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The Journal of Neuroscience, April 15, 1998, 18(8):2974-2981
Evidence That Increased Hippocampal Expression of the Cytokine
Interleukin-1 Is a Common Trigger for Age- and Stress-Induced
Impairments in Long-Term Potentiation
Ciara A.
Murray and
Marina A.
Lynch
Department of Physiology, Trinity College, Dublin 2, Ireland
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ABSTRACT |
Several cytokines and their receptors are identified in brain; one
of these is the proinflammatory cytokine interleukin-1 that is
synthesized and released from neurons and glia in response to stress or
insult. Among the actions of interleukin-1 is its ability to inhibit
long-term potentiation in the hippocampus in vitro, an
action that mimics one of the consequences of stress and age. It has
been shown that the concentration of interleukin-1 in brain tissue
is increased in neurodegenerative conditions, and recent evidence from
our laboratory has indicated an increase in the concentration of
interleukin-1 in the hippocampus of aged rats. These observations
led us to consider that the underlying common cause of impaired
long-term potentiation in aged and stressed rats might be increased
endogenous interleukin-1 concentration in hippocampus. The data
presented here indicate that there was an inverse relationship between
concentration of interleukin-1 in the dentate gyrus and long-term
potentiation in perforant path granule cell synapses in aged rats,
stressed rats, and rats pretreated with interleukin-1. The evidence
suggested that the cytokine induces formation of reactive oxygen
species that triggers lipid peroxidation in vivo, as
well as in vitro, and that these changes lead to
depletion of membrane arachidonic acid that correlates with impaired
long-term potentiation. We propose that three theories of aging, the
glucocorticoid theory, the membrane theory, and the free radical
theory, constitute three facets of age with one underlying trigger: an
increase in the endogenous concentration of interleukin-1 in
hippocampus.
Key words:
long-term potentiation; dentate gyrus; interleukin-1; aging; stress; lipid peroxidation; arachidonic acid; reactive oxygen
species
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INTRODUCTION |
Recent evidence indicates a central
role for the proinflammatory cytokine interleukin-1 (IL-1). For
example, IL-1 modulates gastric function (Mo et al., 1996 ) and appetite
(Plata-Salaman et al., 1988), mediates fever (Fontana et al., 1984;
Dascombe et al., 1989), and stimulates the
hypothalamo-pituitary-adrenal axis (Sapolsky et al., 1987). IL-1 is
synthesized by neuronal (Farrar et al., 1987; Lechan et al., 1990)
and/or glial cells (Giulian et al., 1986; Yao et al., 1992) and is
released in response to injury, insult, and stress (Rothwell and
Hopkins, 1995). It is therefore not surprising that increased
expression of IL-1 has been associated with neurodegenerative
conditions (Griffin et al., 1989).
Autoradiographic (Takao et al., 1990; Ban et al., 1991; Ericsson et
al., 1995), immunohistochemical (Lechan et al., 1990), and in
situ hybridization studies (Cunningham et al., 1992; Parnet et
al., 1994) indicate a high density of IL-1 receptors in hippocampus, and consistent with this are several effects of exogenously applied IL-1; it inhibits calcium influx (Plata-Salaman and ffrench-Mullen, 1994; Murray et al., 1997), protein kinase C (Murray et al., 1997), release of acetylcholine (Rada et al., 1991), and glutamate (Murray et
al., 1997) in hippocampus. IL-1 also inhibits long-term potentiation (LTP) in vitro (Katsuki et al., 1990; Bellinger et al.,
1993; Cunningham et al., 1996), although the mechanism is unknown. One possibility is that IL-1 induces formation of reactive oxygen species
in brain as in other cell types (Nathan and Tsunawaki, 1986; Sumoski et
al., 1989), which is consistent with the finding that free radicals
inhibit LTP (Pellmar et al., 1991). Free radicals also induce oxidation
of polyunsaturated fatty acids, such as arachidonic acid (Nagy, 1994),
decreasing membrane concentrations and thereby inhibiting LTP (Lynch
and Voss, 1994; McGahon et al., 1997).
Plasma levels of corticosterone that increase with age also correlate
with impairments in LTP (Landfield et al., 1978a; Landfield and
Eldridge, 1994; Bodnoff et al., 1995) and with cell loss (Landfield et
al., 1978b). These observations form the basis of the glucocorticoid hypothesis of age-related neurodegeneration (Landfield and Eldridge, 1994). However, free radicals also play a role in neuronal cell death
(Choi and Yu, 1994; Nagy, 1994), supporting the hypothesis that
age-related neuronal deficits result from oxidative damage (Harman,
1956). Aspects of both hypotheses are significant in the context that
IL-1 stimulates secretion of corticotropin-releasing factor from the
hypothalamus (Sapolsky et al., 1987), the hallmark of stress and a
feature of aging (Landfield and Eldridge, 1994), and induces lipid
peroxidation in hippocampal tissue (Lynch, 1997), which may explain the
age-related decrease in membrane arachidonic acid (Lynch and Voss,
1994). In the context of these observations, the finding that there is
an age-related increase in IL-1 concentration in whole hippocampus
(Murray and Lynch, 1997) is particularly salient.
The objective of this study was to investigate the mechanism underlying
the inhibitory effect of IL-1 on LTP and therefore address the
hypothesis that increased concentration of IL-1 might explain the
impairment of LTP in aged and stressed rats.
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MATERIALS AND METHODS |
Male Wistar rats obtained from Charles River were used in these
experiments. Rats were kept at 22-23°C on a 12 hr light/dark cycle
and housed in groups of two or three for aged animals or six for young
animals except in one series of experiments. In this experiment 12 rats
were housed in two groups of six for 10 d, and at the end of this
period six of these rats were separated and individually housed for 1 week before the experiment.
Induction of LTP in perforant pathgranule cell synapses
in vivo. LTP was induced as described previously (McGahon and
Lynch, 1996). Rats were anesthetized by intraperitoneal injection of urethane. The mean ± SEM dose of urethane required to induce
anesthesia (as assessed by the absence of a pedal reflex) in young
animals was 2.09 ± 0.12 gm/kg and 1.70 ± 0.12 gm/kg in aged
animals. Animals were placed in a head holder in a stereotaxic frame.
At this time, some rats were injected intraventricularly with either
IL-1 (10 µl, 3.5 ng/ml) or saline (10 µl). Experiments were not
performed in a double-blind manner. A window of skull was removed to
allow placement of recording and stimulating electrodes in the
molecular layer of the dentate gyrus (2.5 mm lateral and 3.9 mm
posterior to bregma) and perforant path, respectively (angular bundle,
4.4 mm lateral to lambda). The depth of the electrodes was adjusted to
obtain maximal responses in the cell body region, and the stimulus strength chosen was that at which a spike appeared. Test shocks at the
rate of one every 30 sec were delivered for 10 min before tetanic
stimulation, and then LTP was induced unilaterally in perforant
pathgranule cell synapses by delivery of three high-frequency trains
of stimuli (250 Hz for 200 msec) at 30 sec intervals. Stimulation at
test shock frequency resumed, and recordings continued for the
remaining 40 min of the experiment. In the experiments in which rats
received an intraventricular injection, a 30 min period elapsed before
electrophysiological recordings commenced. At the end of the
electrophysiological recording period, rats were killed by
decapitation, and in some experiments blood was collected for analysis
of serum corticosterone. The hippocampus was removed, and the tetanized
and untetanized dentate gyri were dissected on ice and cross-chopped
into slices (350 × 350 µm) using a McIlwain tissue chopper. All
analyses (except circulating corticosteroids) were performed on samples
of dentate gyrus. Individual samples were frozen in 1 ml of Krebs'
solution (in mM: NaCl 136, KCl 2.54, KH2PO4 1.18, MgSO4 · 7
H2O, 1.18, NaHCO3 16, glucose 10, and
CaCl2 1.13) containing 10% dimethylsulfoxide according to
the method of Haan and Bowen (1981). Thawed slices were rinsed three
times in fresh buffer and homogenized in 200 µl of fresh Krebs'
solution for analysis of membrane arachidonic acid concentration, in 40 mM Tris-HCl, pH 7.4, for analysis of reactive oxygen
species production, and in distilled water for analysis of lipid
peroxidation.
Analysis of IL-1 concentration. We used an ELISA for
quantitative analysis of IL-1 (DuoSet ELISA development system,
Genzyme). Ninety-six-well plates were coated with 100 µl of capture
antibody (monoclonal hamster anti-mouse IL-1 antibody; 2.0 µg/ml
final concentration, diluted in 0.1 M sodium carbonate
buffer, pH 9.5) and incubated overnight at 4°C. Samples were washed
several times with PBS containing 0.05% Tween 20 (PBS-Tween) and then
blocked for 2 hr at 37°C with 250 µl of blocking buffer in PBS, pH
7.3 (0.1 M with 4% bovine serum albumin). Blocking buffer
was aspirated, and aliquots (100 µl) of samples or IL-1 standards
(0-1000 pg/ml) were added to each well and incubated for 1 hr at
37°C. Plates were washed, secondary antibody (biotinylated polyclonal
rabbit anti-mouse IL-1 antibody; 100 µl) was added to each well,
and incubation continued for 1 hr at 37°C. The plates were then
washed, and 100 µl of detection agent (horseradish
peroxidase-conjugated streptavidin; 1:1000 dilution in PBS-Tween and
1% bovine serum albumin) was added to each well, incubation continued
for 15 min more at 37°C, and plates were again washed. Aliquots of
substrate (100 µl, tetramethylbenzidine liquid substrate; Sigma,
Poole, UK) were added, and the plates were incubated at room
temperature for 10 min. The reaction was stopped by adding 100 µl of
H2SO4 (1 M), and absorbance was
read at 450 nm within 30 min.
Analysis of lipid peroxidation. Lipid peroxidation was
assessed by measurement of malondialdehyde (MDA), an intermediate in lipid peroxidation, by a modification of the method of Ohkawa et al.
(1979). Briefly, thawed slices were homogenized in ice-cold distilled
water, and aliquots (30 µl) of homogenate were incubated at 37°C
for 60 min, after which time 8.1% SDS (30 µl), 20% acetic acid, pH
3.5 with NaOH (225 µl), and 0.8% (w/v) thiobarbituric acid (225 µl) were added. The volume was adjusted to 600 µl with distilled
water, and the samples were incubated for 60 min more at 95°C. The
samples were cooled, and the absorbance was measured at 532 nm. Lipid
peroxidation was determined from a standard curve of
1,1,3,3-tetramethoxypropane (0-100 µM) and expressed as
nanomoles of MDA per milligram of protein. In the case of aged rats,
values were expressed as micromoles of MDA per milligram of wet weight, because protein concentrations per unit of wet weight were reduced in
aged rats compared with young rats. Protein concentration was analyzed
according to the method of Bradford (1976).
Analysis of arachidonic acid concentration. Arachidonic acid
concentration was assessed as described previously (Miwa et al., 1986).
Briefly, fatty acids were extracted into chloroform/methanol (2:1 v/v;
1 ml) by vigorous shaking for 10 min, followed by centrifugation at
1000 × g for 5 min to separate the phases. The aqueous
layer was discarded, and the chloroform phase was evaporated under
nitrogen and resuspended in ethanol for analysis. Arachidonic acid was analyzed as its 2-nitrophenylhydrazine (NPH) derivative by
reverse-phase HPLC. Fatty acids were derivatized by adding 2-NPH-HCl
solution [0.02 M 2-nitrophenylhydrazine-HCl in 0.25 M HCl-ethanol (1:1, v/v)] and EDC solution
[(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); 0.25 M in ethanol mixed in equal volumes with 3% ethanolic
pyridine] and incubated at 60°C for 20 min. After addition of
potassium hydroxide (15% w/v in methanol/water, 80:20) samples were
incubated at 60°C for 15 min and cooled in running water. Derivatives
were concentrated; n-hexane and phosphate buffer (0.033 M, pH 6.4, in 0.5 M HCl) were added, samples
were vortex-mixed for 30 sec and centrifuged for 5 min at 1500 × g, and the hexane phase was evaporated to dryness under nitrogen. For
HPLC analysis, samples were resuspended in methanol and injected onto a
Intersil C18 column, and fatty acid derivatives were separated in
isocratic mode with a mobile phase of 85% acetonitrile and 15% water
(maintained at pH 4.5 with HCl) and detected by ultraviolet
spectroscopy at 230 nm. Arachidonic acid concentrations were estimated
using the external standard method and were expressed as micromoles per milligram of wet weight.
Analysis of reactive oxygen species formation. Formation of
reactive oxygen species was assessed by the method of Lebel and Bondy
(1990), which relies on the measurement of 2' 7'-dichlorofluorescein (DCF), the oxidized fluorescent product of 2'7'-dichlorofluorescin diacetate (DCFH-DA). Thawed slices were homogenized in 1 ml of ice-cold
Tris buffer (40 mM, pH 7.4) and incubated at 37°C for 15 min in the presence of DCFH-DA (10 µl; 5 µM from a
stock of 500 µM in methanol). To terminate the reaction,
the dye-loaded suspensions were centrifuged at 13,000 × g for 8 min at 4°C, and the pellets were resuspended in 2 ml of ice-cold 40 mM Tris buffer, pH 7.4, and monitored for
fluorescence at 37°C with the excitation wavelength at 488 nm and the
emission wavelength at 525 nm. Reactive oxygen species formation in the
unknown samples was estimated from a DCF standard curve (0.05-1
µM), and results were expressed as nanomoles per
milligram of wet weight per min.
Analysis of circulating corticosterone concentration.
Circulating corticosterone concentration in serum was determined by radioimmunoassay (ImmunoChem double-antibody corticosterone
125I RIA kit, ICN Biomedicals). Serum collected at the end
of the electrophysiological recording period was diluted in
phosphosaline gelatin buffer, pH 7.0, containing rabbit  -globulins,
and incubated with [125I]corticosterone and
anti-corticosterone for 2 hr at room temperature. Antibody-bound
corticosterone was precipitated using polyethylene glycol and goat
anti-rabbit -immunoglobulins in Tris buffer, and samples were
centrifuged at 1000 × g for 15 min and counted in a
gamma counter. [125I]Corticosterone in the samples
was assessed with reference to a standard curve and expressed as
micrograms per milliliter of serum corrected for nonspecific
binding.
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RESULTS |
We analyzed the effect of intraventricularly administered IL-1
(3.5 ng/ml, 10 µl) on synaptic responses and on LTP in perforant pathgranule cell synapses. IL-1 did not affect the mean EPSP slope recorded in the 10 min period before tetanic stimulation (1.22 ± 0.12 mV/msec in saline-treated rats vs 1.23 ± 0.12 mV/msec in IL-1-treated rats; mean ± SEM) but decreased the
amplitude of the response (4.17 ± 0.25 mV vs 3.11 ± 0.08 mV; p < 0.05, Student's t test). Analysis
of the effect of tetanic stimulation indicated that the immediate
increase in EPSP (i.e., in the 2 min period immediately after tetanic
stimulation) was slightly, but not significantly, attenuated by
pretreatment with IL-1 (Fig.
1b). In contrast, intraventricular injection of IL-1 significantly inhibited the long-lasting increase in EPSP; the mean ± SEM percentage changes in EPSP slopes in the last 5 min of the experiment compared with the 5 min period immediately before tetanic stimulation were 120.47 ± 1.39% and 97.27 ± 5.11% in the saline-treated
(n = 6) and IL-1-treated (n = 7)
rats, respectively (p < 0.01, Student's
t test). Figure 1a indicates that
intraventricular injection of IL-1 led to a significant increase in
the concentration of the cytokine in dentate gyrus ~80 min after
injection (p < 0.01, Student's t
test).
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Figure 1.
IL-1 concentration in dentate gyrus is
increased and LTP is compromised after intraventricular injection of
IL-1. a, IL-1 concentration (picograms per
milligrams of tissue) in dentate gyrus was significantly increased
(p < 0.01, Student's t
test; hatched bars) after intraventricular injection of
IL-1 (10 µl, 3.5 ng/ml) 30 min before recording commenced compared
with saline injection (open bars). Values are mean ± SEM. The number of observations is given in
parentheses. b, Induction of LTP was not
significantly affected by pretreatment with IL-1, but EPSP slope
decreased to baseline levels ~20 min after tetanic stimulation
(arrow). Values are given as mean percent of change in
the slope of the EPSP over a 50 min period (n = 7 in both groups). Error bars indicating SE are included at 5 min
intervals; in some instances these are so small as to be obscured by
the symbols.
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We used a similar stimulus strength to evoke responses in aged and
young rats in this series of experiments (mean ± SEM, 3.27 ± 0.21 and 4.29 ± 0.54 V in young and aged rats, respectively; see Materials and Methods). Under these circumstances, there was no
significant difference between the mean EPSP slope recorded in the 10 min period before tetanic stimulation in the young and aged rats
(1.14 ± 0.08 and 0.96 ± 0.09 mV/msec, respectively), but the mean amplitude of the response was significantly decreased with age (3.51 ± 0.26 and 1.90 ± 0.16 mV, respectively;
p < 0.05, Student's t test). Figure
2b shows that the immediate
increase in EPSP slope in the 2 min immediately after tetanic
stimulation was slightly, but not significantly, attenuated in
22-month-old animals compared with 4-month-old animals. There was a
marked age-related effect on maintenance of LTP; the data indicated
that the mean percentage changes in EPSP slope in the last 5 min of the
experiment compared with the 5 min immediately before tetanic stimulation were 134.27 ± 2.67 and 107.99 ± 2.92% in the
4-month-old (n = 7) and 22-month-old (n = 10) rats, respectively (Fig. 2b; p < 0.01, Student's t test). Figure 2a indicates
that IL-1 concentration was significantly increased in dentate gyrus
prepared from 22-month-old compared with 4-month-old rats
(p < 0.01, Student's t test).
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Figure 2.
IL-1 concentration in dentate gyrus is
increased and LTP is compromised in 22-month-old compared with
4-month-old rats. a, IL-1 concentration (picograms
per milligram of tissue) in dentate gyrus was significantly increased
(p < 0.01, Student's t
test; hatched bars) in aged rats compared with young
rats (open bars). Values are mean ± SEM, and the
number of observations is given in parentheses.
b, The mean percent of change in EPSP slope immediately
after the tetanus was similar in both groups of rats, but EPSP slope in
the last 5 min of the experiment was significantly reduced in the aged
rats (n = 10) compared with the young rats
(n = 7; p < 0.01, Student's
t test). Error bars indicating SE are included at 5 min
intervals; in some instances these are so small as to be obscured by
the symbols.
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In a separate series of experiments, six rats were housed as a group,
and another six rats were housed in individual cages, a manipulation
that has been shown to induce mild stress. To confirm that some stress
was incurred in the present experiment, we analyzed circulating
corticosteroid levels in serum obtained from these rats at the end of
the electrophysiological recording period. This analysis indicated a
significant increase in the group of animals housed in isolation,
compared with those housed together (Fig.
3; p < 0.01, Student's
t test). We also observed that there was a significant
increase in circulating corticosteroids in aged rats compared with
young rats (Fig. 3; p < 0.05, Student's t
test). The mean slope of the EPSP in the 10 min period before tetanic stimulation was greater in the individually housed rats compared with
the group-housed rats (1.05 ± 0.07 and 1.52 ± 0.07 mV/msec, respectively; p < 0.05, Student's t test),
but the mean amplitude of the response was similar in both groups
(4.34 ± 0.23 and 4.68 ± 0.18 mV in the individually housed
and group-housed rats, respectively).
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Figure 3.
Circulating corticosteroid concentrations were
elevated in mildly stressed rats and in aged rats. There was a
significant increase in circulating corticosteroids in 22-month-old
compared with 4-month-old rats (p < 0.05, Student's t test) and similarly in rats that had been
housed in isolation, i.e., mildly stressed rats, compared with the
control rats that had been housed as a group
(p < 0.01, Student's t
test). Analysis was made in samples obtained from rats at the end of
electrophysiological recording in all cases. The number of observations
is given in parentheses.
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We report that LTP was impaired in rats that were housed in individual
cages compared with rats that were housed as a group; both induction
and maintenance of LTP were significantly affected. As a measure of
induction of LTP, we assessed the change in EPSP slope in the 2 min
period immediately after tetanic stimulation; these values were
147.38 ± 4.28 and 129.30 ± 4.32% in the group-housed rats
and the individually housed rats, respectively (Fig.
4; p < 0.05, Student's
t test). The data also indicated that maintenance of LTP was
affected; the mean percentage changes in EPSP slope in the last 5 min
of the experiment compared with the 5 min immediately before tetanic
stimulation were 115.42 ± 0.90 and 92.35 ± 0.77% in the
group-housed and individually housed rats, respectively (Fig.
4b; p < 0.05, Student's t
test). Figure 4a indicates that IL-1 concentration was
significantly increased in hippocampus prepared from the individually
housed rats compared with the group-housed rats
(p < 0.01, Student's t test).
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Figure 4.
IL-1 concentration in dentate gyrus is
increased and LTP is compromised in rats that were mildly stressed by
isolation-housing compared with control rats. a, IL-1
concentration (picograms per milligram of tissue) was significantly
increased (p < 0.01, Student's
t test; hatched bars) in mildly stressed
rats compared with control rats (open bars). Values are
mean ± SEM, and the number of observations is given in
parentheses. b, Induction of LTP was
significantly reduced in the mildly stressed rats compared with the
control rats (p < 0.05, Student's
t test), and the mean percent of EPSP slope returned to
baseline levels in ~10 min in the stressed rats
(n = 6), whereas it remained elevated for the
duration of the experiment in the control rats (n = 4). Values are given as mean percent of change in the slope of the EPSP
over a 50 min period. Error bars indicating SE are included at 5 min
intervals; in some instances these are so small as to be obscured by
the symbols.
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The data from these three experiments demonstrate that there is a
negative correlation between IL-1 concentration and LTP in the
dentate gyrus. Analysis of the data from the separate experiments indicate a significant correlation in each case (Fig.
5; p < 0.05 in the
experiments that analyzed changes in aged vs young rats and the
experiment that compared the effect of intraventricular injection of
saline and IL-1; p < 0.001 in the experiments that analyzed difference between individually housed and group-housed rats).
IL-1 concentrations were not obtained in all samples because of
technical difficulties; all the matched paired data that we obtained
for EPSP slopes and IL-1 concentrations are presented. In
vitro analysis revealed that IL-1 induced a significant
increase in lipid peroxidation in hippocampal preparations (Fig.
6; p < 0.001, Student's
t test), and this was reversed by the antioxidant vitamin E,
suggesting that the action of IL-1 might require formation of
reactive oxygen species. Figure 6 also indicates that IL-1 significantly enhanced reactive oxygen species formation in hippocampus in vitro (p < 0.01, Student's
t test). To assess the possibility that increased endogenous
IL-1 might induce lipid peroxidation in vivo, tissue was
prepared from hippocampus of saline-injected and IL-1-injected rats
and analyzed for lipid peroxidation and membrane arachidonic acid
concentration. Figure 7 indicates that intraventricular injection of IL-1 induced a significant increase in
lipid peroxidation (p < 0.05, Student's
t test), and this was coupled with a significant decrease in
membrane arachidonic acid (p < 0.001, Student's t test). A comparison of changes in aged and
young rats indicated significant age-related increases in reactive
oxygen species formation and lipid peroxidation coupled with an
age-related decrease in membrane arachidonic acid concentration (Fig.
8; p < 0.05 in the case
of reactive oxygen species production; p < 0.01 in the
case of lipid peroxidation).
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Figure 5.
Maintenance of LTP is inversely proportional to
IL-1 concentration in hippocampus. Regression analysis on data from
aged rats compared with young (a), individually
housed rats compared with group-housed (b), and
IL-1-pretreated rats compared with saline-pretreated
(c) revealed that there was a statistically
significant correlation between IL-1 concentration and the mean
percent of change in EPSP slope in the last 5 min of the experiment.
Regression (r) and p values are
indicated.
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Figure 6.
IL-1 induces lipid peroxidation and reactive
oxygen species production in hippocampal tissue in
vitro. IL-1 (3.5 ng/ml) significantly increased lipid
peroxidation in hippocampal homogenate (p < 0.001) and this was inhibited by vitamin E (200 µM).
IL-1 also significantly increased formation of reactive oxygen
species (p < 0.01, Student's
t test). The number of observations is given in
parentheses.
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Figure 7.
Intraventricular injection of IL-1 increases
lipid peroxidation and decreases membrane arachidonic acid
concentration in hippocampus. Untetanized dentate gyri from the
saline-injected and IL-1-injected rats were dissected at the end of
the period of electrophysiological recording, stored as described in
Materials and Methods, and analyzed for lipid peroxidation and membrane
arachidonic acid concentration. There was a significant increase in
lipid peroxidation (p < 0.05, Student's
t test), and this was accompanied by a significant
decrease in membrane arachidonic acid concentration
(p < 0.001, Student's t
test). The number of observations is given in
parentheses.
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Figure 8.
Aging was associated with increased reactive
oxygen species formation and lipid peroxidation, coupled with a
decrease in membrane arachidonic acid. Untetanized dentate gyri from 4- and 22-month-old rats were dissected at the end of the period of
electrophysiological recording, stored as described in Materials and
Methods, and analyzed for reactive oxygen species production, lipid
peroxidation, and membrane arachidonic acid concentration. Reactive
oxygen species production and lipid peroxidation were significantly
increased in 22-month-old compared with 4-month-old rats
(p < 0.05 and p < 0.01, respectively, Student's t test). Mean arachidonic
acid concentration was significantly decreased in 22-month-old compared
with 4-month-old rats (p < 0.01, Student's
t test). The number of observations is given in
parentheses.
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DISCUSSION |
Our first objective was to establish whether the impairment
in LTP in perforant pathgranule cell synapses, associated with age,
stress, or IL-1, might be explained by one common underlying factor:
an increase in IL-1 concentration in dentate gyrus. The second
objective was to investigate the mechanism by which this increase in
IL-1 concentration might exert its inhibitory effect.
The data indicate that intraventricular injection of IL-1 had
little effect on induction of LTP in the dentate but inhibited the
persistent increase in EPSP slope. Although an inhibitory effect of
IL-1 has been described in vitro in CA3 (Katsuki et al.,
1990), CA1 (Bellinger et al., 1993), and dentate gyrus (Cunningham et
al., 1996), this is the first indication that intraventricular injection of the cytokine inhibits LTP in any area of the hippocampus in vivo. We found that IL-1 concentration in dentate
gyrus was significantly increased in rats that had received an
intraventricular injection of the cytokine compared with
saline-injected rats. The mechanism by which this change is triggered
is unknown; injected IL-1 may diffuse from the ventricle to the
neighboring dentate gyrus, or it may induce release of IL-1 from
neuronal and/or glial cells in the dentate gyrus, as described
elsewhere in the CNS (Yao et al., 1990). This question remains to be
addressed.
Electrophysiological responses to test shocks were reduced in aged rats
compared with young rats, and we observed that there was an age-related
decrease in the ability to sustain LTP, as reported previously
(Landfield et al., 1978a; Barnes, 1979; Davis et al., 1993; Diana et
al., 1995; Lynch and Voss, 1994; McGahon et al., 1997), although there
was no evidence of an age-related change in EPSP slope immediately
after tetanic stimulation. The age-related impairment of the ability to
sustain LTP correlated with increased IL-1 concentration in dentate
gyrus, and although we have reported an age-related increase in IL-1
in hippocampus (Murray and Lynch, 1997), this is the first report of an
increase in the dentate gyrus and the first evidence that an increase
correlates with impaired LTP.
LTP was impaired in rats that had undergone mild stress compared with
control rats. The stress response stimulates an array of changes,
including the observed increase in circulating corticosterone that
relies on activation of the hypothalamus to increase release of
corticotropin-releasing factor. Hypothalamic release of
corticotropin-releasing factor is stimulated in vitro by
IL-1 (Sapolsky et al., 1987), and because the hippocampus modulates
hypothalamo-pituitary function (Landfield and Eldridge, 1994), we
argued that an increase in IL-1 in hippocampus might underlie the
increase in circulating corticosteroids. We report that in rats that
had undergone a mild stress, the increase in circulating corticosterone
correlated with increased IL-1 concentration in hippocampus. The
observation that circulating corticosteroids and IL-1 concentration
in hippocampus were also increased in parallel, in aged rats compared
with young rats, strengthens the hypothesis that a causal relationship
between the two parameters exists. However, the interplay between
circulating glucocorticoids and cytokines is complex with evidence of
feedback control; thus, whereas IL-1 stimulates the
hypothalamo-pituitary-adrenal axis, the anti-inflammatory actions of
glucocorticoids include inhibition of cytokine synthesis in brain
(Rothwell et al., 1996). The question of the interplay between
circulating corticosteroids and IL-1 concentration in hippocampus
remains to be addressed; the data presented indicate a causal link
between these parameters but do not allow speculation concerning
feedback control.
One consequence of behavioral stress is impaired LTP (Foy et al., 1987;
Diamond et al., 1990; Landfield and Eldridge, 1994; Xu et al., 1997),
which has been attributed to an increase in circulating corticosteroids
and is inversely proportional to ability of rats to sustain LTP (Foy et
al., 1987). In support of these observations, the present findings
indicate an inverse relationship between circulating corticosteroids
and impaired LTP in two groups of animals, i.e., aged rats and
behaviorally stressed rats. It is noteworthy that analysis of the
effect of stress on primed burst potentiation revealed a more
complex relationship between potentiation and circulating
corticosteroids, such that at low concentrations a direct relationship
was observed and at high concentrations an inverse relationship existed
(Diamond et al., 1992).
We demonstrated that increased IL-1 concentration is a common
feature of impaired LTP in three circumstances, after intraventricular injection of IL-1, in aged rats and in stressed rats, but although a
significant inverse correlation between IL-1 concentration and
ability to sustain LTP was observed, it would be unreasonable to
suggest that other factors do not contribute to impairments in LTP in
aged and stressed rats. These data are consistent with the hypothesis
that although limited exposure of tissue to low concentrations of
IL-1 may be neuroprotective, prolonged exposure to higher
concentrations may induce degenerative changes (Hopkins and Rothwell,
1995; Rothwell and Hopkins, 1995). It is of interest that increased
expression of IL-1 has been reported in Alzheimer's disease and
Down's syndrome (Griffin et al., 1989), and it is conceivable that the
age-related increase in IL-1 in dentate gyrus observed in this study
may be indicative of degenerative changes in the rat brain, one
manifestation of which is an impairment in LTP.
Previous reports have indicated that impaired LTP in aged rats was
associated with decreased membrane arachidonic acid concentration (Lynch and Voss, 1994), restoration of which reversed the age-related impairment in LTP (McGahon et al., 1997). Although the trigger responsible for inducing the decrease in membrane arachidonic acid has
not been identified, depletion of polyunsaturated fatty acids is a
known consequence of lipid peroxidation (Rehncrona et al., 1980; Yu et
al., 1992; Nagy, 1994). In vitro analysis indicated that
lipid peroxidation was increased by IL-1, and this effect was
inhibited by the antioxidant vitamin E, suggesting that the
IL-1-induced effect was mediated by production of reactive oxygen
species formation, supporting data previously obtained in macrophages
(Nathan and Tsunawaki, 1986; Harrison and Murphy, 1995). Data presented
here confirm the stimulatory effect of IL-1 on reactive oxygen
species production in hippocampus. We probed the changes that accompany
the increase in IL-1 concentration in vivo and
established that there was a parallel increase in lipid peroxidation
and decrease in membrane arachidonic acid concentration in hippocampus
of aged rats. Similar correlations were observed in rats injected
intraventricularly with IL-1. These observations are consistent with
the hypothesis that IL-1, by inducing lipid peroxidation, may be the
endogenous trigger for the age-related decrease in membrane arachidonic
acid concentration.
Although neuronal damage has been associated with enhanced plasma
levels of corticosterone (Landfield and Eldridge, 1994), accumulation
of oxidative damage by free radicals is readily observed in the aged
brain (Choi and Yu, 1995). Our data indicate that there was an increase
in production of reactive oxygen species in dentate gyrus prepared from
aged rats when compared with young, and in mildly stressed rats when
compared with control. Although IL-1 increases formation of reactive
oxygen species in hippocampus in vitro, we observed that it
also triggers this effect in vivo. This increase in reactive
oxygen species may be a direct effect of IL-1 or it may derive from
IL-1-induced peroxidation of arachidonic acid. Oxygen radicals can
increase IL-1 in hippocampus in vitro (C. A. Murray
and M. A. Lynch, unpublished observations), suggesting that a
positive feedback loop exists in which a spiral of potentially detrimental reactions can be triggered by IL-1. The findings of this
study permit us to propose that the age- and stress-related increase in
reactive oxygen species formation in hippocampus may be consequent on
increased endogenous IL-1, which we propose is the salient feature
of the free radical hypothesis of aging.
The inhibitory effect of IL-1 on LTP in vitro has been
linked with an inhibitory effect on glutamate release (Murray et al., 1997) and protein kinase C (Plata-Salaman and ffrench-Mullen, 1994),
but the molecular mechanism by which IL-1 induces these changes is
unknown. An IL-1-associated decrease in calcium influx in
hippocampus has also been described (Plata-Salaman and ffrench-Mullen, 1994; Cunningham et al., 1996), although recent evidence has indicated that higher concentrations of IL-1 increased intracellular calcium concentrations (Campbell and Lynch, 1998). This finding is of interest because there is a good deal of evidence suggesting that intracellular calcium concentration is increased in hippocampal neurons
of aged rats (e.g., Landfield and Eldridge, 1994). We propose that
IL-1 induces lipid peroxidation that decreases membrane arachidonic
acid, consequently affecting membrane fluidity that will impact on
membrane-associated functions, and may therefore explain the
IL-1-induced effects on transmitter release, channel activity, and
enzyme activity. We propose that the changes leading to increased
membrane rigidity are triggered by IL-1, and therefore, that the
age-related increase in endogenous IL-1 in hippocampus may be the
precursor to the membrane hypothesis of aging. Our findings directly
couple increased circulating corticosterone in two physiological
conditions, age and stress, with increased endogenous IL-1
concentration in hippocampus, and therefore provide evidence that the
glucocorticoid theory of aging might also be explained by elevated
levels of the cytokine.
| |
FOOTNOTES |
Received Oct. 29, 1997; revised Jan. 30, 1998; accepted Feb. 5, 1998.
This work was supported by the Health Research Board of Ireland,
Forbairt, and the Provost's Academic Development Fund, Trinity College.
Correspondence should be addressed to Dr. Lynch at the above address.
| |
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Abstract
Introduction
