature, others (17 of 58) did
not respond with such a decrease until the temperature had changed by
>5°C. The cumulative histogram was approximately linear over this
range, indicating that there was a nearly uniform distribution of
thresholds across this range. The median temperature change required to
elicit a 20% decrease in the fEPSP was 3.4°C.
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Mechanisms underlying the inhibitory response to
temperature increases
Because various other kinds of metabolic stresses, such as hypoxia
and hypoglycemia (Fowler, 1993; Lloyd et al., 1993), increase the
extracellular adenosine concentration in brain slices and because
adenosine is a well characterized inhibitory presynaptic modulator at
this synapse, we determined the effects of adenosine receptor
antagonists on the response to temperature increases. Pretreatment with
theophylline (200 µM) antagonized the reduction in
synaptic responses normally seen with the increased temperature (Fig.
5A,C; n = 5;
p < 0.05). Similarly, once maximal inhibition of the
fEPSP was achieved during a period of increased temperature, theophylline was able to reverse the inhibition completely (Fig. 5B; n = 4). Because these experiments
suggested that adenosine was responsible for the inhibition and
presynaptic adenosine A1 receptors have been linked to the
inhibitory modulation of this synapse (Dunwiddie and Fredholm, 1989),
the effect of an A1-selective antagonist, DPCPX (1 µM), was examined also. Pretreatment with DPCPX was
as effective as theophylline in preventing the fEPSP decrease (Fig.
5C; n = 4; p < 0.005).
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Figure 5.
Effects of pharmacological antagonists on
thermally induced synaptic depression (32.538.5°C).
A, Pretreatment with theophylline (Theo;
200 µM) caused an initial increase in the fEPSP
attributable to antagonism of the inhibitory effect of endogenous
adenosine. Continued superfusion of theophylline during the temperature
increase caused a significant reduction in the inhibition that
typically is seen with the temperature increase (compare with Figs.
1-3). B, Theophylline not only reduced the depression
of synaptic responses when it was superfused before a temperature
increase (A, B) but also fully reversed
the effect of the temperature increase on the fEPSP amplitude once the
depression had occurred (n = 4). C,
In each slice the initial response to the increased temperature was
recorded and compared with the effect of increased temperature in the
same slice during drug superfusion (statistical comparisons were with
paired Student's t tests). The average control
depression in all 32 slices tested with drugs is shown as
control. The nonselective adenosine receptor antagonist
theophylline (Theo; 200 µM), the adenosine
A1 receptor antagonist DPCPX (1 µM), and
adenosine deaminase (ADA; 0.8-1.6 U/ml) all increased the
baseline fEPSP (Theo, 136.2 ± 15.1%; DPCPX, 114.1 ± 3.8%;
ADA, 122.9 ± 12.8%), and all three significantly decreased the
degree of synaptic depression as compared with that observed during an
initial temperature increase (*p < 0.05;
**p < 0.005). However, application of either the
competitive NMDA receptor antagonist APV (50 µM) or the
noncompetitive antagonist MK801 (10 µM) had no
significant effect on the synaptic depression.
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These results suggested that adenosine was most likely responsible for
the synaptic inhibition induced by temperature increases. However, it
was possible that another purine that is active at A1
receptors might mediate the inhibitory response. To demonstrate that
adenosine per se was responsible for the synaptic depression, we
superfused slices with adenosine deaminase (ADA; 0.8-1.6 U/ml), which rapidly converts extracellular adenosine to inosine, which is
essentially inactive at adenosine receptors. Pretreatment for 15 min
before and during the temperature increase with this concentration of
ADA significantly reduced the degree of inhibition when compared with
the inhibition seen during the initial temperature increase in the same
set of slices (Fig. 5C; n = 9;
p < 0.05).
Mechanisms underlying increases in extracellular adenosine
The preceding results point to an increased activation of
A1 receptors by endogenous adenosine as mediating the
temperature-induced fEPSP decrease. There are multiple mechanisms that
can lead to increases in extracellular adenosine, including efflux of
cAMP from cells followed by extracellular conversion to adenosine
(Rosenberg et al., 1994), direct efflux of adenosine itself (Lloyd et
al., 1993), and an unknown mechanism that is initiated by activation of
the NMDA receptors (Craig and White, 1992; Manzoni et al., 1994). NMDA
receptor activation increases adenosine efflux from both cortical and
hippocampal slices and is thought to result from the release of an
unknown adenine nucleotide, followed by extracellular conversion to
adenosine (Craig and White, 1993). However, application of neither the
competitive NMDA receptor antagonist APV (50 µM;
n = 3, n.s.), nor the noncompetitive antagonist MK801
(10 µM; n = 11, n.s.) before and during
the temperature increase had any effect on the synaptic inhibition
(Fig. 5C). Previous studies in other regions have suggested
that activation of the NMDA receptors occurs even with relatively low
rates of stimulation one stimulus every 20 sec; Harvey and Lacey, 1997) and is sufficient to initiate adenosine release. Therefore, experiments were conducted in which the synaptic stimulation was terminated before
the temperature increase and then started at a time when the maximal
change in the fEPSP would be expected. With this protocol the responses
were inhibited maximally immediately on restarting the stimulation
(decreased by 56.4%; n = 2), providing further evidence that the activation of glutamate receptors by the synaptic stimulation was not involved in the increased concentrations of adenosine.
To evaluate the role of cAMP efflux as the source of extracellular
adenosine, we inhibited the conversion of cAMP to 5'-AMP (which
subsequently can be dephosphorylated into adenosine) with the type IV
phosphodiesterase inhibitor Ro 20-1724 (200 µM; Bonci and
Williams, 1996). In a separate set of experiments we blocked cAMP
efflux directly by inhibiting the transporter that is primarily responsible for its release with probenecid (0.2-2 mM).
Neither Ro 20-1724 nor probenecid had a significant effect on the
synaptic inhibition produced by the temperature increase (control
inhibition vs Ro 20-1724 treatment, 80.4 ± 10.9 vs 70.4 ± 20.9%, respectively; n = 3; control inhibition vs
probenecid treatment, 53.3 ± 10.4 vs 58.9 ± 11.4%,
respectively; n = 6; both effects were nonsignificant by paired t tests).
Because adenine nucleotides can be released and subsequently can be
converted to adenosine via ectonucleotidases (Zimmerman, 1996), we
evaluated the role of extracellular nucleotides as a source for
extracellular adenosine. Slices were superfused with the ecto-ATPase
inhibitor FPL 67156 (200 µM) to prevent the conversion of
extracellular ATP into ADP, which could undergo subsequent dephosphorylation into adenosine. We also used a combination of AOPCP
and GMP (500 µM and 2 mM, respectively) to
inhibit the ecto-5'-nucleotidase that converts AMP into adenosine.
Neither treatment affected the synaptic inhibition produced by the
temperature increase (control inhibition vs FPL 67156 treatment,
73.2 ± 17.4 vs 75.7 ± 9.7%, respectively;
n = 2; control inhibition vs AOPCP/GMP treatment, 52.2 ± 9.3 vs 32.1 ± 9.7%, respectively; n = 6, n.s.). Although not significant, the AOPCP/GMP data showed a trend
toward a reduced effect of temperature after pretreatment with this
cocktail. Because superfusion with AOPCP/GMP resulted in a large
decrease in the EPSP (most probably because of the inhibitory effect of
AOPCP that has been noted by other groups as well; Cunha et al., 1998), the somewhat reduced response to the temperature increase might have
been the consequence of a "floor" effect. To control for this, we
performed three additional experiments in which the stimulus amplitude
was increased after superfusion with AOPCP/GMP to return the synaptic
response to the baseline amplitude; under these conditions the
inhibition in the presence of AOPCP/GMP (60.8 ± 14.8%) was nearly identical to the control inhibition in the same slices (60.2 ± 13.8%). These results suggest that extracellular
conversion of nucleotides is not the major source of adenosine in the
current paradigm. However, with the inhibitors that are currently
available, it is difficult to establish the complete inhibition of the
ectoenzymes or phosphodiesterases targeted here, so these negative
results should be interpreted with caution.
To determine the contribution of direct adenosine efflux as a source of
extracellular adenosine, we used dipyridamole (5 µM) to
block the primary adenosine transporter found in the hippocampus (Deckert et al., 1988). Superfusion of dipyridamole had two effects on
the synaptic inhibition consequent to the temperature increase. Dipyridamole increased the latency and decreased the rate of onset of
the depression of synaptic responses (Fig.
6), suggesting that a
dipyridamole-sensitive transporter mediates adenosine release during
the temperature increase. If adenosine efflux were occurring via a
nontransporter-dependent mechanism, the rate of inhibition would be
expected to increase, rather than decrease, because extracellular adenosine generated via such mechanisms would accumulate faster when
uptake through the transporter is inhibited. Ultimately, dipyridamole
did not reduce the maximal effect of the temperature increase; rather,
it significantly augmented the extent of depression of the fEPSP
response (control inhibition vs dipyridamole treatment, 60.2 ± 10.3 vs 83.6 ± 1.6%, respectively; n = 9;
p < 0.05). This is likely attributable to dipyridamole
blocking the usual role of this bidirectional nucleoside transporter in
removing adenosine from the extracellular space; without the
significant reuptake of adenosine, more adenosine accumulates under
equilibrium conditions, although the rate of efflux may be
reduced.
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Figure 6.
Effect of dipyridamole (dipyr) on
latency of onset and rate of synaptic depression. Response amplitudes
are shown for a single slice during an initial control temperature
increase (filled circles) and during a second
temperature increase in which dipyridamole superfusion was begun
immediately before the temperature increase (open
diamonds). Dipyridamole delayed the onset of the depression and
slowed the rate at which the synaptic response decreased during the
temperature increase. Because dipyridamole alone results in a slow
continuous decline in the fEPSP in most slices (Dunwiddie and Diao,
1994), statistical analyses of the time course of averaged responses
were problematic, because most slices showed significant effects of
dipyridamole alone. This individual slice was one in which the
dipyridamole-induced decline was minimal at the time that the
temperature was increased. Despite the possible additional effect of
dipyridamole contributing to a decrease in the EPSP, there was instead
a delay in the onset and a slowing of the rate of the inhibition.
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Because the metabolic energy requirements of tissue generally are
increased at higher temperatures and because ischemia and hypoxia are
well known stimulators of adenosine release, we considered the
possibility that increases in temperature were making the slices
hypoxic by increasing their metabolic rate. Previous studies with both
hypoxia and ischemia have demonstrated that the ability of synaptic
transmission to recover is a function of the duration of exposure and
that prolonged treatment (>10 min and up to 1 hr, depending on the
severity of the insult) leads to irreversible loss of, or a significant
decrease in, subsequent synaptic transmission. Therefore, we determined
the effects of maintaining slices for longer durations (45-65 min) at
increased temperatures on the magnitude of the fEPSP inhibition as well
as the extent of subsequent recovery. When the temperature increase was
maintained for a longer duration (see, e.g., Fig. 3B), the
synaptic response remained depressed for the duration of the
temperature increase and recovered after this extended temperature
increase. In nine slices tested for >45 and up to 65 min with the
32.5°38.5° protocol, the inhibition of the fEPSP was maintained
throughout the increased temperature, but, more importantly, the fEPSP
amplitude after recovery was nearly identical to the original baseline
(0.754 ± 0.050 vs 0.753 ± 0.067 mV; n = 9, n.s.). Finally, if the adenosine release were secondary to
hypoxia/ischemia, the extent of recovery would be expected to be least
complete in those slices that were most affected by the increased
temperature. However, there was no correlation between the percentage
of inhibition at increased temperature and the degree of recovery after
the inhibition (r = 0.133, n.s.).
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DISCUSSION |
The results of this study demonstrate that increasing the
temperature of hippocampal slices results in a profound decrease in the
amplitude of synaptically evoked CA1 field potentials. The inhibition
of synaptic transmission is fully reversible on cooling to the initial
recording temperature, repeatable in individual slices, and mediated
primarily by adenosine A1 receptors. Increasing the
temperature from 32.5 to 38.5°C produced a decrease in fEPSP amplitude in 80% of the slices that were tested. If similar changes in
adenosine levels occur concurrently with endogenous temperature fluctuations in vivo, this suggests that there may be an
important temperature dependency to the ongoing modulatory role of
adenosine in the hippocampus and in the nervous system in general.
Given that normal endogenous fluctuations in brain temperature are
somewhat less than the standard protocol used here, it is unclear
whether these effects are relevant to more modest physiological temperature increases likely to occur in intact animals. Some slices
did respond to temperature changes of <2°C, and, in vivo, body temperature changes of this magnitude can be initiated in a
variety of ways, such as by increased physical activity, stress, or
increased ambient temperature (Anderson et al., 1983; Kluger, 1991;
Rothwell, 1994). However, all of the responses tested in the present
experiments were from a hypothermic baseline (usually 32.5°C), so it
is unclear whether similar changes would ever be seen in
vivo from a normothermic baseline. Although some studies provide
support for the hypothesis that similar responses might occur in
vivo [for example, Porkka-Heiskanen et al. (1997) have shown that
brain adenosine levels are elevated at a time when circadian body
temperatures are high (Refinetti and Menaker, 1992)], other studies
suggest the opposite conclusion; (e.g., increased brain temperatures
are associated with increased field potential amplitudes
in vivo; Moser et al., 1993). Thus, future studies are
required to establish the physiological relevance of this phenomenon.
Although the conditions studied here are not pathological, adenosine
release has been established as a common way that cells respond to
metabolically stressful conditions. The release of adenosine during
cell stress has been shown to be neuroprotective, because adenosine
receptor antagonists exacerbate neuronal cell loss initiated by hypoxia
or ischemia (Rudolphi et al., 1987). Although adenosine release confers
neuroprotection against pathological insults, its neuroprotective
ability may be simply an extension of its more normal role in
neuromodulation. Correlative evidence obtained in vivo
suggests that an increase in body temperature is associated with an
increase in brain adenosine levels. In many mammals the highest body
temperature is found before sleep onset (Refinetti and Menaker, 1992),
when extracellular adenosine in brain is highest (Porkka-Heiskanen et
al., 1997). Extracellular adenosine levels in the basal forebrain
increase during periods of sleep deprivation and drop during sleep
(Porkka-Heiskanen et al., 1997), at which time the body temperature
usually drops as well (Refinetti and Menaker, 1992). Similarly, Huston
et al. (1996) demonstrated an increase in adenosine specific to the
hippocampus as rats approach sleep, start to exhibit sleep-like
behaviors, and decrease other behaviors. These researchers suggest that
promotion of the restorative phase of sleep by adenosine may be related to the well characterized neuroprotective role of adenosine.
In addition to altering overall metabolic rate, temperature changes are
known to influence the dynamics of agonist and antagonist binding to
receptors. With this in mind, we considered whether the inhibition
observed here might be merely a result of an increased responsiveness
to adenosine at higher temperatures rather than a change in adenosine
concentrations. However, several studies suggest that this is not the
case. Spangenberger et al. (1995) found that rat hippocampal slices
were less sensitive to adenosine at increased temperatures
and thus could not provide an explanation for our observations. In
addition, although binding thermodynamics at A1 receptors
results in a small increase in agonist affinity with increased
temperature, this effect reverses at temperatures above 35°C (Borea
et al., 1995). Even if agonist affinity continued to increase linearly
at temperatures above 35°C, this could account only for ~10% of
the observed decrease in the fEPSP (Borea et al., 1991; Dunwiddie and
Diao, 1994). Thus, changes in adenosine receptor sensitivity cannot
account for the large synaptic depression observed in these experiments.
The mechanism underlying the adenosine increases observed in these
studies is unclear. Although activation of the NMDA receptors is one
potential mechanism that can result in an increase in extracellular adenosine in hippocampal slices (Manzoni et al., 1994), the lack of
effect of NMDA receptor antagonists suggests that this mechanism was
not involved. Adenosine concentrations appeared to change independently
of direct electrical stimulation of the Schaffer collateral/commissural
pathway, suggesting that postsynaptic glutamate receptor activation is
not required. In addition, the potential role of adenine nucleotide
release and its subsequent conversion to adenosine seems unlikely;
however, the efficacy of inhibitors, particularly for 5'-nucleotidase,
is somewhat problematic, so it is difficult to make definitive
conclusions based on this kind of negative data. However, the delay in
the onset of the inhibition with dipyridamole is consistent with the
hypothesis that adenosine per se is released from cells within the
brain slice. In biochemical studies the inhibitors of adenosine
transport reduce the efflux of adenosine itself (Lloyd et al., 1993)
because the transporters are essential for adenosine flux in either
direction across cell membranes. The modest delay and slowed rate of
onset of the hyperthermic response suggest that adenosine efflux
through a dipyridamole-sensitive transporter is the most likely source
for the increase in extracellular adenosine.
Whatever mechanism accounts for the release of adenosine, the stimulus
required is not hyperthermia per se, because the temperatures that were
used were not hyperthermic; the average core temperature of a rat is
between 37 and 38°C (Refinetti and Menaker, 1992), which were the
highest temperatures tested. Moreover, we observed adenosine-mediated
changes in synaptic transmission at temperatures that were in the
hypothermic range (2332°C). A recent paper by Gabriel et al.
(1998) also demonstrated an adenosine-mediated synaptic depression in
rat hippocampal slices increased from 22 to 29°C. Taken together,
this evidence emphasizes that the stimulus responsible for adenosine
release is an increase in temperature but that hyperthermia itself is
not involved.
Although the definitive mechanism underlying the
temperature-related increase in extracellular adenosine is unclear, the
results presented here have interesting implications for clinical
as well as nonclinical situations. For instance, fever is associated
with a general decrease in locomotor activity, an increase in sleep, and a reduced cognitive ability (Hart, 1988). Such symptoms are consistent with the known behavioral manifestations of increased extracellular adenosine and may be mediated at least in part via the
increased activation of adenosine receptors. As noted, the endogenous
circadian rhythm of body temperature and adenosine levels is consistent
with the current results both the highest body temperature and the
highest adenosine concentrations are found before sleep onset
(Refinetti and Menaker, 1992; Porkka-Heiskanen et al., 1997). It also
should be noted that adenosine itself induces hypothermia and that
stable A1 receptor agonists can induce profound hypothermia
in vivo (Dunwiddie and Worth, 1982). Finally, these results
are relevant to brain slice studies, because changing temperatures even
by several degrees may have significant functional consequences related
to altered adenosine levels in the extracellular space.
Increased temperature may be similar to some pathological conditions in
that it might be expected to produce an imbalance between metabolic
requirements and the supply of oxygen and glucose to the tissue.
However, in contrast to hypoxia or ischemia models, we observed full
recovery of function even after 1 hr at increased temperatures. Such
recovery would not be possible consequent to these other insults,
although the adenosine A1 receptor-mediated synaptic
inhibition is similar. Studies with mild to moderate hypoxia are
perhaps most similar to those described here, insofar as the effects
appear to be partially reversible even after 1 hr (Arlinghaus and Lee,
1996). However, temperature changes sufficient to alter extracellular
adenosine seem much more likely to occur both under normal
physiological conditions and under more common conditions, such as
during illness or exercise, than do other types of metabolic stress
that have been shown to alter adenosine levels.
In conclusion, in this study we demonstrate that a mild form of
stressmoderate temperature increasecauses a profound and sustained
synaptic depression mediated by adenosine A1 receptor activation in the CA1 region of the hippocampus. The characteristics of
this synaptic depression include its full reversibility on cooling to
the initial temperature and its repeatability in an individual slice.
In this regard, an increase in temperature represents a nonpathological
manipulation that markedly affects synaptic transmission via adenosine.
| |
FOOTNOTES |
Received Sept. 14, 1998; revised Dec. 15, 1998; accepted Dec. 22, 1998.
This work was supported by Grant R01 NS 29173 from the National
Institute of Neurological Disorders and Stroke and the Veterans Administration Medical Research Service. We acknowledge the
contribution of Daniel Lopez.
Correspondence should be addressed to Dr. Susan A. Masino, Neuroscience
Program, University of Colorado Health Sciences Center, Neuroscience
B138, 4200 East Ninth Avenue, Denver, CO 80262.
| |
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Top
Abstract
Introduction
