Hippocampal long-term potentiation (LTP) is a form of synaptic plasticity used as a cellular model of memory. Beta amyloid (Aβ) is involved in Alzheimer's disease (AD), a neurode-generative disorder leading to cognitive deficits. Nicotine is also claimed to act as a cognitive enhancer. Aβ is known to bind with high affinity to the α7-nicotinic acetylcholine receptor (nAChR). Here we have investigated the effect of intracerebroventricular (icv) injection of the endogenous peptide Aβ1–40 on LTP in area CA1 of urethananesthetized rats. We also examined the effect of Aβ12–28 (icv), which binds with high affinity to the α7-nAChR and the specific α7-nAChR antagonist methyllycaconitine (MLA) on LTP. We found that Aβ12–28 had no effect on LTP, whereas MLA depressed significantly LTP, suggesting that activation of the α7-nAChR is a requirement for LTP. Within the in vivo environment, where other factors may compete with Aβ12–28 for binding to α7-nAChR, it does not appear to modulate LTP. To determine if the depressive action of Aβ1–40 on LTP could be modulated by nicotine, these agents were also co-applied. Injection of 1 or 10 nmol Aβ1–40 caused a significant depression of LTP, whereas nicotine alone (3 mg/kg) had no effect on LTP. Co-injection of nicotine with Aβ1–40 1 h prior to LTP induction caused a further significant depression of LTP compared with Aβ1–40 alone. These results demonstrate that nicotine enhances the deficit in LTP produced by Aβ1–40. This then suggests that nicotine may exacerbate the depressive actions of Aβ on synaptic plasticity in AD.
Beta amyloid peptide (Aβ) is known to be involved in Alzheimer's disease (AD), a neurodegenerative disorder. The cognitive impairments associated with AD have also been linked to a decline in cholinergic function (McGeer et al. 1984) with a reported decrease in the number of nicotinic acetylcholine receptor (nAChRs) in the hippocampus and cortex of AD brains (for review, see Schroder and Wevers 1998). Nicotine has been reported to act as a memory enhancer in both humans (Warburton et al. 1992) and animals (Abdulla et al. 1993). A negative correlation between cigarette smoking and the onset of AD has been reported (Brenner et al. 1993); however, smokers have also been reported to have a higher incidence of AD (Shalat et al. 1987). The effectiveness of nAChRs as a therapeutic target for the treatment of AD is therefore controversial but potentially beneficial (Kem 2000). Aβ-induced amnesia in mice is attenuated by nicotine administration (Maurice et al. 1996) whereas nicotine injected intravenously has been shown to enhance memory in AD patients (Newhouse et al. 1988). However, improved attention and awareness, without a corresponding enhancement of memory, has also been reported (Jones et al. 1992).
High-affinity binding between the α7-nAChR and Aβ has been reported in vitro with the binding domain between amino acids 12–28 (Wang et al. 2000a,b). Aβ1–42 and Aβ12–28 have also been shown to reduce carbachol-induced currents in stratum radiatum interneurons in hippocampal slices (Pettit et al. 2001) via a decrease in the probability of nAChR-gated channel opening. Aβ1–42 has also been shown to reduce α7-nAChR-mediated current in cultured cortical neurons (Liu et al. 2001).
Long-term potentiation (LTP) is regarded as a cellular model for learning and memory (Bliss and Collingridge 1993). Administration of Aβ peptides (intracerebroventricularly, icv) is known to impair LTP in vivo (Cullen et al. 1997; Freir et al. 2001). Nicotinic agonists have also been shown to facilitate LTP in area CA1 in vitro (Fujii et al. 1999; Hunter et al. 1994), whereas nicotine has been shown to enhance synaptic transmission in area CA3 (Gray et al. 1996). Intraperitoneal injection of nicotine also produces a form of LTP in mouse dentate gyrus in vivo (Matsuyama et al. 2000). The aim of our study was to investigate the effects of nicotine, the endogenous peptide Aβ1–40, and agents that bind to the α7-nAChR including Aβ12–28 (Wang et al. 2000a) and the specific α7-antagonist methyllycaconitine (MLA) on synaptic transmission and plasticity in the form of LTP. Part of this work has been presented previously in abstract form (Freir and Herron 2001).
In vivo preparation
All experiments were carried out in accordance to guidelines under license from the Department of Health, Ireland (86/609/EEC). Male Wistar rats (175–200 g) were surgically prepared for acute electrophysiological recordings. Briefly, rats were anesthetized with intraperitoneal injections of 1.5 g/kg urethan (ethyl carbamate), and supplementary injections (0.2–0.5 g/kg) were given when necessary to ensure full anesthesia. Heating pads (Braintree scientific) were used to maintain the temperature of the animals at 36.5 ± 0.5°C. Deep body temperature was recorded throughout the experiment using a rectal thermometer (Precision Instruments). Small holes were drilled in the skull at the positions of the reference stimulating and recording electrodes. Additionally, in some experiments, a separate hole was drilled to introduce a guide cannula for icv injection of drug/vehicle. The cannula was secured using acrylic dental cement (Emperor CC) to avoid interference with the stimulating/recording electrodes. Animals were placed in a stereotaxic frame for all recordings. The recording electrode was positioned in the stratum radiatum of area CA1 (3 mm posterior, 2 mm lateral to bregma). A bipolar stimulating electrode was placed in the Scheffer-collateral/commissural pathway distal to the recording electrode (4 mm posterior, 3 mm lateral to bregma). The cannula was positioned above the lateral ventricle in the opposite hemisphere to that of the recording/stimulating electrodes (1 mm posterior, 1.2 mm lateral to bregma).
In vivo electrophysiology
Physiological and stereotactic indicators were used to lower the electrodes through the cortex and into area CA1 of the hippocampus. Test stimuli were delivered to the Schaffer-collateral/commissural pathway every 30 s (0.033Hz). Electrodes were positioned to record a maximal field excitatory postsynaptic potential (EPSP). Baseline EPSPs were recorded at 35–40% of maximal response. LTP was induced using a high-frequency stimulus protocol (HFS: 10 trains of 10 stimuli at 200 Hz, intertrain interval of 2 s) at a stimulus intensity that evoked a field EPSP of approximately 80% of maximum response. This protocol was repeated three times to produce a robust LTP. Field EPSPs were evoked in the CA1 region using low-frequency stimulation (0.033 Hz). Baseline synaptic potentials were recorded for ≥30 min prior to injection of drug/vehicle to ensure a steady state response. Rats were injected with nicotine (3 mg/kg ip) or MLA (5 mg/kg ip). Aβ1–40 (1 or 10 nmol in 5 μl distilled water) and Aβ12–28 (10 nmols in 5 μl distilled water) were injected icv 1 h prior to tetanus. After injection of drug/vehicle, baseline recordings were monitored for a further period of 30–60 min (depending on the experimental protocol). A series of high-frequency stimuli (HFS) were delivered to induce a long-lasting potentiation of the synaptic response. Low-frequency stimulation (0.033 Hz) was then used to evoke EPSPs for a further period of 1 h to record any changes in synaptic response. Paired-pulse facilitation (PPF) with an interstimulus interval of 50 ms was also examined, before, and 1 h after injection of drug/vehicle and measured using the percent change in EPSP amplitude after the second stimulus.
EPSPs were amplified (100 times), filtered at 5 kHz, digitized, and recorded using MacLab software acquisition system. The EPSP slope was used to measure synaptic efficacy. EPSPs are expressed as a percentage of the mean initial slope measured during the last 10 min of the preinjection baseline-recording period. LTP data were analyzed using ANOVA measured over a 5-min period (55–60 min) after the induction of LTP. PPF was analyzed prior to and 1 h after drug/vehicle injection using a paired Student's t-test. The significance level was set at P < 0.05. Error bars on the graphs shown represent the standard error of the mean (SE). Data points in each figure were an average of four consecutive EPSPs taken at 30-s intervals to present our findings in a clear and concise manner. Sample traces are averages of four consecutive EPSPs recorded at the time indicated on each graph.
Materials and chemicals
Rats were obtained from the Biomedical Facility, University College Dublin, Belfield. Stimulating (bi-polar stainless steel; 0.125 mm diam) and recording (mono-polar stainless steel; 0.125 mm diam) electrodes were obtained from Plastics One. Aβ1–40 and Aβ12–28 were purchased from Biosource; nicotine and methyllycaconitine (MLA) were obtained from Sigma (RBI) (Ireland). All other reagents were obtained from Sigma (Ireland). All drugs were dissolved in distilled water and stock solutions were maintained at -20°C.
Aβ1–40 causes a depression of LTP
The endogenous peptide Aβ1–40 was injected icv at a concentration of 1 or 10 nmol in 5 μl distilled water. Aβ1–40 had no significant effect on baseline synaptic transmission monitored for 1 h postinjection. After icv injection with 5 μl distilled water (vehicle) LTP was measured (167 ± 3%, n = 12) 1 h post tetanus (Fig. 1). LTP was depressed significantly in animals treated with 1 nmol (141 ± 6%, n = 6, P < 0.01) or 10 nmol (123 ± 8%, n = 6, P < 0.001) Aβ1–40, compared with control values (Fig. 1).
Effect of nicotine on synaptic transmission and LTP
Animals were injected intraperitoneally with distilled water/nicotine after a baseline recording period, and EPSPs were monitored for a further 60 min postinjection (Fig. 2). The nicotine-treated groups (3 mg/kg in 0.5 ml distilled water) showed no significant change in baseline response ≤1 h post injection (Fig. 2). LTP was induced 1 h after injection of nicotine/vehicle, and EPSPs were monitored for a further period of 1 h post-HFS. Vehicle-injected animals (0.5 ml distilled water ip) showed stable LTP measured 1 h post-tetanus (164 ± 3%, n = 10). Nicotine (3 mg/kg) delivered 60 min prior to tetanus produced no significant change in LTP when compared with control values (153 ± 4%, n = 5; Fig. 2).
Co-injection of nicotine and Aβ1–40
The effects of co-applying Aβ1–40 (1 nmol icv) and nicotine (3 mg/kg ip) 1 h prior to the induction of LTP were examined (Fig. 3). Drug applications did not alter baseline synaptic transmission; however, a significant depression of LTP was noted in the co-injected group (121 ± 6%, n = 5, P < 0.01) when compared with vehicle controls (167 ± 3%, n = 12, icv; Fig. 3). The depression of LTP reported in the co-injected group was significantly greater than that previously recorded in animals injected with Aβ1–40 (1 nmol) alone (141 ± 6%, n = 6, P < 0.05). This demonstrates that co-injection of nicotine and Aβ1–40 1 h prior to LTP induction caused a further significant impairment of LTP compared with either agent alone.
Effect of Aβ12–28 on LTP
Injection of 10nmol Aβ12–28 1 h prior to induction of LTP had no effect on baseline synaptic transmission (Fig. 4). There was also no significant reduction in LTP observed in animals treated with Aβ12–28 (157 ± 3%, n = 6) compared with the icv control group (167 ± 3%, n = 12; Fig. 4)
MLA depresses LTP
Injection of the α7nAChR antagonist, MLA (5 mg/kg ip), 1 h prior to LTP induction did not alter baseline synaptic transmission. Application of HFS produced LTP (144 ± 5%, P < 0.05) that was depressed significantly compared with control values (164 ± 3%, n = 10; ip controls, Fig. 5)
Nicotine and Aβ12–28 increase PPF
Nicotine (3 mg/kg) produced an increase in PPF when measured 60 min post-injection (P < 0.05; Fig. 5A). Aβ12–28 caused an increase in PPF; however, Aβ1–40 had no effect (Fig. 5B). An increase in PPF is usually associated with a decrease in presynaptic transmitter release.
We reported previously that the shorter fragment Aβ25–35 depresses LTP in vivo (Freir et al. 2001). Here we demonstrate that the endogenous peptide, Aβ1–40, which is neurotoxic in cell culture (Yankner et al. 1990), also depresses LTP in vivo. These results are consistent with in vitro studies showing that amyloid peptides cause impairment of hippocampal LTP (Itoh et al. 1999). LTP is also depressed in transgenic mice that overexpress the mutated human APP gene (Chapman et al. 1999). What then are the potential mechanisms involved in this impairment of LTP by Aβ peptides?
Nicotinic agonists can improve performance in memory-linked behavioral tasks (Abdulla et al. 1993; Levin 1992) and the nAChR antagonist mecamylamine causes impairment of memory in humans (Newhouse et al. 1992). Aβ is also known to bind with picomolar affinity to the α7-nicotinic acetylcholine receptor (nAChR), with the binding domain contained within amino acid sequence 12–28 of the full-length peptide (Wang et al. 2000a,b). We investigated therefore a possible interaction between Aβ and the α7-nAChR.
In the hippocampal CA1 region, α7-nAChRs are present on GABAergic inhibitory interneurons and the soma of CA1 pyramidal cells (Jones and Yakel 1997). Stimulation of presynaptic α7-nAChRs can enhance the release of glutamate due to the high calcium permeability of the α7-gated channel (Gray et al. 1996). Nicotine, at concentrations found in plasma from cigarette smokers (50–500 nM), has been shown to desensitize nAChRs on CA1 GABAergic interneurons (Alkondon et al. 2000), leading to pyramidal cell disinhibition (Dani et al. 2000). Aβ1–40, Aβ1–42, and Aβ12–28 can antagonize reversibly α7- and other nAChR-mediated currents in vitro. (Liu et al. 2001; Pettit et al. 2001). Nicotine (Shimohama and Kihara 2001), cytisine, (α4β2 agonist) (Kihara et al. 1998), and the α7-agonist, DMXBA, (Li et al. 1999) have also been shown to inhibit Aβ-mediated toxicity in cultured neurons.
To examine a possible role for the α7-nAChR in LTP, because α7-agonists are known to facilitate LTP (Hunter et al. 1994), we investigated the effects of the specific α7-nAChR antagonist MLA. We found that MLA caused a significant depression of LTP when administered 1 h pretetanus. The concentration of MLA used in our study (5 mg/kg) delivered intraperitoneally should result in a concentration of 50–100 nM in rat brain (Turek et al. 1995). This concentration range (50–100 nM) has been shown to block completely the activation of α7-nAChRs in cultured hippocampal neurons (Alkondon et al. 1992).
Because Aβ12–28 was reported to cause a block of the α7-channel (Pettit et al. 2001) in a similar manner to Aβ1–42, we investigated the effects of Aβ12–28 on LTP. We found that injection of Aβ12–28 icv had no significant effect on baseline synaptic transmission or LTP. This suggests that binding of Aβ to the α7-nAChR does not modulate the induction of LTP in CA1 in vivo.
In addition, in view of the reported benefits of nicotine as a cognitive enhancer, we examined the effects of nicotine alone and combined with Aβ1–40 to determine if nicotine could modulate the Aβ1–40-induced depression of LTP. The concentration of nicotine used (3 mg/kg) was similar to that shown to cause a form of LTP in mouse dentate gyrus in vivo (Matsuyama et al. 2000). We did not, however, observe any change in baseline synaptic transmission or LTP after the injection of nicotine. Co-injection of nicotine and Aβ1–40 1 h pre-HFS caused a further significant impairment of LTP compared with Aβ alone.
Our result suggests that activation of α7-nAChRs is involved in LTP in the CA1 region in vivo. Although both Aβ1–40 and Aβ12–28 have been shown to bind to the α7 receptor with high affinity in vitro, in our study these peptides had differing effects on LTP in vivo. Assuming a role for α7-nAChRs in LTP, because MLA reduces LTP, it is difficult to interpret the depression of LTP by Aβ1–40 in terms of an interaction with the α7-binding site. Although Aβ1–42 at picomolar concentrations was shown to inhibit binding of MLA to synaptic membranes (Wang et al. 2000a), there was no interaction unless the membranes had been washed extensively. This suggests the possible existence of a soluble endogenous factor/protein that binds to either Aβ1–42 or the α7-nAChR. It has been shown that a variety of albumins can bind to and potentiate α7-nAChR-mediated responses in vitro (Conroy et al. 2003; Gurantz et al. 1993). Aβ1–40 injected in vivo may not bind substantially to the α7-receptor in view of competitive binding of endogenous proteins present, although this was not investigated in the present study and remains somewhat speculative. Our results, however, suggest that Aβ1–40-induced depression of LTP in vivo is not likely due to an interaction with the α7-nAChR.
We have also examined neurotransmitter release in the form of PPF in area CA1. Injection of nicotine (3 mg/kg) 60 min pretetanus caused a significant increase in PPF. An increase in PPF is usually associated with a decrease in presynaptic neurotransmitter release. An increase in neurotransmitter release due to activation of calcium permeable presynaptic α7 nAChR channels on mossy-fiber terminals has been demonstrated (Gray et al. 1996). Desensitization of these receptors, however, may lead to decreased neurotransmitter release. GABAergic inhibition may also play a role in apparent alteration of PPF. Because long-term exposure to nicotine will cause desensitization of the α7nAChRs (Alkondon et al. 2000) on inhibitory interneurons, this is likely to lead to increased excitability in the CA1 region and subsequent increases in amplitude of the second EPSP that is evoked at a 50-ms interval. We found that Aβ12–28 caused a significant increase in PPF indicative of decreased neurotransmitter release. This correlates with a report demonstrating that Aβ12–28 reduces Ca2+ influx via the α7nAChR channel (Pettit et al. 2001). Blockade of presynaptic α7-nAChRs onto CA3 pyramidal neurons, similar to nicotine desensitization of these receptors, may explain reduced transmitter release onto CA1 excitatory neurons. This result would not agree however with the existence of a soluble endogenous factor that competes for Aβ binding and is under further investigation.
These results suggest that although Aβ1–40 causes an impairment of LTP in vivo, the effects mediated via binding to nAChRs may be reduced due to endogenous proteins present in cerebrospinal fluid. Surprisingly, because nicotine has been reported to act as a cognitive enhancer, we found that co-application of nicotine and Aβ1–40 produced a level of LTP significantly depressed compared with that recorded in Aβ1–40 alone. This observation, which is under further investigation, indicates that nicotine can exacerbate the effects of Aβ and may account for the slightly higher incidence of AD among smokers (Shalat et al. 1987).
This research was supported by Enterprise Ireland, Department of Physiology University College Dublin (UCD), and UCD Presidents Research Award 2001.
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- Copyright © 2003 by the American Physiological Society