Lipopolysaccharide inhibits long term potentiation in the rat dentate gyrus by activating caspase-1.

Lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, may be responsible for at least some of the pathophysiological sequelae of bacterial infections, probably by inducing an increase in interleukin-1beta (IL-1beta) concentration. We report that intraperitoneal injection of lipopolysaccharide increased hippocampal caspase-1 activity and IL-1beta concentration; these changes were associated with increased activity of the stress-activated kinase c-Jun NH(2)-terminal kinase, decreased glutamate release, and impaired long term potentiation. The degenerative changes in hippocampus and entorhinal cortical neurones were consistent with apoptosis because translocation of cytochrome c and poly(ADP-ribose) polymerase cleavage were increased. Inhibition of caspase-1 blocked these changes, suggesting that IL-1beta mediated the lipopolysaccharide-induced changes.

correlated with neurodegenerative disorders such as Down syndrome, Alzheimer's disease (6), and Parkinson's disease (7), whereas in experimental models, IL-1␤ is considered to be responsible for the cell damage associated with ischemia (8) and excitotoxicity (9) and is increased after experimental traumatic lesions (10). A striking example of a neuronal deficit induced by IL-1␤ is the impairment in long term potentiation (LTP) in the hippocampus in vitro (11)(12)(13) and in vivo (14 -16).
IL-1␤ is produced by glia (17,18) and neurones (19,20) in response to tissue stress. It is cleaved from the inactive percursor, pro-IL-1␤, by the action of caspase-1, a member of a large family of cysteine proteases that have been implicated in apoptotic cell death (21)(22)(23)(24)(25). It might be predicted therefore that any trigger such as LPS, which induces an increase in IL-1␤, will do so by increasing activity of caspase-1.
Our objective was to investigate the cellular consequences of an increase in IL-1␤ concentration in hippocampus in an effort to establish the mechanism by which IL-1␤ inhibits LTP in dentate gyrus. Intraperitoneal injection of LPS stimulated caspase-1 activity and induced an increase in IL-1␤ concentration, and these changes were paralleled by an increase in activity of the stress-activated protein kinase c-Jun NH 2 -terminal kinase (JNK), a decrease in glutamate release, and inhibition of LTP in perforant path granule cell synapses. These changes, and the degenerative changes in neurones of the hippocampus and entorhinal cortex, were reversed by caspase-1 inhibition.

EXPERIMENTAL PROCEDURES
Induction of LTP in Vivo-Six groups of six male Wistar rats (250 -350 g), obtained from the BioResources Unit, Trinity College Dublin, were anesthetized by intraperitoneal injection of urethane (1.5 g/kg). All rats groups received 1 ml of saline or 1 ml of LPS (200 g/kg) intraperitoneally; four groups were pretreated either with an intracerebroventricular injection of 5 l of saline or 5 l of the caspase-1 inhibitor (10 pmol of Ac-YVAD-CMK, 2.5 mm posterior to Bregma, 0.2 mm lateral to midline, 3.5-mm depth) prior to the intraperitoneal injection and monitored for 3 h. A bipolar stimulating electrode and a unipolar recording electrode were placed in the perforant path (4.4 mm lateral to Lambda) and in the dorsal cell body region of the dentate gyrus (2.5 mm lateral and 3.9 mm posterior to Bregma), respectively, and 0.033-Hz test shocks were given for 10 min before, and 40 min after, tetanic stimulation (three trains of stimuli delivered at 30-s intervals, 250 Hz for 200 ms (26)). Rats were killed by cervical dislocation; cross-chopped slices (350 ϫ 350 m) were prepared from ipsilateral and contralateral dentate gyri, entorhinal cortex, and hippocampus and used to prepare dissociated cells (see below) or frozen separately in Krebs solution containing 10% dimethyl sulfoxide (27) and stored at Ϫ80°C. For analysis, slices were thawed rapidly and rinsed in fresh oxygenated Krebs solution before preparation of homogenate or the crude synaptosomal pellet P 2 (26).
Analysis of Reactive Oxygen Species Formation-Formation of reactive oxygen species was assessed by measuring 2Ј7Ј-dichlorofluorescein (DCF), the oxidized, fluorescent product of 2Ј7Ј-dichlorofluorescein diacetate (DCFH-DA (28)). Synaptosomes prepared from hippocampal slices were incubated at 37°C for 15 min in the presence of 10 l of 5 M DCFH-DA (from a stock of 500 M) in methanol and centrifuged at 13,000 ϫ g for 8 min at 4°C to yield pellets that were resuspended in 2 ml of ice-cold 40 mM Tris buffer, pH 7.4, and monitored for fluorescence at 37°C (excitation, 488 nm; emission, 525 nm).
Analysis of Caspase-1 Activity-Cleavage of the caspase-1 substrate (YVAD peptide, Alexis Corporation) to its fluorescent product was used as a measure of caspase-1 activity. Slices of tissue were washed, homogenized in 400 l of lysis buffer (25 mM HEPES, 5 mM MgCl 2 , 5 mM dithiothreitol, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml pepstatin, pH 7.4), subjected to four freezethaw cycles, and centrifuged at 15,000 rpm for 20 min at 4°C. 90-l samples of supernatant were added to 10 l of 500 M YVAD peptide and incubated at 37°C for 60 min. 900 l of incubation buffer (100 mM HEPES containing 10 mM dithiothreitol, pH 7.4) was added, and fluorescence was assessed (excitation, 400 nm; emission, 505 nm).
Analysis of JNK Phosphorylation, Cytochrome c Translocation, and Poly(ADP)-ribose Polymerase (PARP) Cleavage-JNK phosphorylation was analyzed in samples prepared from hippocampal tissue; cytochrome c translocation and PARP cleavage were analyzed in samples prepared from entorhinal cortex. In the case of JNK and PARP, tissue homogenates were diluted to equalize for protein concentration (29), and 10-l aliquots (1 mg/ml) were added to 10 l of sample buffer (0.5 mM Tris-HCl, pH 6.8, 10% glycerol, 10% SDS, 5% ␤-mercaptoethanol, 0.05% bromphenol blue, w/v), boiled for 5 min, and loaded onto gels (10% SDS for PARP and 12% for JNK). In the case of cytochrome c, the cytosolic fraction was prepared by homogenizing slices of entorhinal cortex in lysis buffer (composition in mM: 20 HEPES, pH 7.4, 10 KCl, 1.5 MgCl 2 , 1 EGTA, 1 EDTA, 1 dithiothreitol, 0.1 phenylmethylsulfonyl fluoride, 5 g/ml pepstatin A, 2 g/ml leupeptin, 2 g/ml aprotonin), incubating for 20 min on ice, and centrifuging (15,000 ϫ g for 10 min at 4°C). The supernatant (i.e. cytosolic fraction) was suspended in sample buffer (150 mM Tris-HCl, pH 6.8, 10% glycerol v/v, 4% SDS w/v, 5% ␤-mercaptoethanol v/v, 0.002% bromphenol blue w/v) to a final concentration of 300 g/ml, boiled for 3 min, and loaded (6 g/lane) onto 12% gels. In all cases proteins were separated by application of a 30-mA constant current for 25-30 min, transferred onto nitrocellulose strips (225 mA for 75 min), and immunoblotted with the appropriate antibody. To assess JNK activity, proteins were immunoblotted with an antibody that specifically targets phosphorylated JNK (Santa Cruz Biotechnology, Inc.; 1:2,000 in PBS and 0.1% Tween 20 containing 2% non-fat dried milk) for 2 h at room temperature. Immunoreactive bands were detected using peroxidase-conjugated anti-mouse IgG (Sigma) and enhanced chemiluminescence (Amersham Pharmacia Biotech). To assess cleavage of PARP, we immunoblotted with an antibody (1:2,000) raised against the epitope corresponding to amino acids 764 -1014 of PARP of human origin (Santa Cruz Biotechnology Inc.), and immunoreactive bands were detected using peroxidase-conjugated anti-rabbit IgG (Sigma) and enhanced chemiluminescence. To assess cytochrome c, a rabbit polyclonal antibody raised against recombinant protein corresponding to amino acids 1-104 of cytochrome c (Santa Cruz Biotechnology Inc.) was used. Immunoreactive bands were detected using peroxidase-conjugated anti-rabbit antibody (Sigma) and enhanced chemiluminescence.
Release of Glutamate-Synaptosomal tissue prepared from untetanized and tetanized dentate gyrus was resuspended in ice-cold Krebs solution containing 2 mM CaCl 2 , aliquotted onto 0.45-m Millipore filters, and rinsed under vacuum. Tissue was incubated in 250 l of oxygenated Krebs solution Ϯ 40 mM KCl at 37°C for 3 min, and the filtrate was collected and stored. To analyze glutamate concentration, triplicate 50-l samples or 50-l glutamate standards (50 nM to 10 M in 100 mM Na 2 HPO 4 buffer, pH 8.0) were added to 320-l glutaralde-hyde (0.5% in 100 mM NaH 2 PO 4 buffer, pH 4.5)-coated 96-well plates and incubated for 60 min at 37°C (30). 250 l of ethanolamine (0.1 M in 100 mM Na 2 HPO 4 buffer) and 200 l of donkey serum (3% in PBS-T) were used to bind unreacted aldehydes and to block nonspecific binding, respectively. Samples were incubated overnight at 4°C in the presence of 100 l of anti-glutamate antibody (raised in rabbit, 1:5,000 in PBS-T, Sigma), washed with PBS-T, and then incubated for 60 min at room temperature with 100 l of anti-rabbit horseradish peroxidase-linked secondary antibody (1:10,000 in PBS-T, Amersham Pharmacia Biotech). 100 l of 3,3Ј,5,5Ј-tetramethylbenzidine liquid substrate was added, incubation continued for exactly 60 min, 30 l of 4 M H 2 SO 4 was added to stop the reaction, and optical densities were determined at 450 nm.
Dissociation of Cells and Analysis of Cell Viability-350-m slices prepared from entorhinal cortex and hippocampus were equilibrated in oxygenated Krebs solution for 30 min at 30°C and then incubated in Krebs solution containing 1 mg/ml protease X, 1 mg/ml protease XIV, and 1,600 Kunitz Dnase for 30 min at 30°C. Washed slices were resuspended in 1 ml of prewarmed Dulbecco's modified essential medium containing 1,600 Kunitz Dnase, triturated with a glass Pasteur pipette, and passed through a nylon mesh filter to remove tissue clumps. 30-l aliquots were plated out on poly-L-lysine-coated 11-mm round glass coverslips, placed in a 5% CO 2 incubator at 37°C for 1 h, and fixed in 4% paraformaldehyde (30 min at room temperature). Coverslips were stored at 4°C in PBS until use (31).
Cells were stained using the Rapi-diff II staining procedure (Di-aCheM International Ltd., Lancastershire, U. K,) and viewed under ϫ 100 magnification. Cells displaying degenerative features (e.g. shrinkage and membrane blebbing) were counted and expressed as a percentage of the total number of cells examined (80 -100/coverslip in the case of entorhinal cortex and 100 -200 in the case of the hippocampus).
Statistical Analysis-A one-way analysis of variance (ANOVA) was performed to determine whether there were significant differences between conditions. When this analysis indicated significance (at the 0.05 level), post hoc Student Newmann-Keuls test analysis was used to determine which conditions were significantly different from each other. Student's t test was used to establish statistical significance in some cases; for example, when analysis was performed on tissue prepared from untetanized and tetanized tissue obtained from the same rat.

LPS Blocks LTP by Increasing IL-1␤ Concentration in the
Hippocampus and Activating JNK-Tetanic stimulation delivered to the perforant path 3 h after intraperitoneal injection of LPS resulted in an increase in the mean slope of the population epsp; the mean percentage increase in the 2 min immediately following tetanic stimulation (Ϯ S.E., compared with the 5 min immediately before tetanic stimulation) was 133.58 (Ϯ 3.48), The LPS-induced attenuated LTP was associated with a significant increase in reactive oxygen species production, caspase-1 activity, IL-1␤ concentration, and JNK activity (*p Ͻ 0.05, ** p Ͻ 0.01, Student's t test for independent means, Fig.  2, a-d; n ϭ 6 in all cases) in hippocampus. The stimulatory effect of LPS on JNK activity was mimicked by the addition of IL-1␤ to hippocampal tissue in vitro (p Ͻ 0.01, Student's t test for paired means, Fig. 2e). Fig. 2f indicates that endogenous glutamate release was increased significantly by the addition of 40 mM KCl to synaptosomes prepared from untetanized dentate gyrus of saline-pretreated rats (*p Ͻ 0.05, Student's t test for paired means), but this effect was enhanced in synaptosomes prepared from tetanized dentate gyrus (**p Ͻ 0.01, Student's t test for paired means). In contrast, KCl failed to stimulate glutamate release in synaptosomes prepared from untetanized dentate gyrus of LPS-pretreated rats, although release was increase in tetanized tissue (*p Ͻ 0.05, Student's t test for paired means) albeit to an attenuated degree.
Inhibition of Caspase-1 Blocks the Effects of LPS-These data suggested that the LPS-induced effect on LTP may be a consequence of its ability to increase activity of caspase-1 and thence IL-1␤ concentration and to determine whether this was the case, rats were injected intracerebroventricularly with 5 l of a caspase-1 inhibitor peptide (Ac-YVAD-CMK) or with 5 l of saline prior to LPS or saline treatment. Fig. 3a indicates that although LTP was inhibited by LPS, this effect was blocked by the caspase-1 inhibitor. Thus the mean percentage change in population epsp slope (Ϯ S.E.) in the 2 min immediately after tetanic stimulation was 177.77 Ϯ 15.34 in the control group (treated with saline intracerebroventricularly and intraperitoneally) compared with 118.92 Ϯ 3.35 in the group treated with saline intracerebroventricularly and LPS intraperitoneally. In the last 5 min of the experiment the values were 123.86 Ϯ 2.14 and 96.21 Ϯ 1.14, respectively. Injection of the caspase-1 inhibitor partially reversed the inhibitory effect of LPS on the early changes induced by the tetani and completely blocked the LPS-induced inhibition of the later phase of LTP; the mean percentage changes were 147.32 Ϯ 9.23 and 123.54 Ϯ 6.33 in the first 2 min after tetanic stimulation and in the last 5 min of the experiment, respectively. However, LTP was similar in the control rats and the group of rats injected the caspase-1 inhibitor intracerebroventricularly and saline intraperitoneally; in the latter group, the mean percentage changes in population epsp slope were 174.57 Ϯ 16.24 and 142.07 Ϯ 7.42 in the 2 min after tetanic stimulation and the last 5 min of the experiment, respectively (Fig. 3a, n ϭ 6 in all groups). mM KCl to synaptosomes prepared from untetanized dentate gyrus of saline-injected rats significantly increased glutamate release (*p Ͻ 0.05, ANOVA, panel f), but this effect was enhanced in synaptosomes prepared from tetanized dentate gyrus (**p Ͻ 0.01, ANOVA). The addition of KCl to synaptosomes prepared from untetanized dentate gyrus of LPS-injected rats failed to enhance release, whereas the response was attenuated in synaptosomes prepared from tetanized tissue. Fig. 3b shows that intraperitoneal injection of LPS (in rats treated with saline intracerebroventricularly) significantly increased IL-1␤ concentration in hippocampus (* p Ͻ 0.05, Student's t test for independent means, n ϭ 6) and that this effect was inhibited by pretreatment with the caspase-1 inhibitor. Similarly, JNK activity was enhanced significantly in hippocampal tissue prepared from LPS-treated rats (p Ͻ 0.05, Student's t test for independent means), but this effect was also inhibited by the caspase-1 inhibitor (Fig. 3c).

Inhibition of Caspase-1 Blocks LPS-induced Degenerative Changes in Hippocampus and
Entorhinal cortex-Acutely dissociated cells were prepared from hippocampal tissue obtained from rats in each of the four treatment groups. LPS treatment significantly increased the number of degenerating cells, with evidence of an increased number of cells displaying degenerative features such as shrinkage and blebbing of the plasma membrane (Fig. 4b). This contrasts with cells prepared from saline-treated rats (panel a) and rats treated only with caspase-1 inhibitor (panel c). Treatment with the caspase-1 inhibitor (panel d) partially reversed the effects of LPS with fewer cells displaying degenerative changes. Fig. 4e shows that the percentage of cells which showed degenerative changes was enhanced significantly in the LPS-treated group compared with any of the other groups (p Ͻ 0.05, Student's t test for independent means) and indicates that the caspase-1 inhibitor reversed the degenerative effect of LPS.
In an effort to account for the compromise in transmitter release observed in dentate gyrus synaptosomes prepared from LPS-treated rats, we analyzed caspase-1 activity and IL-1␤ concentrations in tissue prepared from entorhinal cortex and found that both measures were increased significantly after LPS treatment (*p Ͻ 0.05, Student's t test for independent means), but these effects were both attenuated by the caspase-1 inhibitor (Fig. 5, a and b, n ϭ 6). In parallel with the observations in hippocampus, we observed that there was an LPS-induced increase in the number of degenerating cells changes (** p Ͻ 0.01, Student's t test for independent means, Fig. 6a), with evidence of cell shrinkage and membrane blebbing. Pretreatment with the caspase-1 inhibitor blocked these LPS-associated changes (Fig. 6, a and b). Consistent with the evidence of cell degeneration in hippocampus, we observed that cytochrome c translocation was increased markedly in tissue prepared from entorhinal cortex of LPS-treated rats (p Ͻ 0.01, Student's t test for independent means), whereas there was a decrease in expression of the 116-kDa fragment of (PARP, * p Ͻ 0.05, Student's t test for independent means); both of these LPS-associated changes were attenuated by pretreatment with the caspase-1 inhibitor (Fig. 6, c and d; n ϭ 6). DISCUSSION We set out to investigate the effect of an intraperitoneal injection of LPS on synaptic function in hippocampus because LPS is considered to contribute significantly to the neuropathological effects associated with Gram-negative bacterial infections probably by increasing IL-1␤ concentration in brain. The evidence presented indicates that the LPS-induced increase in IL-1␤ concentration, consequent on increased caspase-1 activity, leads to activation of JNK which may underlie the observed decrease in transmitter release in dentate gyrus, degenerative changes in hippocampus and entorhinal cortex, and inhibition of LTP.
Intraperitoneal injection of LPS inhibited LTP in perforant path granule cell synapses; to our knowledge this effect of LPS has not been shown previously. The current data present at least two possible mechanisms that might underlie the effects. First, we observed that LPS induced an increase in reactive oxygen species production in hippocampus, and the attenuated LTP may arise, directly or indirectly, from this. Such an effect of oxygen radicals has been reported in CA1 in vitro (32), and we have recently observed that LTP in dentate gyrus in vivo was inhibited by hydrogen peroxide. 2 A second possibility is that the impairment in LTP is a consequence of the LPSinduced increase in IL-1␤ concentration in hippocampus. We have observed that intracerebroventricular injection of IL-1␤ inhibits LTP in perforant path-granule cell synapses in vivo (14,15,33) and that LTP was also compromised in aged and stressed rats, in which hippocampal IL-1␤ concentration is increased (14). The inhibitory effect of IL-1␤ on LTP in vitro has also been documented; thus IL-1␤-induced attenuation of LTP in CA1 (11), CA3 (12) and dentate gyrus (13) has been reported.
Peripheral injection of LPS induced an increase in IL-1␤ concentration in hippocampus, which supports earlier reports of a similar change in hippocampus and cortex (3,34), cerebellum (4), and in whole brain (2). These data are backed up by several observations of LPS-induced increases in IL-1␤ concentrations or IL-1␤ mRNA in cultured glial cells (4,35), which, together with other data indicating that IL-1␤ is synthesized in glia (17,18) and neurons (19,20), suggests that IL-1␤ may be produced locally. We have observed that the blood-brain barrier is not affected by intraperitoneal injection of 200 g/kg LPS within the time frame of the experiment described here, 3 suggesting that the increase in hippocampal IL-1␤ is unlikely to be peripheral in origin. We cannot rule out the possibility that LPS gains access to the brain after intraperitoneal injection and stimulates production of IL-1␤ directly, or indirectly, for example through production of tumor necrosis factor or another cytokine. The evidence presented suggests that the increase in IL-1␤ concentration in hippocampus is a consequence of an increase in caspase-1 activity, which cleaves pro-IL-1␤ to yield the active cytokine. Cleavage may occur by an autocatalytic process, or it may involve other proteases (21), or, because caspases are cysteine-dependent enzymes and are redox-sensitive (36), they may be stimulated by reactive oxygen species (37). The parallel LPS-induced increases in reactive oxygen species production and caspase-1 activity observed here might indicate a causal interaction between these two parameters. The LPS-induced increase in caspase-1 activity is in contrast to a previous finding in which intraperitoneal injection of 2 mg/kg LPS (compared with 200 g used here) triggered caspase-1 activation in pituitary gland, but not in hippocampus or hypothalamus (38), although mRNA for caspase-1 was increased in all regions.
JNK phosphorylation was increased significantly in hippocampus of LPS-treated rats, and although a similar effect has been reported in neutrophils (39), macrophages (39,40), and cultured rat microglia (41), we believe that this is the first indication that such an effect occurs in brain in vivo. A few groups have reported that LPS stimulates tyrosine kinase activity, for example in macrophages (42) and in cultures of glial cells (35); this probably represents one of the earliest signaling events stimulated by LPS and explains the observed increase in phosphorylation of mitogen-activated protein kinases, JNK, and p38 (39,40). The stimulatory effect of LPS on JNK activation in vivo was mimicked in vitro by IL-1␤ as reported by us previously (16). Consistent with the evidence presented here, we have observed that LTP in dentate gyrus was attenuated when JNK activity was enhanced in hippocampus, for example in aged rats (16) or rats injected intracerebroventricularly with IL-1␤ (56).
Several reports indicate that LTP in dentate gyrus is accompanied by an increase in release of glutamate (26,(43)(44)(45). The present data provide further direct evidence of the coupling between LTP and enhanced glutamate release and indirect evidence of a causal relationship between these parameters 3  because both were attenuated after LPS treatment. The mechanism underlying the LPS-induced decrease in release was not addressed directly in this study but is paralleled by increased IL-1␤ concentration and increased JNK activation. An inhibitory effect of IL-1␤ on glutamate release in hippocampus has been reported previously (46), whereas release has also been shown to be attenuated when IL-1␤ concentration and JNK activation are increased in hippocampus, for example in aged rats (16).
If increased IL-1␤ concentration and/or increased JNK activation in hippocampus is primarily responsible for the LPSinduced impairment in LTP, it follows that LTP would not be affected if these changes were inhibited; we argued that this inhibition might be achieved by pretreating rats with the caspase-1 inhibitor Ac-YVAD-CMK. We report that the inhibitor blocked each of these effects of LPS, providing evidence that they are causally linked and permitting us to restate our proposal that LTP is impaired when IL-1␤ concentration is increased (14,15) and adding support to the argument that increased JNK activation is coupled with impaired LTP (16).
There was an increase in the number of cells exhibiting evidence of degeneration in both entorhinal cortex and hippocampus prepared from LPS-treated rats compared with saline-treated controls. Because this was reversed by the caspase-1 inhibitor, it seems likely that one of the consequences of increased activity of caspase-1, for example increased concentration of IL-1␤ or increased activation of JNK, mediates the effect. A role for the caspases in cell death has been described (21), and increased concentrations of IL-1␤ have been closely linked with neuronal degeneration (6 -10, 47). Similarly, JNK activation has been shown to be a component of the cell death pathway in PC12 cells (48 -50) and is required for apoptosis during early brain development (51).
Accompanying the LPS-associated neuronal degeneration in entorhinal cortex, we observed an increase in caspase-1 activity and IL-1␤ concentration, both of which were attenuated in tissue prepared from rats pretreated with the caspase-1 inhibitor. Evidence of increased cytochrome c translocation and cleavage of the DNA repair enzyme, PARP, were also observed after LPS treatment. Cytochrome c translocation from the mitochondria to the cytosol, which may trigger activity of certain caspases (52), has been shown to be associated with apoptosis (53). This was confirmed by the recent demonstration that injection of cytochrome c (but not an inactive cytochrome c) resulted in apoptotic morphology in a variety of cells (54) and also by the finding that hydrogen peroxide induces apoptosis by triggering cytochrome c release from mitochondria (55). Similarly, cleavage of PARP, a substrate for caspases, has also been considered to be a reliable indicator of apoptosis (21). It is significant, though not perhaps surprising, that the LPS-induced increases in cytochrome c translocation and PARP cleavage were inhibited in tissue prepared from rats pretreated with the caspase-1 inhibitor. These data therefore suggest a role for caspase-1 in neuronal degeneration.
We attribute the decrease in LTP after injection of LPS described here to degenerative changes in neuronal cells in both the hippocampus and entorhinal cortex. Because these changes are blocked by a caspase-1 inhibitor, it seems that  (panel b, iv). The mean data were obtained by counting 80 -100 cells on each coverslip (*p Ͻ 0.05, Student's t test for independent means, n ϭ 6). Expression of the 116-kDa subunit of PARP (panel c) was decreased significantly and cytochrome c translocation (panel d) was increased significantly in tissue prepared from entorhinal cortex of LPS-treated rats (*p Ͻ 0.05, Student's t test for independent means, n ϭ 6). These effects were blocked in rats pretreated with the caspase-1 inhibitor. Sample immunoblots demonstrate these effects of LPS (lanes 2 and 4) compared with control (lanes 1 and 3) in saline-pretreated (lanes 1 and 2) and Ac-YVAD-CMK-pretreated (lanes 3 and 4) rats.
IL-1␤ plays a pivotal role. The evidence suggests that LPS injection increases caspase-1 activity and thence IL-1␤ expression in hippocampus and entorhinal cortex. We propose that one downstream effect of increased IL-1␤ concentration in hippocampus is an increase in JNK phosphorylation, which directly or indirectly inhibits glutamate release (Fig. 7); however, the data presented are consistent with the view that increased IL-1␤ concentration in hippocampus and entorhinal cortex leads to degenerative changes consistent with apoptosis, and these changes are likely to contribute to the impaired LTP exhibited by LPS-treated rats. FIG. 7. Scheme suggesting cascade of events leading to the LPS-induced impairment in LTP. Intraperitoneal injection of LPS leads to an increase in reactive oxygen species (ROS) production, caspase-1 activity, and IL-1␤ concentration in hippocampus and entorhinal cortex. We propose that the increase in IL-1␤ leads to an increase in activity of JNK, which inhibits glutamate release and thereby LTP. The evidence presented suggests that increased IL-1␤ concentration also leads to degenerative changes in both hippocampus and entorhinal cortex, which is likely to contribute to the impairment in LTP.