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Variability in cognitive functioning increases markedly with age, as does cognitive vulnerability to physiological and psychological challenges. Exploring the basis of this vulnerability may provide important insights into the mechanisms underlying aging-associated cognitive decline. As we have previously reported, the cognitive abilities of aging (24-month-old) F344xBN rats are generally good, but are more vulnerable to the consequences of a peripheral immune challenge (an i.p. injection of live E. coli) than those of their younger (3-month-old) counterparts. Four days after the injection, the aging, but not the young rats show profound memory deficits, specific to the consolidation of hippocampus-dependent memory processes.
Here, we have extended these observations, using hippocampal slices to examine for the first time the combined effects of aging and a recent infection on several forms of synaptic plasticity. We have found that the specific deficit in long-lasting memory observed in the aged animals following infection is mirrored by a specific deficit in a form of long-lasting synaptic plasticity. The late-phase long-term potentiation (L-LTP) induced in area CA1 using theta burst stimulation is particularly compromised by the combined effects of aging and infection – a deficit that can be ameliorated by intra-cisterna magna administration of the naturally occurring anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra). These data support the idea that the combination of aging and a negative life event such as an infection might produce selective, early-stage failures of synaptic plasticity in the hippocampus, with corresponding selective deficits in memory.
Although it is not clear that a decline in the ability to learn and remember is a normal feature of aging, it is clear that variability in cognitive functioning increases with aging in humans (Laursen, 1997; Unverzagt et al., 2001) and animals (Gage et al., 1984; Barnes and McNaughton, 1985; Deupree et al., 1991; Gallagher et al., 2003). An intriguing clue about sources of this variability comes from the observation that aging increases cognitive vulnerability to challenging life events such as infection (Wofford et al., 1996), surgery (Bekker and Weeks, 2003), heart attack and psychological stress (VonDras et al., 2005).
Because little is known about the mechanisms that mediate aging-associated increases in cognitive vulnerability, we have developed a rodent model to study them (Barrientos et al., 2006). 24-month-old Fischer344/Brown Norway rats generally do not display significant physical or cognitive impairments prior to a brief infection produced by an intraperitonneal injection of Escherichia coli. However, after recovering from the active infection, the aged animals show significant impairment in hippocampus-dependent memory tasks (e.g. contextual fear and place learning); the young animals generally do not (Barrientos et al., 2006).
Data from conventional aging models examining variability in cognitive functioning with aging per se suggest that when age-related deficits in hippocampus-dependent learning occur, they do not arise from a loss of hippocampal neurons or synapses (Rapp and Gallagher, 1996; Geinisman et al., 2004), but rather from more subtle alterations in synaptic efficacy (Rapp et al., 1999; Smith et al., 2000). Not surprisingly, some age-related neurodegenerative disorders (e.g. Alzheimer’s Disease) first manifest themselves as disorders of synaptic plasticity, prior to the onset of overt cellular pathology (Selkoe, 2002). Thus, synaptic plasticity, particularly LTP, has been extensively studied in aging and disease models (Barnes and McNaughton, 1985; Deupree et al., 1991; Diana et al., 1995; Bach et al., 1999; Tombaugh et al., 2002) (Martin et al., 2000; Bliss et al., 2003).
These earlier studies have reinforced the idea that synaptic plasticity has multiple forms: short-term forms (e.g., early-phase LTP or E-LTP) involving the covalent modification of existing proteins, and long-lasting forms (e.g. late-phase LTP or L-LTP) that require transcription and translation (Bliss et al., 2007). It has also become apparent that different stimulation paradigms can evoke similarly sized, and similarly enduring, manifestations of synaptic plasticity – for example L-LTP -that nonetheless arise from distinct biochemical processes, and may reflect different information storage processes with differential vulnerabilities to disruption (e.g. Woo et al., 2000; Patterson et al., 2001). Thus, examining the impact of aging and immune challenge on these processes should ultimately provide mechanistic insights into aging-associated cognitive vulnerability.
In the present study, we have examined several forms of synaptic plasticity in hippocampal slices from young and aged rats, with and without a recent history of E. coli infection as a first step in utilizing the slice system to examine the cellular and molecular mechanisms underlying the memory deficits evoked by immune challenge in aged animals.
The rats were 3- and 24-month old male Fischer344/Brown Norway F1 crosses from the NIA Aged Rodent Colony. Animals were pair housed, on a 12-hr light dark cycle, with ad libitum access to food and water, and were allowed to acclimate to the animal facility for two weeks before experiments were begun. All experiments were conducted in accordance with protocols approved by the University of Colorado Animal Care and Use Committee.
One day prior to the start of experimentation, stock E. coli cultures (ATCC 15746; American Type Culture Collection, Manassas, VA) were thawed and cultured overnight (15–20 h) in 40 mL of brain-heart infusion (BHI; DIFCO Laboratories, Detroit, MI) in an incubator (37 °C, 95% air + 5% CO2). The number of bacteria in individual cultures was quantified by extrapolating from previously determined growth curves. Cultures were then centrifuged for 15 min at 3000 rpm, the supernatants were discarded, and the bacteria were resuspended in sterile phosphate buffered saline (PBS), to achieve a final dose of 2.5 × 109 CFU in 250μl.
All animals received an intraperitonneal (i.p.) injection of 250μl of either E. coli or the vehicle (sterile PBS).
Cisterna magna, rather than intra-hippocampal or ICV injections were utilized because this procedure does not require surgery - which can itself produce memory impairments in aging animals. Twenty-four month old rats were briefly anesthetized using halothane, and a 27- gauge needle connected to a 25 μl Hamilton syringe via PE50 tubing was inserted into the cisterna magna. Interleukin-1 specific receptor antagonist (IL-1Ra) or vehicle (endotoxin-free saline from Abbott Laboratories, North Chicago, IL, USA) was then injected into the cisterna magna. The IL-1Ra (112 μg; Amgen, Thousand Oaks, CA) was administered in a total volume of 3 μl. Immediately after this procedure, the rats received an i.p. injection of either E. coli or vehicle.
Physiology experiments were performed 4–5 days after the initial infection. This time point was chosen based on several observations: (1) all of the animals have completely recovered from the acute infection after four days (symptoms such as fever are gone within 3 days); (2) the 24-month-old rats, but not the 3-month-old rats show a significant impairment in long-term hippocampus-dependent memory four days after the E. coli infection (Barrientos et al., 2006); and (3) levels of IL-1 protein in the hippocampus are still significantly elevated in the 24-month-old rats, but not in the 3-month-old rats, 4–5 days after the infection (Barrientos et al., 2009).
Experiments on hippocampi from young and aged, saline or E. coli injected animals were interleaved. Hippocampi were collected from rats following decapitation. Transverse hippocampal slices (400 μm) were prepared using conventional techniques (e.g. (Patterson et al., 1992; Patterson et al., 1996). Slices were maintained in an interface chamber at 28°C, and perfused with an oxygenated saline solution (in mM: 124.0 NaCl, 4.4 KCl, 26.0 NaHCO3, 1.0 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4, 10 glucose). Slices were permitted to recover for at least 90 minutes before recording. Field excitatory postsynaptic potentials (fEPSP) were recorded from Schaffer collateral–CA1 synapses by placing both stimulating and recording electrodes in the stratum radiatum. All stimuli were delivered at intensities that evoked fEPSP slopes equal to 1/3 of the maximum in each slice. Test stimuli were delivered once every minute, and test responses were recorded for 15–30 minutes prior to beginning the experiment to assure stability of the response.
Slices were tetanized using one of three protocols: one, 1 sec train at 100 Hz; four, 1 sec trains, at 100 Hz, delivered 5 min apart; or 12 bursts, of 4 pulses at 100 Hz, delivered 200 msec apart (theta frequency). The 1-Train protocol was used to induce E-LTP. The 4-Train and Theta Burst protocols were used to induce L-LTP. The same stimulus intensity was used for tetanization and evoking test responses.
Data were analyzed using factorial ANOVA, followed by Fisher’s PLSD post hoc tests.
We began seeking functional deficits associated with aging and or a recent history of infection by examining basal synaptic transmission at the Schaffer collateral-CA1 synapse in hippocampal slices (Fig. 1A). To provide an initial indication of possible differences in the response to stimuli of a given intensity, we generated input-output curves from slices from young and aged rats injected 4 days earlier with either E. coli or saline. We found that these curves were not significantly altered by aging, or infection.
We next examined paired-pulse facilitation (Fig. 1B). PPF is a presynaptic form of short-term plasticity in which the synaptic response to the second of a pair of closely spaced stimuli is increased. This is thought to reflect residual Ca2+ in the presynaptic nerve terminal from the first stimulus adding to the influx of Ca2+ evoked by the second stimulus, with a resulting increase in presynaptic neurotransmitter release (Katz and Miledi, 1968). Neither aging nor a history of infection had a significant effect on PPF across a range of inter-stimulus intervals.
In order to examine possible alterations in short-term synaptic plasticity, we tetanized the slices using one high frequency stimulus train: 1 sec, at 100 Hz - a protocol frequently used to induce early-phase LTP lasting approximately 1–2 hours in slices from naive rats (Fig. 2). Neither age nor a history of infection had a significant effect on post-tetanic potentiation (% baseline, immediately after the stimulus train: young/vehicle = 235 ± 20%, young/E coli = 214 ±17%, aged/vehicle = 220 ± 24%, and aged/E coli = 223 ± 16%; P(age) = 0.581, and P(infection) = 0.290), or 1-Train E-LTP (% baseline, measured 90 minutes after the stimulus train: young/vehicle = 163 ± 23%, young/E coli = 134 ± 13%, aging/vehicle = 143 ± 13%, and aging/E coli = 141 ± 5%; P(age) = 0.373, and P(infection) = 0.250).
Different stimulus protocols produce long-lasting forms of synaptic plasticity with somewhat different molecular requirements (e.g. (Kang et al., 1997; Patterson et al., 2001). Thus, for the experiments reported here, slices were tetanized using one of two protocols: either four trains of high frequency stimulation or theta-burst stimulation. Both of these protocols induce late-phase LTP in animals of the hybrid strain used here. The high frequency 4-train protocol produces a robust activation of many, though not all, plasticity-related signaling cascades (reviewed in (Bliss et al., 2007). The theta burst protocol is more naturalistic - designed to mimic the burst firing of CA1 pyramidal cells at theta frequency recorded in vivo from awake behaving animals during spatial exploration (reviewed in (O’Keefe, 2007) – and has proven to be a sensitive indicator of alterations in mnemonic processes associated with aging (reviewed in Lynch et al., 2006) or pharmacological or genetic manipulation of the substrates for memory.
When we examined the effects of aging and infection on the L-LTP evoked by the 4-train protocol (Fig. 3A), we found no significant effects on post-tetanic potentiation (% baseline: young/vehicle = 247 ± 23%, young/E coli = 247 ± 18%, aged/vehicle = 253 ± 42%, and aged/E coli = 280 ± 27%; P(age) = 0.857, and P(infection) = 0.420) or 3 hours after tetanus (% baseline: young/vehicle = 204 ± 20%, young/E coli = 178 ± 13%, young/vehicle = 202 ± 32%, and young/E coli = 184 ± 16%; P(age) = 0.970, and P(infection) = 0.408).
The effects of the theta burst stimulation were more complex (Fig. 3B). Under the conditions used, age did not have a significant effect on post-tetanic potentiation (P(age) = 0.703) or L-LTP three hours after the tetanus (P(age) = 0.307). In contrast, E coli infection had no effect on post-tetanic potentiation (P(infection) = 0.534%; baseline: young/vehicle = 236 ± 19%, young/E coli = 238 ± 13%, aged/vehicle = 255 ± 14%, and aged/E coli = 232 ± 21%), but resulted in significantly smaller L-LTP in slices from young rats (P(infection in young rats) = 0.010), and profoundly reduced L-LTP in slices from aged animals (P(infection in aged rats) = 0.006; % baseline, 3 hours after tetanus: young/vehicle = 184 ± 11%, young/E coli = 147 ± 5%, aged/vehicle = 169 ± 12%, and aged/E coli = 113 ± 5%).
The pro-inflammatory cytokine interleukin-1beta (IL-1beta) is a major mediator of inflammatory responses in the brain. We have previously found that IL-1beta is elevated in the hippocampi of aged rats with a recent history of peripheral E. coli infection (Barrientos et al., 2006). This elevation parallels the E. coli evoked deficits in hippocampus-dependent long-term memory (Barrientos et al., 2006), and blocking IL-1 signaling in the brain with the naturally occurring interlukin-1 receptor antagonist (Dinarello, 1997) blocks the memory deficit (Frank et al., 2010). We therefore set out to determine if blocking hippocampal IL-1beta signaling with IL-1Ra would also block the E. coli evoked deficit in theta burst L-LTP in aged animals.
As before, E. coli infection greatly reduced theta burst L-LTP in the aged animals (P(peripheral E. coli) = 0.002), but we found that this reduction could be blocked by central administration of IL-1Ra (P(peripheral E. coli + CNS IL-1Ra) = 0.001), which had no significant effect on L-LTP in the absence of infection (P(peripheral vehicle + CNS IL-1Ra) = 0.69; % baseline 3 hours after tetanus: vehicle/vehicle = 156± 15%, E. coli/vehicle = 104± 3%, E coli/IL-1Ra = 143 ± 3%, and vehicle/IL-1Ra = 166± 17%). We found no significant effects of E. coli or IL-Ra on post-tetanic potentiation (% baseline: E. coli/vehicle = 199± 13%, E coli/IL-1Ra = 237 ± 26%, vehicle/vehicle = 241 ± 16%, and vehicle/IL-1Ra = 228 ± 22%; P(peripheral injection) = 0.417, and P(CNS injection) = 0.476).
In the experiments presented here, we examined for the first time the effects of aging combined with a secondary experimental insult - a peripheral immune challenge - on synaptic function in area CA1 of the hippocampus. Our principle findings are that the E. coli infection (1) had no significant effects on basal synaptic transmission or short-term synaptic plasticity in slices from young or aged rats; (2) had no significant effects on a form of late-phase LTP evoked by high-frequency stimulation in slices from young or aged rats; but (3) significantly reduced a form of L-LTP evoked by theta burst stimulation in slices from young rats and essentially abolished it in slices from aged rats. Interestingly, we were able to block the reduction in theta burst L-LTP in aged animals by blocking IL-1 signaling in the brain with the anti-inflammatory cytokine IL-Ra.
These results are consistent with the results of prior behavioral studies indicating that the E. coli infection does not compromise the initial learning of the test tasks, or the formation of short-term memories in any of the animals, but instead, produces profound deficits specific to the consolidation of hippocampus-dependent memory in aged, but not in young rats (Barrientos et al., 2006). The physiology experiments presented here add support to the idea that the infection does not produce large-scale, non-specific disruptions in hippocampal function. Instead, they suggest that limited and relatively subtle synaptic deficits might give rise to the selective memory deficits associated with the combined effects of aging and infection.
A number of studies have examined the impact of aging alone on learning and memory and synaptic plasticity - often with mixed result. As noted earlier, the range of cognitive and synaptic function grows wider with increasing age - an observation consistent with the idea that age is not the only important variable in aging-associated cognitive decline. Aging is often, but not always, associated with some cognitive impairment, and with deficits in the induction and or maintenance of hippocampal LTP (Gage et al., 1984; Barnes and McNaughton, 1985; Deupree et al., 1991; Gallagher et al., 2003). At Schaffer collateral-CA1 synapses, the available data suggest that the basic mechanisms for producing LTP remain intact into old age but are somewhat less likely to be recruited by naturalistic patterns of stimulation or by patterns of afferent activity associated with normal behavior – in contrast, age-related impairments tend to be masked by high frequency stimulation protocols (reviewed in (Lynch et al., 2006).
Our results are largely in line with these earlier findings, but may also offer insight into secondary events that can interfere with production of long-lasting plasticity in aging. In the absence of immune challenge, the aging Fischer Brown Norway rats did not show overt cognitive deficits or impairments in synaptic function. This is not particularly surprising, as we elected to use the aging, but pre-senescent 24-month-old F344xBN rats in order to minimize basal differences in memory functions between young and aged rats. However, following the immune challenge, the aged animals showed dramatic deficits in consolidation of hippocampus-dependent memories (Barrientos et al., 2006), and, as reported here, in theta burst L-LTP. The association of these deficits is intriguing since it has been suggested that the formation of stable spatial memories may require selective strengthening of synapses in hippocampal area CA1 in response to short bursts of action potentials at theta frequency (Buzsaki, 2002) – an idea supported by the observation that deficits in theta-frequency LTP in area CA1 distinguish cognitively impaired from unimpaired aged Fischer 344 Rats (Tombaugh et al., 2002).
How might aging render hippocampal memory processes vulnerable to the deleterious effects of a peripheral infection? One possibility is suggested by the fact that products of peripheral immune activation can communicate with the brain both via circulatory and neural routes, leading to a cascade of CNS effects including microglial activation and subsequent production of pro-inflammatory cytokines such as interleukin-1beta (reviewed in (Maier et al., 2001; Konsman et al., 2002). Numerous studies have provided evidence that elevated levels of pro-inflammatory molecules such as IL-1beta may sometimes impair cognition and synaptic plasticity. Conditions or treatments likely to lead to aberrant increases in brain levels of pro-inflammatory cytokines (e.g. autoimmune diseases) are intermittently associated with problems in memory, learning and concentration. Experimentally elevated levels of IL-1beta in the hippocampus impair the formation of long lasting memory in hippocampus-dependent tasks (Oitzl et al., 1993; Gibertini et al., 1995; Pugh et al., 1999; Barrientos et al., 2002; Yirmiya et al., 2002), and inhibit LTP in several regions of the hippocampus (e.g. (Katsuki et al., 1990; Bellinger et al., 1993; Coogan and O’Connor, 1997) in young adult animals. Thus, individuals with exaggerated brain inflammatory responses to peripheral immune challenge might be more vulnerable to challenge-evoked disruptions of hippocampal memory systems.
Interestingly, aging sensitizes the hippocampal inflammatory response to peripheral E. coli (Frank et al., 2006; Chen et al., 2008). We have previously reported that basal levels of IL-1beta protein in the hippocampus are low in our F344xBN rats, and do not differ significantly between 3- and 24-month-old animals (Barrientos et al., 2006). However, when levels of hippocampal IL-1beta protein were examined 4 days after infection with E. coli, IL-1beta was markedly increased in the older animals, but not in the younger (Barrientos et al., 2006). This is not because the dose of E. coli used failed to induce an inflammatory response in the younger animals. Rather, both the magnitude and the duration of the inflammatory response were increased in the older animals (Barrientos et al., 2009). Not surprisingly, we have very recently found that blunting this response in the brain using the IL-1 receptor antagonist IL-1Ra largely prevents the E. coli induced impairment in hippocampus-dependent memory in the aged rats (Frank et al., 2010), and as shown here, blocks the deficit in theta burst L-LTP.
It is not yet clear how aberrant elevation of IL-1beta impairs synaptic plasticity and learning and memory. Potential mechanisms may involve activation of p38MAPK, c-jun NH2-terminal kinase (JNK), caspase 1, and NFkB (Vereker et al., 2000b; Vereker et al., 2000a; Curran et al., 2003; Kelly et al., 2003), and down regulation of brain-derived neurotrophic factor (BDNF) (Guan and Fang, 2006).
The aging and immune challenge model provides an excellent system for exploring these questions. The relatively physiological E. coli infection has been shown to produce selective deficits in hippocampus-dependent memory. Here we extend these results, demonstrating that the interaction between aging and peripheral infection also produces selective effects on synaptic plasticity. Since these behavioral and physiological deficits occur in a predictable time-frame, and are not confounded by genetic manipulation, the aging induced vulnerability model may be especially tractable for examining the cellular and molecular basis of the initial events (e.g. early failures of synaptic plasticity) giving rise to a form of memory disruption that mimics many aspects of human pathology.
This work was supported by an Innovative Seed Grant Award from the University of Colorado (to SLP) and National Institute on Aging Grants 1R21AG031467 (to SLP), and 1R01AG02827 (to RMB and SFM). We thank A. M. Hein, N. M. Kim and J. Hoover for skillful technical assistance.