Diabetes onset influences hippocampal synaptic plasticity in streptozotocin-treated rats

Neuroscience 227 (2012) 293–304

DIABETES ONSET INFLUENCES HIPPOCAMPAL SYNAPTIC PLASTICITY IN STREPTOZOTOCIN-TREATED RATS S. SASAKI-HAMADA, H. SACAI AND J.-I. OKA *

intelligence (Ack et al., 1961; Holmes and Richman, 1985; Ryan et al., 1985). Epidemiological studies have revealed about 50–60% of patients with type 1 DM to be younger than 16–18 years old. Moreover, there is a current trend toward decreasing age at presentation, in particular, in children younger than 5 years old (for review, Daneman, 2006). We thus focused on synaptic transmission related to the age of onset of diabetes. Long-term potentiation (LTP) and long-term depression (LTD) are primary experimental models for investigating the synaptic basis of learning and memory (Bliss and Lomo, 1973; Bliss and Collingridge, 1993; Kandel, 2001). Previous studies show that hippocampal LTP and LTD as well as performance in learning behavior tests are severely affected in rats with streptozotocine-induced diabetes (STZ-rats), an established model of type 1 DM (Biessels et al., 1996; Kamal et al., 2000). We showed that LTD was impaired in juvenile-onset STZ-rats compared with juvenile control rats (3–4 weeks) (Iwai et al., 2009). Since LTD is more reliably obtained with low-frequency stimulation and greater magnitude in 2–3 week-old rats than in more mature animals (Dudek and Bear, 1993; Wagner and Alger, 1995), we should examine whether the impaired LTD of 3–4 week-old STZ-rats was similar in adulthood (5–6 month) or not. In contrast, LTP was impaired in young adult-onset STZ-rats compared with adult control rats (Biessels et al., 1996; Chabot et al., 1997; Kamal et al., 1999; Artola et al., 2005). The effect of the age of diabetes onset on LTP and LTD is still unknown. The principal responses are mediated by glutamate binding to postsynaptic ionotropic AMPA and N-methyl-D-aspartate (NMDA) receptors. In the hippocampus of young adult-onset STZ-rats, the calcium-induced upregulation of AMPA receptor binding was impaired (Chabot et al., 1997), and the functional activities of AMPA receptors were reduced (Kamal et al., 2006). On the other hand, NMDA receptor-dependent EPSPs decreased due to a reduction in NMDA receptors with the NR2B subunit (Di Luca et al., 1999; Gardoni et al., 2002) or not (Chabot et al., 1997). However, no study has examined the interaction between AMPA and NMDA receptor-mediated transmission at Schaffer collateral-CA1 pyramidal (SC-CA1) synapses in STZ-rats. In this study, we demonstrated that diabetes-induced synaptic changes depend on the age of diabetes onset. Here, extracellular and whole-cell patch-clamp recordings were used to investigate synaptic transmission at SC-CA1 synapses in two ages of diabetes onset (juvenile-onset and young adult-onset STZ-rats)

Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

Abstract—Children with type 1 diabetes mellitus (DM) are at risk of developing cognitive difficulties. Although a diabetes onset of patient influences cognitive difficulties, synaptic properties related to the age of diabetes onset remain unknown. Here we showed that synaptic plasticity including long-term potentiation (LTP) or long-term depression (LTD), and excitatory synaptic transmission at Schaffer collateralCA1 (SC-CA1) synapses in hippocampal slices were affected by age of onset in rats with streptozotocin-induced diabetes (STZ-rats), compared with age-matched control rats. LTP was impaired and the ratio of AMPA receptor-mediated EPSCs relative to N-methyl-D-aspartate (NMDA) receptormediated EPSCs (the AMPA/NMDA ratio) decreased in young adult-onset STZ-rats, whereas LTD was impaired and both AMPA receptor-mediated and NMDA receptormediated EPSCs increased in juvenile-onset STZ-rats. Furthermore, impaired LTD of juvenile-onset STZ-rats was restored with an NMDA receptor antagonist. These results suggest that the pathophysiology of diabetes-induced cognitive difficulties varies with the age of diabetes onset. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: diabetes, hippocampal synaptic plasticity, AMPA receptors, NMDA receptors, CA1.

INTRODUCTION Many organ systems are affected by diabetes mellitus (DM), including the brain. Cognitive deficits, such as impaired learning, memory, problem solving, and mental flexibility, are more common in patients with type 1 DM than in age-matched non-diabetic patients (Ryan et al., 1985; Biessels et al., 2008). Also, children who develop diabetes early in life, before 5–7 years of age, have been found in some cases to score lower in tests of overall *Corresponding author. E-mail address: [email protected] (J.-I. Oka). Abbreviations: ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; APV, D-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxialine-2,3-dion; DM, diabetes mellitus; EGTA, ethylene glycol tetraacetic acid; fEPSPs, field EPSPs; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; HFS, high-frequency stimulation; LFS, low-frequency stimulation; LTD, long-term depression; LTP, long-term potentiation; NMDA, N-methyl-Daspartate; PPF, paired-pulse facilitation; SC-CA1, Schaffer collateralCA1; STZ, streptozotocine; STZ-rats, streptozotocine-treated diabetic rats; TBS, Tris–buffered saline; Vmr, resting membrane potential.

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.09.081 293

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compared with age-matched control rats. The juvenileonset STZ-rats were treated with STZ at 17 days old (Iwai et al., 2009) and the young adult-onset STZ-rats were treated with STZ at 10 weeks old (Chabot et al., 1997; Kamal et al., 1999, 2000). We found that the age of onset resulted in differences in the impairment of synaptic plasticity, and enhancement or impairment of excitatory synaptic transmission. The age of onset might be considered a factor for improving cognitive function in patients with diabetes.

EXPERIMENTAL PROCEDURES Chemicals Streptozotocine (STZ), phosphocreatine, lidocaine N-ethylbromide (QX-314), and picrotoxin were obtained from Sigma–Aldrich (St. Louis, MO, USA). 6-Cyano-7D-2-amino-5nitroquinoxialine-2,3-dion (CNQX) and phosphonopentanoic acid (APV) were obtained from Tocris Cookson (Bristol, UK). All other chemicals were from Wako Pure Chemical Industries (Osaka, Japan).

Animals All experimental protocols were approved by the Institutional Animal Care and Use Committee at Tokyo University of Science, and were conducted according to the guidelines of the National Institute of Health and Japan Neuroscience Society. We used Wistar rats (SLC, Shizuoka, Japan), and tried to minimize the number of animals used and animal pain and distress. DM was induced by STZ (85 mg/kg, i.p.) in 17-day-old rats (juvenile-onset STZ-rats) or 10-week-old rats (young adultonset STZ-rats). The dose of STZ was based on previous studies (Piotrowski et al., 2001; Iwai et al., 2009). To confirm the induction of DM, we measured glucose levels in venous blood samples using a glucose test meter (GUNZE Co., Kyoto, Japan) before the experiment. Animals were considered diabetic when glucose levels were >300 mg/dl. Age-matched and vehicle-treated rats were used as controls. All animals were kept in a controlled environment, with a 12:12-h light schedule, temperature of 23 ± 1 °C, and relative humidity of 55 ± 5%, and provided ad libitum access to food and water.

Slice preparation Hippocampal slices were prepared from 22- to 24-week-old rats. The animals were anesthetized with diethyl ether and decapitated. Transverse hippocampal slices (300-lm thick) were cut using a vibratome (DTK-1000, Dosaka, Kyoto, Japan) in icecold dissection buffer (in mM; 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 10 MgSO4, 26 NaHCO3 and 11 D-glucose) saturated with 95% O2 and 5% CO2. The slices were allowed to recover at 36 °C for 50 min in normal artificial CSF (ACSF) (in mM; 124 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.24 KH2PO4, 10 D-glucose and 26 NaHCO3) saturated with 95% O2 and 5% CO2, and thereafter maintained at room temperature. Slices were left for a minimum of 30 min at room temperature before being transferred to a submerged recording chamber for experiments.

Extracellular field potential recordings Extracellular field EPSPs the hippocampal CA1 micropipettes filled with were maintained while

(fEPSPs) from the stratum radiatum of region were recorded using glass ACSF. During the recordings, slices being perfused at 3–4 ml/min with

ACSF. Test stimuli (80 ls duration) were delivered once per 60 s by a bipolar tungsten electrode (150-lm pole separation, WPI, Sarasona, FL), which was placed on the afferent fibers of the stratum radiatum. Evoked fEPSP was generated by an electric stimulator (SEN-3301; Nihon Koden, Tokyo, Japan) and an isolater (SS-202J; Nihon Koden), and was recorded with an amplifier (CEZ-2400; Nihon Koden), filtered at 5 kHz, digitized at 20 kHz, and analyzed in a personal computer using a PowerLab/4s system (AD Instruments, Castle Hill, Australia). For induction of LTP or LTD, the stimulation intensity was set to elicit 40–50% or 70–80% of the maximal fEPSP. After stable baseline recording at 0.017 Hz for at least 15 min, LTP was induced by high-frequency stimulation (HFS; 100 stimuli at 100 Hz for 1 s), whereas LTD was induced by low-frequency stimulation (LFS; 900 stimuli at 1 Hz for 15 min). The responses were recorded for another 60 min. The statistical analysis for the normalized fEPSP amplitude at fixed times after the HFS or LFS was conducted with a two-factor (animal group  time) repeated measures analysis of variance (ANOVA). For measuring the paired-pulse ratio, paired pulses were delivered through a single stimulation electrode at varying inter pulse intervals. The first pulse was set to elicit 50% of the maximal fEPSP. The various inter-stimulus intervals between successive pulses were 30, 50, 80, 120, 200, and 500 ms. The ratio of the maximum negative slope of the second pulse to the slope of the first pulse was computed as the paired-pulse ratio. The slope was measured by 30–80% of fEPSP peak amplitude. The presynaptic fiber volley was recorded in the presence of 4 lM CNQX to avoid contamination of the fEPSP. The amplitude of fiber volley was measured as a difference between the initial positive and the following negative peak.

Whole-cell recordings A whole-cell recording was made from CA1 pyramidal cells using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The slices were visualized using an upright microscope equipped with a x40 water immersion objective (BX50WI, Olympus, Tokyo, Japan) and an infrared differential interference contrast (DIC) video system (C2700-79H, Hamamatsu Photonics, Hamamatsu, Japan). Patch pipettes were made from borosilicate capillaries (1.5-mm outer diameter and 0.9-mm inner diameter; Narishige). The resistance of patch pipettes was 4–7 MX when filled with the internal solution. The internal solution for membrane potential contained (mM): 130 K-gluconate, 6 NaCl, 0.2 EGTA, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na2 and 10 phosphocreatine (adjusted to pH 7.3 with KOH). The internal solution for EPSC contained (mM): 135 Cs-methylsulfate, 8 NaCl, 0.5 EGTA, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na2 and 5 QX-314 (adjusted to pH 7.2 with CsOH). QX-314 (final 5 mM) was added to prevent Na+ spikes. EPSCs were evoked by electrical stimulation (200 ls, 0.2 Hz) of axons in the stratum radiatum, using an electronic stimulator (SEN3301; Nihon Koden) and an isolator (SS-202J; Nihon Koden) and bipolar tungsten electrodes. The access resistance for recording was below 15 MX and compensated by 70–80%. Cells were rejected if the access resistance increased above 15 MX. The current signals were filtered at 2 kHz and digitized at 10 or 20 kHz. These electrophysiological recordings were performed at a bath temperature of 30–32 °C, and monitored and recorded using pClamp through a Digidata 1320 interface (Molecular Devices). The values of EPSC peak amplitudes were obtained from averages of 20–30 consecutive traces. To calculate the weighted decay time constant (sw), Igor Pro software (Wavemetrics, Lake Oswego, OR, USA) was used to fit exponential decay curves to averaged NMDA-mediated EPSCs using the formula: y = A1exp( inv s1x) + exp( inv s2x), where s1 and s2 are the decay time constants of the fast and slow components, and A1 and A2 are their respective amplitudes. The weighted time constant of decay (sw) was calculated as: sw = s1 (A1/(A1 + A2)) + s2(A2/(A1 + A2)).

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SDS–PAGE and Western blotting The CA1 region of the hippocampus was dissected out under the microscope. These tissue samples were suspended in lysis buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 2% Triton X-100, 0.25%, v/w, protease inhibitor cocktail (Sigma–Aldrich). Protein content was determined with the BCA Protein Assay Kit (PIERCE, Rockford, IL, USA). Four micrograms of tissue sample was loaded and separated on a 10% SDS– polyacrylamide gel. After transfer to a polyvinylidene difluoride membrane, blots were blocked overnight with Tris–buffered saline (TBS) containing 5% nonfat dried milk and 0.1% Tween 20 at 4 °C and then probed with anti-NR1(1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-NR2A (1:500; Santa Cruz Biotechnology Inc.), anti-NR2B (1:5000; BD Biosciences, Bedford, MA), anti-GluR1(1:1000; Millipore Bioscience Research Reagents, Temecula, CA), anti-GluR2 (1:1000; Millipore Bioscience Research Reagents) or anti-b-actin (1:500; Cell Signaling Technology, Beverly, MA) antibody at room temperature for 2 h. After three washes with TBS containing 0.05% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated bovine anti-goat IgG, antimouse IgG secondary antibody (Santa Cruz Biotechnology Inc.) or goat anti-rabbit IgG secondary antibody (BD Biosciences) diluted 1:5000 with TBS containing 0.1% Tween 20 at room temperature for 1 h, followed by detection using enhanced chemiluminescence (Millipore). The intensities of the bands were semiquantified using the public domain NIH Image program (ver. 1.6, NIH, USA).

Data analyses and statistics Data analyses were performed using Igor Pro. All values are given as means ± SEMs. Statistical comparisons between control and STZ-rats were performed with Student’s t-test (⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001) or a two-way repeated measures ANOVA. The data from extracellular field potential recordings were analyzed by two-factor (animal group, time/ stimulus intensity/inter-stimulus interval) repeated measures ANOVA. In all cases, significance was set at p < 0.05. Statistical analyses were performed using Graphpad Prism 4 (Graphpad Software, San Diego, CA, USA).

RESULTS Effect of diabetes onset on LTP and LTD Previous studies using young adult-onset STZ-rats showed impaired LTP (Biessels et al., 1996; Chabot et al., 1997; Kamal et al., 1999; Artola et al., 2005) or enhanced LTD (Kamal et al., 1999; Artola et al., 2005) at Schaffer collateral synapses in hippocampal CA1 slices. However, we found that juvenile-onset STZ-rats, 3- to 4-week-old rats, showed impaired LTD, but not impaired LTP, compared with age-matched control rats (Iwai et al., 2009). These results might depend on developmental stage. Here we showed the relationship between the age of diabetes onset and synaptic plasticity using adult age-matched rats (22–24 weeks old). Control rats had normal blood glucose levels (138.2 ± 3.0 mg/dl, N = 48), and all STZ-injected rats showed hyperglycemia (584.7 ± 15.9 mg/dl, N = 47) (Table 1). As indicated in Table 1, plasma glucose levels were significantly elevated in juvenile-onset STZrats (595 ± 21.4 mg/dl versus 136.3 ± 4.4 mg/dl for control rats, both N = 23; ⁄⁄⁄p < 0.001, Student’s t-test)

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and young adult-onset STZ-rats (574.8 ± 23.8 mg/dl, N = 24, versus 139.9 ± 4.2 mg/dl for control rats, N = 25; ⁄⁄⁄p < 0.001, Student’s t-test). There was no difference in resting membrane potential (Vmr) in CA1 pyramidal neurons between STZrats and adult age-matched control rats ( 62.0 ± 1.17 mV for control rats, n = 23; 61.8 ± 0.74 mV for juvenile-onset STZ-rats, n = 24; p > 0.05, Student’s t-test) ( 62.7 ± 1.17 mV for control rats, n = 24; 61.6 ± 0.77 mV for young adult-onset STZrats, n = 24; p > 0.05, Student’s t-test) (Table 1). According to young adult-onset STZ-rats, the result is consistent with one previous study (Artola et al., 2005), but not another (Heng et al., 2011). We compared LTP and LTD between juvenile-onset and young adult-onset STZ-rats. LTP of CA1 fEPSPs was induced by a single episode of HFS (100 Hz, 1s), and LTD was induced by LFS (1 Hz, 15 min). The average of fEPSP amplitude during the 15 min before HFS or LFS induction defined as 100%. After 15 min of baseline recording, HFS induced LTP for 60 min in both control and juvenile-onset STZ-rats (control: 129.0 ± 8.4%; juvenile-onset STZ-rats: 128.1 ± 12.2%, both n = 7), but a modest level of LTP in young adultonset STZ-rats (105.9 ± 9.5%, n = 7). We found that young adult-onset STZ-rats showed significantly impaired LTP (F(1,12) = 6.213, p < 0.05, two-way repeated measures ANOVA) (Fig. 1A2), but not juvenile-onset STZ-rats (F(1,12) = 1.202, p = 0.29, twoway repeated measures ANOVA) (Fig. 1A1) compared with age-matched control rats. Moreover, LTP was impaired in the first 15 min after its induction in young adult-onset STZ-rats (F(1,12) = 5.612, p < 0.05, two-way repeated measures ANOVA). On the other hand, LFSinduced LTD for 60 min in both control and young adultonset STZ-rats (control: 72.5 ± 2.1%; young adult-onset STZ-rats: 74.2 ± 4.8%, both n = 6), but a modest level of LTD in juvenile-onset STZ-rats (88.3 ± 5.7%, n = 11). Juvenile-onset STZ-rats showed significantly impaired LTD (F(1,20) = 5.787, p < 0.05, two-way repeated measures ANOVA) (Fig. 1B1), but not young adult-onset STZ-rats (F(1,10) = 0.296, p = 0.43, two-way repeated measures ANOVA) (Fig. 1B2) compared with age-matched control rats. LTD in juvenile-onset STZrats was impaired 15 min after its induction (F(1,20) = 5.401, p < 0.05, two-way repeated measures ANOVA) through 60 min. In young adult-onset STZ-rats, these results are consistent with previous studies (Biessels et al., 1996; Chabot et al., 1997). We found impaired LTD of juvenile-onset STZ-rats in not only the developmental stage (3–4 weeks) (Iwai et al., 2009), but also adulthood (5–6 month). These results demonstrated that the age of diabetes onset in STZ-rats had significant effects on synaptic plasticity. Synaptic transmission at the SC-CA1 pathway To investigate why the age of onset affected hippocampal synaptic plasticity, we measured excitatory synaptic transmission by making extracellular recordings at SC-CA1 synapses in juvenile-onset and young adult-onset STZ-rats, compared with age-matched control rats. The

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Table 1. Body weight, blood glucose and resting membrane potential in STZ-rats and age-matched control rats

Control (N = 23) Juvenile-onset (N = 23) Control (N = 25) Young adult-onset (N = 24)

Weight (g)

Blood glucose (mg/dl)

324 ± 18 141 ± 12⁄⁄⁄ 360 ± 18 178 ± 13⁄⁄⁄

136.3 ± 4.4 595.0 ± 21.4⁄⁄⁄ 133.9 ± 4.2 574.8 ± 23.8⁄⁄⁄

Vmr (mV) 62.0 ± 1.17 61.8 ± 0.74 62.7 ± 1.17 61.6 ± 0.77

(n = 23) (n = 24) (n = 24) (n = 24)

The reduction in body weight and elevation of blood glucose levels in STZ-rats were highly significant (N = 23, control rats; N = 23, juvenile-onset STZ-rats; ⁄⁄⁄p < 0.001, Student’s t-test) (N = 25, control rats; N = 24, juvenile-onset STZ-rats; ⁄⁄⁄p < 0.001, Student’s t-test). On the other hand, no significant difference in resting membrane potential (Vmr) was detected between CA1 pyramidal cells (n = 23–24, from 8 to 9 rats, p > 0.05, Student’s t-test). All resting membrane potentials were measured after withdrawal of the pipette. (N) means the number of animals and (n) means the number of cells. Data are shown as the mean ± SEM.

relation of fEPSP slope to stimulus intensity (input–output relation) of synaptic transmission increased significantly in juvenile-onset STZ-rats (F(1,59) = 4.338, p < 0.05, twoway repeated measures ANOVA) (Fig. 2A1), but not in young adult-onset STZ-rats (F(1,56) = 3.526, p > 0.05, two-way repeated measures ANOVA) (Fig. 2A2), compared with the control rats. Paired-pulse facilitation (PPF) is a facilitation of a second response when a synapse is stimulated twice with a short inter-stimulus interval (30–500 ms). This phenomenon is attributed to an increase in the amount of neurotransmitter released in response to the second stimulus (Katz and Miledi, 1968; Zucker, 1989). The strength of PPF did not change in STZ-rats compared with age-matched control rats (F(1,25) = 1.154, p > 0.05, two-way repeated measures ANOVA for juvenile-onset STZ-rats; F(1,25) = 1.931, p > 0.05, two-way repeated measures ANOVA for young adult-onset STZ-rats) (Fig. 2B1 and B2). Moreover, STZ-rats showed no change in presynaptic fiber volley amplitude plotted against stimulus intensity (F(1,21) = 0.002, p > 0.05, two-way repeated measures ANOVA for juvenile-onset STZ-rats; F(1,21) = 0.058, p > 0.05, two-way repeated measures ANOVA for young adult-onset STZ-rats) (Fig. 2C1 and C2). These results suggest that the electrophysiological properties of the presynaptic fibers in STZ-rats did not differ from those in control rats.

Enhanced AMPA and NMDA receptor-mediated EPSCs in juvenile-onset STZ-rats We next examined AMPA and NMDA postsynaptic responses, because the input–output relation of synaptic transmission in juvenile-onset STZ-rats was significantly different from that in age-matched control rats. Wholecell voltage-clamp recordings were made of CA1 pyramidal neurons in hippocampal slices derived from adult rats. AMPA receptor-mediated EPSCs (AMPAEPSCs) were evoked by stimulation of Schaffer collaterals in the presence of the GABAA receptor antagonist picrotoxin (100 lM) and NMDA receptor antagonist AP5 (50 lM), when membrane potentials were held at 70 mV (Vh = 70 mV). The peak amplitude of AMPA-EPSCs against stimulus intensity increased significantly in juvenile-onset STZ-rats (F(1,22) = 4.467, p < 0.05, two-way repeated measures ANOVA) (Fig. 3A1), but not in young adult-onset STZrats (F(1,20) = 0.380, p > 0.05, two-way repeated measures ANOVA) (Fig. 3A2).

We further recorded NMDA receptor-mediated EPSCs (NMDA-EPSCs) in the presence of picrotoxin (100 lM) and CNQX (10 lM), when membrane potentials were held at + 40 mV (Vh = + 40 mV). The peak amplitude of NMDA-EPSCs against stimulus intensity increased significantly in juvenile-onset STZ-rats (F(1,22) = 6.899, p < 0.05, two-way repeated measures ANOVA) (Fig. 3B1), but not in young adult-onset STZ-rats (F(1,20) = 0.142, p > 0.05, two-way repeated measures ANOVA) (Fig. 3B2). A possible mechanism for the enhancement of NMDA receptor function was a change in subunit composition (i.e., NR2B-containing NMDA receptors have slower kinetics and mediate a greater amount of current). We thus analyzed the decay time constant of NMDA-EPSCs, which was best fitted into a double exponential function. However, a significant change in the decay time of NMDA-EPSCs was not observed in juvenile-onset STZ-rats (48.3 ± 3.5 ms, n = 15, versus 42.6 ± 5.3 ms in control rats, n = 9; p >0.05, Student’s t-test), or young adult-onset STZ-rats (48.3 ± 2.9 ms versus 40.5 ± 3.7 ms in control rats, both n = 9; p > 0.05, Student’s t-test).

Reduced AMPA/NMDA ratio of excitatory transmission in young adult-onset STZ-rats The relative contribution of AMPA receptors and NMDA receptors to excitatory synaptic currents is thought to play a role in synaptic integration and plasticity, and is an indicator of long-term synaptic plasticity (Malinow and Malenka, 2002). We measured the ratio of AMPA and NMDA receptor-mediated EPSCs (the AMPA/ NMDA ratio of EPSCs) through the whole-cell recordings. AMPA-EPSCs and NMDA-EPSCs were evoked by the stimulation (averaged stimulus intensity; 53 ± 7 lA, n = 61) of Schaffer collaterals in the presence of picrotoxin (100 lM), and were measured at 70 and +40 mV, respectively. To ensure that the NMDA-EPSCs at +40 mV were free of AMPA receptor contamination, the currents were measured 100 ms after the stimulation. No-responses were not included. Interestingly, the AMPA/NMDA ratio of EPSCs at SC-CA1 synapses was significantly decreased (about 40%) in young adult-onset STZ-rats (4.19 ± 0.46, n = 14, versus 6.92 ± 1.22 in control rats, n = 12; ⁄ p < 0.05, Student’s t-test) (Fig. 4A2), but not in juvenile-onset STZ-rats (5.13 ± 0.74, n = 18, versus 6.53 ± 1.36 in control rats, n = 15; p > 0.05, Student’s t-test) (Fig. 4A1).

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Fig. 1. Impaired LTP in young adult-onset and impaired LTD in juvenile-onset STZ-rats. Hippocampal slices were obtained from control rats (white symbols), juvenile-onset STZ-rats (black symbols) or young adult-onset STZ-rats (gray symbols) at 22–24 weeks. LTP was induced by HFS stimulation (arrow). (A1) The magnitude of LTP in juvenile-onset STZ-rats was not significantly affected (n = 7 in control rats, n = 7 in juvenileonset STZ-rats; p > 0.05, two-way repeated measures ANOVA). (A2) The magnitude of LTP in young adult-onset STZ-rats was significantly impaired (n = 7 in control rats, n = 7 in young adult-onset STZ-rats; p < 0.05, two-way repeated measures ANOVA). LTD was induced by LFS stimulation (dashed line). (B1) The magnitude of LTD in juvenile-onset STZ-rats was significantly impaired (n = 11 in control rats, n = 11 in juvenile-onset STZ-rats; p < 0.05, two-way repeated measures ANOVA). (B2) The magnitude of LTD in young adult-onset STZ-rats was not significantly affected (n = 6 in control rats, n = 6 in young adult-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA). Representative traces at the time points are shown on the top. Data are shown as the mean ± SEM.

Effect of diabetes onset on the expression of AMPA and NMDA receptor subunits Next we investigated the expression of AMPA and NMDA receptor subunits in the hippocampal CA1 region by Western blot analysis, because of the increased AMPA and NMDA receptor-mediated EPSCs in juvenile-onset STZ-rats and the reduced AMPA/NMDA ratio of EPSCs in young adult-onset STZ-rats. The quantity of immunoreactive bands of AMPA and NMDA receptor subtype (GluR1, GluR2, NR1, NR2A and NR2B) and

b-actin proteins was normalized by comparing with their expression levels in control rats. In juvenile-onset STZrats (Fig. 5A1 and A2), no significant changes were observed for the GluR1 subunit (136 ± 14% of control), GluR2 subunit (120 ± 7% of control), NR2A subunit (130 ± 13% of control), NR2B subunit (117 ± 19% of control) and NR1 subunit (120 ± 13% of control). In young adult-onset STZ-rats (Fig. 5B1 and B2), no significant changes were observed for the GluR1 subunit (88 ± 6% of control), GluR2 subunit (79 ± 5% of control), NR2A subunit (98 ± 6% of control), NR2B

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Fig. 2. The influences of diabetes onset on basal synaptic transmission of SC-CA1 synapses. (A) The input–output relation was obtained by plotting the fEPSP slope against the intensity of stimulation. The insets show typical traces of fEPSP at 35 lA. The input–output relation increased significantly in juvenile-onset STZ-rats (A1), but not in young adult-onset STZ-rats (A2) compared with control rats (n = 32 in control rats, n = 29 in juvenile-onset STZ-rats; p < 0.05, two-way repeated measures ANOVA) (n = 30 in control rats, n = 28 in young adult-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA). (B) Facilitation ratios were plotted against inter-stimulus intervals. (B1, B2) PPF did not change significantly in STZ-rats (n = 13 in control rats, n = 14 in juvenile-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA) (n = 13 in control rats, n = 14 in young adult-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA). The insets show typical traces of fEPSP. (C) Presynaptic fiber volley amplitude was plotted against the intensity of stimulation. (C1, C2) These presynaptic fiber volley amplitudes did not change significantly in STZ-rats (n = 11 in control rats, n = 12 in juvenile-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA) (n = 11 in control rats, n = 12 in young adult-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA). Data are shown as the mean ± SEM.

subunit (86 ± 8% of control) and NR1 subunit (121 ± 10% of control) either. Western blot analysis demonstrated that total levels of GluR1, GluR2, NR2A, NR2B or NR1 did not significantly change in STZ-rats, compared to age-matched control rats (Fig. 5).

Impaired LTD caused by the activation of NMDA receptor in juvenile-onset STZ-rats Previous studies in the CA1 of the hippocampus found that LTD, which was induced by LFS, was dependent on the activation of NMDA receptor (Dudek and Bear,

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Fig. 3. The influences of diabetes onset on AMPA- and NMDA-EPSCs of SC-CA1 synapses. Synaptic currents evoked by stimulation of Schaffer collaterals were recorded from CA1 pyramidal cells in control rats (white), juvenile-onset STZ-rats (black) or young adult-onset STZ-rats (gray) at 22–24 weeks. (A) AMPA-EPSCs recorded at a holding potential of 70 mV in the presence of picrotoxin (100 lM) and APV (50 lM). The peak amplitude of AMPA-EPSCs was plotted against the intensity of stimulation. (A1, A2) The amplitude increased significantly in juvenile-onset STZ-rats (n = 10 in control rats, n = 14 in juvenile-onset STZ-rats; p < 0.05, two-way repeated measures ANOVA), but not in young adult-onset STZ-rats compared with control rats (n = 10 in control rats, n = 12 in young adult-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA). (B) NMDA-EPSCs recorded at a holding potential of +40 mV in the presence of picrotoxin (100 lM) and CNQX (10 lM). The peak amplitude of NMDAEPSCs was plotted against the intensity of stimulation. (B1, B2) The amplitude increased significantly in juvenile-onset STZ-rats (n = 10 in control rats n = 14 in juvenile-onset STZ-rats; p < 0.05, two-way repeated measures ANOVA), but not in young adult-onset STZ-rats compared with control rats (n = 10 in control rats, n = 12 in young adult-onset STZ-rats; p > 0.05, two-way repeated measures ANOVA). The insets show EPSC traces at 50 lA. Data are shown as the mean ± SEM.

1992; Mulkey and Malenka, 1992; Norris et al., 1996). In juvenile-onset STZ-rats, enhanced NMDA receptormediated transmission might contribute to the expression of LFS-induced LTD. To test this hypothesis, we used a low concentration of an NMDA receptor antagonist, APV (0.5 lM), during the LTD induction protocol. As shown in Fig. 6, the magnitude of LTD in APV-treated slices was significantly different from those in APV-untreated slices from juvenile-onset STZ-rats (APV-untreated slices: 90.3 ± 5.1%; APV-treated slices: 69.5 ± 6.7%, both n = 7, F(1,12) = 9.174, p < 0.05, two-way repeated measures ANOVA). The concentration of APV was based on previous studies (Cummings et al., 1996; Nishiyama et al., 2000; Liu et al., 2004), in which LTD was shown to be sensitive to the partial NMDA receptor blockade, produced with low concentrations of APV.

DISCUSSION Diabetes induces a number of neurological changes that may make the hippocampus more susceptible to agerelated structural and functional deficits. There are some reports of cognitive differences related to onset age of type 1 DM (Ferguson et al., 2005; Gaudieri et al., 2008). However, no study has examined the synaptic plasticity and transmission related to the age of diabetes onset in animal models of type 1 DM. In the present study, we conducted an analysis of hippocampal synaptic properties related to age at diabetes onset using STZtreated diabetic rats. The principal findings of this study were a significantly impaired LTD in juvenile-onset STZ-rats, and a significantly impaired LTP in young adult-onset STZrats, compared with age-matched control rats, at

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Fig. 4. The AMPA/NMDA ratio was lower in young adult-onset STZ-rats, but not in juvenile-onset STZ-rats. The insets show compound EPSCs at 70 mV (lower traces) and +40 mV (upper traces) in the presence of picrotoxin (100 lM). (A1) Averaged AMPA/NMDA ratio showed no significant difference between control rats (white) and juvenile-onset STZ-rats (black) (n = 15 in control rats, n = 18 in juvenile-onset STZ-rats; n.s., nonsignificant; p > 0.05, Student’s t-test). (A2) Averaged AMPA/NMDA ratio decreased significantly in young adult-onset STZ-rats (gray) compared with control rats (n = 12 in control rats, n = 14 in young adult-onset STZ-rats; ⁄p < 0.05, Student’s t-test). Data are shown as the mean ± SEM.

SC-CA1 synapses (Fig. 1). LTP and LTD are not unitary phenomena. Their mechanisms vary depending on the synapses and neural circuit in which they operate. To examine whether the age of diabetes onset had an effect on SC-CA1 synaptic transmission or not, we performed several experiments and obtained the following results: (1) The relation of fEPSP slope to stimulus intensity of synaptic transmission increased significantly in juvenile-onset (Fig. 2A1), but not in young adult-onset STZ-rats (Fig. 2A2). (2) PPF, a presynaptic phenomenon, in STZ-rats was not significantly different from that in control rats (Fig. 2B1 and B2). (3) The relationship between the amplitudes of presynaptic fiber volley and stimulus intensity did not change (Fig. 2C1 and C2). These results suggest that neurotransmitter release from presynaptic terminals does not change in STZ-rats. Next we examined postsynaptic transmission that was discriminated between AMPA and NMDA components, and obtained the following results: (4) AMPA and NMDA receptormediated transmission increased significantly in juvenile-onset STZ-rats (Fig. 3). (5) On the other hand, the AMPA/NMDA ratio of excitatory synaptic transmission decreased significantly in young adultonset STZ-rats (Fig. 4). (6) In the hippocampal CA1 region, there were no differences in total levels of GluR1, GluR2, NR2A, NR2B, or NR1 between STZ-rats and age-matched control rats (Fig. 5). (7) Finally, impaired LTD in juvenile-onset STZ-rats was rescued with an NMDA receptor antagonist (Fig. 6). All results indicate that significant enhancement of excitatory postsynaptic transmission, in particular, NMDA receptor-

mediated transmission, affects the plastic change in CA1 pyramidal neurons of juvenile-onset STZ-rats. In young adult-onset STZ-rats, our results imply disproportionate numbers of AMPA and NMDA receptors on the membrane of CA1 pyramidal neurons. Taken together, we conclude that the age of diabetes onset should be considered in the treatment of diabetic cognitive difficulties. LTP and diabetes onset in STZ-rats Here we demonstrated that LTP was impaired in young adult-onset STZ-rats but not juvenile-onset STZ-rats. A previous report showed a progressive impairment in LTP in SC-CA1 synapses after 6–8 weeks of diabetes in young adult-onset STZ-rats. The impaired LTP reached a maximum after 12 weeks of diabetes and remained stable thereafter (Kamal et al., 1999). At least two explanations could account for these results. First, Hebb’s idea has been strengthened by the finding that many synapses undergo LTP and that this process requires both presynaptic activity and strong postsynaptic depolarization. According to presynaptic activity, young adult-onset STZ-rats may be functional. Our results indicated that the excitability of presynaptic fibers was unchanged in young adult-onset STZ-rats. Moreover, a previous report showed that PPF was similar in control and young adult-onset STZ-rats (Biessels et al., 1996), which was consistent with our results, but another report showed that PPF in young adult-onset STZ-rats was decreased (Artola et al., 2005). PPF is thought to result from a higher probability

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Fig. 5. Western blot analysis of control- and STZ-rats with respective antibodies (GluR1, GluR2, NR2A, NR2B, NR1 and b-actin). In each lane, 4 lg protein of CA1 crude fraction was loaded. (A1, B1) Immunoblots with antibodies to GluR1, GluR2 and b-actin (upper panel) from control and juvenileonset STZ-rats (A1) and from control and young adult-onset STZ-rats (B1). (A2, B2) Immunoblots with antibodies to NR2A, NR2B, NR1 and b-actin (upper panel) from control and juvenile-onset STZ-rats (A2) and from control and young adult-onset STZ-rats (B2). Summary graphs indicate density of immunoreactivity in STZ-rats normalized to that in control for each molecule (lower panel) (N = 6, respectively).

of transmitter release to the second pulse attributed to the residual presynaptic Ca2+ concentration left from the first pulse. Artola et al. (2005) suggested that these differences depended on the Ca2+/Mg2+ ratio. A lower Ca2+/Mg2+ ratio leads to less probability of transmitter release. PPF would thus be similar in control and young adult-onset STZ-rats at the same Ca2+/Mg2+ ratio (2.5/ 1.3), but presynaptic Ca2+ dynamics might change in young adult-onset STZ-rats with a lowering of the probability of transmitter release. Second, postsynaptic malfunction may induce impaired LTP in young adult-onset STZ-rats. Because there was no change of the total expression of AMPA and NMDA subunits in the CA1 region of young adultonset STZ-rats, it is possible that young adult-onset STZ-rats may affect on postsynaptic transmission. LTP requires the activation of NMDA receptors and is

caused by an enhancement of AMPA receptor-mediated transmission (Malinow and Malenka, 2002; Bredt and Nicoll, 2003; Collingridge et al., 2004; Malenka and Bear, 2004). In young adult-onset STZ-rats, there was no change of AMPA- and NMDA-EPSCs, but a significant decrease of AMPA/NMDA ratio of EPSCs. These two experiments were used by different stimulus intensities (AMPA- and NMDA-EPSCs evoked by 20– 50 lA; AMPA/NMDA ratio of EPSCs evoked by 53 ± 7 lA). AMPA- or NMDA-EPSCs in young adultonset STZ-rats would thus change if more strong stimulation was applied. Moreover, previous studies showed that AMPA receptor binding was impaired (Chabot et al., 1997) and the functional activities of AMPA receptors were reduced (Kamal et al., 2006) in young adult-onset STZ-rats. The trafficking or synaptic targeting of AMPA-receptors is one of the expression

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Fig. 6. Impaired LTD of juvenile-onset STZ-rats was rescued in the presence of APV. LTD was induced by LFS stimulation (dashed line). The magnitude of LTD in APV-treated slices was significantly different from that in APV-untreated slices from juvenile-onset STZrats (n = 7 in APV-untreated slices, n = 7 in APV-treated slices; p > 0.05, two-way repeated measures ANOVA). Representative traces at the time points are shown on the top. Data are shown as the mean ± SEM.

mechanisms for LTP (Malinow and Malenka, 2002; Collingridge et al., 2004). In further studies, we would examine concerning mechanisms involved the trafficking or synaptic targeting of glutamate receptors in young adult-onset STZ-rats. Moreover, recent studies showed that in young adultonset STZ-rats, membrane excitability and mitochondorial function of CA1 pyramidal neurons were affected at 5 weeks of STZ-injection (Heng et al., 2011; Ye et al., 2011). However, another study (Artola et al., 2005) and we showed that there was no change in resting membrane potential using young adult-onset STZ-rats after 12–14 weeks of STZ-injection. Diabetes-induced changes may depend on the duration of diabetes.

LTD and diabetes onset in STZ-rats In contrast to LTP, LTD was impaired in juvenile-onset STZ-rats but not young adult-onset STZ-rats. In juvenile-onset STZ-rats, LTD was affected 4 days after STZ-treatment (Iwai et al., 2009) and remained impaired thereafter (24 weeks). What is the influence of diabetes on the developing brain? During the early stages of postnatal development, glutamatergic synapses change with the number and type of receptor. For example, there is an increase in the strength of AMPAR-mediated transmission (Wu et al., 1996; Petralia et al., 1999) and a switch in the subunit composition of synaptic NMDA receptors (Bellone and Nicoll, 2007). In juvenile-onset STZ-rats, AMPA- and NMDA-EPSCs increased, but the AMPA/NMDA ratio of EPSCs did not change. However, juvenile-onset STZrats showed no detectable effect on the level of overall

expression of AMPA and NMDA receptors. Therefore these results imply that juvenile-onset STZ-rats may be affected by the number of AMPA and NMDA receptors in the synaptic cleft. However, previous reports showed that LTD was enhanced in young adult-onset STZ-rats (Artola et al., 2005) and LTD was similar in control and young adultonset STZ-rats (Chabot et al., 1997). Our result was similar with Chabot et al., but not with Artola et al. These differences may be due to the induction protocols. In the present study, we used a typical protocol (900 stimuli at 1 Hz) for inducing LTD (Dudek and Bear, 1992, 1993). This number of stimulations was thought to be reduced if the postsynaptic neuron is modestly depolarized to relieve the Mg2+ block of the NMDA receptor (Selig et al., 1995). Moreover, we showed that impaired LTD of juvenileonset STZ-rats was rescued with an NMDA receptor antagonist. Although juvenile-onset STZ-rats showed an increase in AMPA as well as NMDA currents, these results have led us to hypothesize that impaired LTD in juvenile-onset STZ-rats might be a specific consequence of enhanced NMDA receptor function at hippocampal CA1 synapses. In further studies, we would examine hippocampus-dependent learning behavior in juvenile-onset STZ-rats treated with NMDA receptor antagonists. Modified membrane excitability or altered spike frequency accommodation in hippocampal neurons might also influence the magnitude and type of plasticity in juvenile-onset STZ-rats. Previous studies showed voltage-dependent thresholds for inducing LTD and LTP (Artola et al., 2005) and frequency-dependent properties in synaptic plasticity (Kamal et al., 2000) in young adultonset STZ-rats. There was no difference in resting membrane potential between juvenile-onset STZ-rats and age-matched control rats. To address the properties of synaptic plasticity in juvenile-onset STZ-rats, further studies are necessary, particularly on activity-dependent synaptic plasticity. In addition, a recent study showed that elevated glucocorticoid levels contributed to the impairment of hippocampal synaptic plasticity and neurogenesis in young adult-onset STZ-rats (Stranahan et al., 2008). Moreover cognitive performance of young adult-onset STZ-mice was improved during chronic treatment with the glucocorticoid receptor antagonist mifepristone (RU486) (Revsin et al., 2009). The hippocampus is enriched with glucocorticoid receptors and plays a crucial role in the effect of stress on synaptic plasticity and memory (Shors et al., 1989; Oitzl and de Kloet, 1992; Pavlides et al., 1996; Xu et al., 1997; de Quervain et al., 1998; McEwen, 1999; Kim and Diamond, 2002). Since glucocorticoid facilitates hippocampal glutamate transmission (for review; Popoli et al., 2011), we should examine the effect of glucocorticoid in juvenile-onset STZ-rats. Functional implications The present study first showed that the age of onset of diabetes had different influences on synaptic

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LTP (60 min) LTP (60 min) Input–output relation PPF Fiber volley amplitude AMPA-EPSCs NMDA-EPSCs AMPA/NMDA ratio Expression of receptor subunits (GluR1, GluR2, NR1, NR2A, NR2B)

Juvenile-onset STZ-rats versus control rats

Young adult-onset STZ-rats versus control rats

n.s. impaired increased n.s. n.s. increased increased n.s. n.s.

impaired n.s. n.s. n.s. n.s. n.s. n.s. decreased n.s.

n.s., non-significant.

transmission and plasticity at SC-CA1 synapses (Table 2). In young adult-onset STZ-rats, LTP was impaired and the AMPA/NMDA ratio of EPSCs decreased. The present results, together with other studies (Chabot et al., 1997; Kamal et al., 2006), imply that the functional change in AMPA receptors might contribute the expression mechanisms for LTP in young adult-onset STZ-rats. Artola et al. (2005) reported that LTP was obtained at more depolarized membrane potential in young adult-onset STZ-rats compared with control rats. In this study, the movement of cations through the AMPA receptors during HFS in young adultonset STZ-rats might be attenuated, so that activation of postsynaptic NMDA receptors became insufficient to induce LTP. On the other hand, LTD was impaired and AMPA- and NMDA-EPSCs increased in juvenile-onset STZ-rats. LFS-induced LTD requires modest but not large depolarization (for review; Bear and Abraham, 1996). In this study, LFS-induced depolarization might be large enough to inhibit LTD in juvenile-onset STZrats. Since an NMDA receptor antagonist rescued the impairment of LTD, the increase in NMDA receptormediated currents is presumably responsible for impaired LTD in juvenile-onset STZ-rats. By the age of diabetes onset, STZ-rats could experience an alteration in the hyperglycemia process. Changes in SC-CA1 synaptic plasticity and transmission appeared after 4 days in juvenile-onset STZ-rats and at 6–8 weeks in young adult-onset STZ-rats. These differences might reflect developmental and/or activitydependent synaptic maturation in STZ-rats. Further studies on diabetes onset-dependent synaptic maturation by genetic and pharmacological manipulation would be necessary to improve diabetic cognitive difficulties. In this study, we used a typical LTP or LTD induction protocol, which is not physiological. The use of spiketiming dependent plasticity protocols that are based on behavior in vivo would more accurately allow comparison to animal behavior (Kampa et al., 2007; Caporale and Dan, 2008). Moreover, extrinsic modulators of somatodendritic voltage-gated channel activity such as acetylcholine, serotonin, and noradrenaline will, by affecting a postsynaptic cell’s propensity to produce action potentials, alter not only neuronal throughput but also spike backpropagation

(Tsubokawa and Ross, 1997; Sandler and Ross, 1999) and thresholds for inducing LTP and LTD, a phenomenon called metaplasticity (for review; Abraham and Bear, 1996). To elucidate these differences between young adult-onset and juvenile-onset STZ-rats, more studies are required.

CONCLUSION In the present study, it should be noted that the age of diabetes onset is important to choose an appropriate therapy for central neurological diseases. For example, NMDA receptor antagonists might be suitable for the treatment of cognitive decline in patients with type 1 DM appearing in childhood, but not in adolescence.

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(Accepted 28 September 2012) (Available online 13 October 2012)