Aβ25-35 induced depression of long-term potentiation in area CA1 in vivo and in vitro is attenuated by verapamil

The effect of intracerebroventricular (i.c.v.) injection of Aβ25-35 and/or intraperitoneal (i.p.) application of the L-type calcium channel (VDCC) blockers verapamil or diltiazem were examined in vivo. In order to by-pass possible systemic actions of these agents their effects on LTP in the CA1 region of the in vitro hippocampal slice preparation were also examined. Application of Aβ25-35 (10nmol in 5μl, i.c.v.) significantly impaired LTP in vivo, as did i.p. injection of verapamil (1 or 10mg/kg) or diltiazem (1mg or 10mg/kg). In the in vitro slice preparation, LTP was also depressed by prior application of either Aβ2535 (500nmol), verapamil (20 μM) or diltiazem (50μM). Combined application of Aβ25-35 and verapamil in either the in vivo or in vitro preparation resulted in a significant reversal of the LTP depression observed in the presence of either agent alone. Co-application of diltiazem and Aβ2535 however failed to attenuate the depression of LTP observed in the presence of either agent alone in vivo or in vitro. Since LTP is a cellular correlate of memory, and Aβ is known to be involved in Alzheimer’s disease (AD) these results indicate that verapamil, a phenylalkylamine may be useful in the treatment of cognitive deficits associated with AD. INTRODUCTION Deposition of beta-amyloid (Aβ) is recognised as an early (Lippa et. al, 1998) and critical event in the pathogenesis of Alzheimer’s disease (AD) (Selkoe, 1997). Amyloid deposits are found in cortical regions including the hippocampus, an area known to play a role in memory processing. Aβ peptides have been shown to be neurotoxic in cultured cells (Yankner et. al., 1990) and can lead to apoptosis in cultured hippocampal (Mattson et. al, 1998) and cortical neurones (Yan et. al, 1999). Aβ has also been shown to cause disruption of calcium homeostasis. Aβ1-40 can enhance calcium influx in rat cortical synaptosomes and cultured neurons through Land N-type voltage dependent calcium channels (VDCCs) (MacManus et. al, 2000) and through L-type VDCCs in PC12 cells (Green and Peers, 2001). Indirect activation of L-type VDCCs by Aβ2535 has also been reported via activation of MAP kinases (Ekinci et. al., 1997) or generation of free radicals (Ueda et. al, 1997). Post-mortem analysis of the hippocampus of AD patients has revealed an increase in the density of L-type VDCCs in both the dentate gyrus and area CA1 when compared to aged-matched controls (Coon et. al, 1999). These findings suggest that L-type VDCCs may play a role in the pathogenesis of AD. Aβ peptides have been shown to impair hippocampal synaptic plasticity in the form of long-term potentiation (LTP) in vitro and in vivo. (Chen et. al, 2000; Freir et. al, 2001; Freir and Herron, 2002). In addition, studies have shown a correlation between impaired synaptic plasticity and memory deficits following the generation of Aβ aggregates in the rat hippocampus (Stephan et. al, 2001). Here we have investigated whether the depression of hippocampal LTP reported previously (Chen et. al., 2000; Freir et. al., 2001; Freir and Herron, 2002) may be linked to a disruption of postsynaptic calcium influx, a critical event in LTP induction (Malenka et. al, 1988). We therefore examined the effect of reducing the activity of L-type VDCCs via application of VDCC blockers in the presence of Aβ2535 in vivo and in vitro. Part of this work has been presented previously in abstract form (Freir and Herron, 2001; Costello and Herron, 2002). METHODS In vivo preparation: All experiments in vivo were performed in accordance with guidelines and under licence from the Department of Health, Ireland (86/609/EEC). Male Wistar rats, 175-200g (8-10 weeks old) were surgically prepared for acute recordings. Briefly, rats were anaesthetised with an intraperitoneal (i.p.) injection of 1.5g/kg urethane (ethyl carbamate) and supplementary injections (0.2-0.5g/kg) were given when necessary to ensure full anaesthesia. Deep body temperature was recorded throughout the experiment and heating pads (Braintree scientificTM) were used to maintain the temperature of the animals at 36.5 ± 0.5C. 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 i.c.v. injection of drug/vehicle. Animals were placed in a stereotactic frame for recording. The recording electrode was positioned in the stratum radiatum of area CA1 (3mm posterior, 2mm lateral to bregma). A bipolar electrode was placed in the Schaffer-collateral/commissural pathway distal to the recording electrode (4mm posterior, 3mm lateral to bregma). The cannula was positioned above the lateral ventricle in the opposite hemisphere to that of the electrodes (1mm posterior, 1.2mm lateral to bregma). Electrophysiological recordings: Stimulating (bi-polar stainless steel; 0.125 mm diameter) and recording electrodes (mono-polar stainless steel; 0.125 mm diameter) obtained from Plastics OneTM were lowered through the cortex and into area CA1 of the hippocampus using both physiological and stereotactic indicators. Test stimuli were delivered to the Schaffer-collateral/commissural pathway every 30 s (0.033Hz). Electrodes were positioned to record a maximal field excitatory post-synaptic potential (EPSP). Baseline EPSPs were recorded at 35-40% of maximal response. LTP was induced using a high-frequency stimulus protocol (HFS: 3 x 10 trains of 10 stimuli at 200Hz) at a stimulus intensity that evoked a field EPSP of approximately 80% of maximum response. Field EPSPs were evoked in the CA1 region using low frequency stimulation (0.033Hz.). Extracellular field potentials were amplified (x10), filtered at 5 kHz, digitised, and recorded using Mac Lab software acquisition system. Baseline recordings were taken for at least 30 minutes prior to injection of drug/vehicle to ensure a steady state response. Following injection of drug/vehicle, baseline recordings were monitored for a further period of one hour to monitor normal synaptic transmission. At a time of one hour post-injection, a series of high frequency stimuli were delivered to induce a potentiation of the synaptic response. Low frequency stimulation was then used to evoke EPSPs for a further period of one hour to record any changes in synaptic response. Data points displayed on graphs are an average of four consecutive recordings. Drug application: Aβ25-35 (10nmol in 5μl distilled water) was injected i.c.v. (over a period of 2-3 mins) using a microsyringe (Hamilton) 1 hour prior to high frequency stimulation. Rats were injected i.p. with diltiazem (1 or 10mg/kg in 0.5ml distilled water) or verapamil (1 or 10mg/kg in 0.5ml distilled water) one hour prior to high frequency stimulation.. In experiments involving the co-application of Aβ25-35 with verapamil or diltiazem, both drugs were injected 1 hour pre-tetanus. Hippocampal slice preparation Male Wistar rats, weighing 50–100g (4-6 weeks old) were decapitated under anaesthesia. The brains were rapidly removed and immersed in chilled, oxygenated artificial cerebrospinal fluid (aCSF). Brains were dissected and transverse slices, 350μm in thickness, were cut using a vibrotome (Campden Instruments, U.K.). The slices were transferred to a holding chamber and incubated at room temperature in oxygenated aCSF for at least 1 hour. The aCSF was composed of (mM): 120 NaCl, 2.5 KCl, 2.0 MgSO4 2.0 CaCl2, 26 NaCO3, 1.25 NaH2PO4 and 10 D-glucose and oxygenated with 95%O2 and 5%CO2. Slices were then transferred to a submerged recording chamber, superfused with oxygenated aCSF at a rate of 7ml/min and maintained at a temperature of 29–30°C. Electrophysiological recording: Field excitatory postsynaptic potentials (fEPSPs) were evoked using stimulating (~1MΩ) and recording (~2 MΩ) glass capillary microelectrodes (Harvard Apparatus Ltd., U.K.) filled with aCSF. The stimulating electrode was placed in the Schaffer collateral/Commissural pathway of the CA1 and recordings were made from the CA1 stratum radiatum. Stimulus frequency was 0.033Hz, duration 0.1ms and intensity 2-8V. The stimulation intensity was set at ~40% of maximal EPSP amplitude as determined from an input-output curve for each experiment. Stable baseline recordings were made for at least 20 minutes prior to application of drug/ or LTP induction. EPSPs obtained were amplified x100 using a Brownlee Precision (model 410) instrumentation amplifier, displayed on an Iso-tech ISP622 oscilloscope and recorded and analysed using software supplied by Dr. J. Dempster (Strathclyde University). LTP was induced by 3 bursts of high frequency stimulation (10 trains of 10 pulses at 200Hz) given at 20second intervals, with no change in stimulation intensity. Drugs were applied via the perfusion media. Verapamil (20μM) and diltiazem (50μM) were added 20 minutes pre-HFS. Aβ2535 (500nM) was added to the perfusate 1 hour pre-tetanus. Co-application of both Aβ25-35 and verapamil/diltiazem took place 1 hour pre-tetanus. Drugs were then maintained within the perfusate for the duration of each experiment. Data analysis 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 minutes of the baselinerecording period prior to LTP induction. LTP data was analysed using a 2-way analysis of variance (ANOVA), which examined all data recorded between 55-60 minutes post HFS. The significance level was set at p<0.05. Error bars on the graphs shown represent the standard error of the mean (SEM). Data insets are an average of four consecutive EPSPs recorded at the time indicated on the graph. Control experiments in vitro and in vivo were performed between test experiments. RESULTS Aβ25-35 caused a depression of LTP Administration of Aβ25-35 or vehicle (distilled water) had no significant effect on baseline synaptic transmission, in vivo or in vitro (Fig 1a, b). In the hippocampal slice preparation, prior administration of 500nM Aβ25-35 caused a significant impairment of LTP (139 ± 5%, n=12, P<0.001, F=110.5) with respect to vehicle controls (169 ± 7%, n=9, Fig.1a). Injection of 10nmol Aβ25-35 (i.c.v.) 1 hr. prior to HFS however, caused a significant depression of LTP in vivo (128 ± 10%, n=6, P<0.001, F=97.98) compared to control values (176 ± 10%, n=6, Fig.1b). Verapamil impairs LTP In vitro: In the slice preparation, administration of verapamil (20μM) had no significant effect on baseline responses however, following HFS in the presence of verapamil, LTP was depressed significantly (132 ± 4%, n=6, P<0.001, F=125) compared to the control value (169 ± 7%) (Fig.2a). In vivo: In control experiments, injection of distilled water vehicle (0.5ml i.p.) had no effect on baseline synaptic transmission and following a high frequency stimulus robust LTP was produced in the control group (178 ± 11%, n=7). Injection of either 1 or 10mg/kg verapamil (i.p.) also had no significant effect on baseline recordings when measured up to one hour post-injection. Both concentrations however significantly depressed LTP when measured 1 hour post-HFS compared to control, (139 ± 5%, n=5, P<0.001, F=62.06) and (127 ± 10, n=6, P<0.001, F=96.10) respectively (Fig. 2b). Diltiazem reduces LTP In vitro: In the slice preparation, diltiazem (50μM), had no effect on baseline synaptic transmission when administered 20 minutes prior to HFS, however diltiazem caused a significant impairment of LTP (123 ± 4%, n=7, P<0.001, F=236.3) compared to vehicle controls (169 ± 7%)(Fig. 3a). In vivo: Following injection (i.p.) of a second class of Ltype calcium channel antagonist, diltiazem, at 1 or 10mg/kg there was no significant effect on baseline EPSPs. LTP was depressed significantly however (1mg; 146 ± 10, n=6, P<0.001, F=34.72) and (10mg; 130 ± 10%, n=6, P<0.001, F=57.38) when compared to control values (178 ± 11%, n=7) (Fig.3b). Verapamil attenuates the Aβ25-35-induced depression of LTP In vivo: Aβ2535 (10nmol, i.c.v.) and verapamil (i.p.) at concentrations of 1 or 10mg/kg were co-injected 1 hr. prior to LTP induction. Co-application of A β25-35 and verapamil did not alter baseline synaptic transmission (Fig. 4a, b). Injection of Aβ25-35 with verapamil (1mg/kg) produced a similar LTP (176 ± 7%, n=6, P=0.947, F=0.0052) to that seen for controls (178 ± 11%; Fig. 4a). The level of LTP produced in this group was significantly different to that observed following application of verapamil (1mg/kg) (139 ± 5%) or Aβ25-35 (128 ± 10%) alone. Co-administration of Aβ2535 and a higher concentration of verapamil (10mg/kg) also produced a smaller, yet significant reversal of the Aβ25-35-induced depression of LTP (169 ± 11%, n=5, P=0.06, F=4.33) (Fig.4b). In vitro: Using the slice preparation, verapamil (20μM) and Aβ25-35 (500nM) were superfused for 1 hour prior to the induction of LTP. In the presence of both agents the level of LTP produced (159 ± 7%, n=6) was similar to that recorded under control conditions (169 ± 7%, P=0.0993, F=2.759), yet significantly different to that produced following application of Aβ25-35 (139 ± 5%, n=12, P<0.001, F=58.8) or verapamil (132 ± 4%, P<0.001, F= 107) alone (Fig.5) Diltiazem failed to reverse the Aβ25-35 -induced impairment of LTP In vivo: Diltiazem at concentrations of 1 or 10mg/kg were co-applied with Aβ25-35 (10nmol) 1 hour pre-tetanus. However, unlike verapamil, co-administration of Aβ25-35 and 1mg/kg diltiazem produced an LTP (149 ± 11%, n=6) that was significantly different to that seen in controls (178 ± 11%, P<0.001, F=25.11) (Fig.6a). Similarly co-application of the higher dose of diltiazem (10mg/kg) failed to reverse the Aβ25-35 induced depression of LTP (135 ± 5%, n=6, P<0.001, F=94.57) (Fig.6b). In vitro: In the slice preparation we also found that co-administration of Aβ25-35 and diltiazem produced a significant depression of LTP (98 ± 7%, n=5, P<0.001, F=404) when compared to control values (169 ± 7%). The degree of potentiation produced was, however, significantly lower than seen following application of diltiazem (123 ± 4%, P<0.001, F=108.8) or Aβ25-35 (139 ± 5%, P<0.001, F=189.5) alone (Fig.7).