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Effects of treadmill running onshort-termpre-synaptic plasticityat dentate gyrus of streptozotocin-induced diabetic rats

Parham Reisia,b,⁎, Shirin Babria,b, Hojjatallah Alaeic, Mohammad Reza Sharific,Gisue Mohaddesb, Reza Lashgarid,e

aLaboratory of Physiology, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, IranbDepartment of Physiology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, IrancDepartment of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IrandCenter for Neurobiology and Behavior and Department of Psychiatry, Columbia University, New York, NY 10032, USAeImam Bagher(as) Institute of Science and Technology, Tehran, Iran

A R T I C L E I N F O

⁎ Corresponding author. Fax: +98 411 3364664E-mail address: parhamzh@gmail.com (P.Abbreviations: ANOVA, Analysis of Varianc

dorsal ventral; DM, diabetes mellitus; EPSP, efPPF, field paired pulse facilitation; fPS, fieldhertz; h, hour; I/O, Input/output; i.p., intra-pmilligram per deciliter; mg/kg, milligram pecentigrade; PPF, paired pulse facilitation; PPDsecond; S.E.M, standard error of mean; STZ,

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.03.024

A B S T R A C T

Article history:Accepted 13 March 2008Available online 21 March 2008

Previous studies indicated that diabetes mellitus leads to impairments in hippocampalsynaptic plasticity and defects in learning and memory. Although diabetes affects synaptictransmission in the hippocampus through both pre- and post-synaptic influences, it is notclear if the defects are pre- or post-synaptic or both; and whether these are prevented byrunning. The aim of this study was to evaluate the effects of treadmill running on short-termplasticity in inhibitory interneurons in the dentate gyrus of STZ-induced diabetic rats.Experimental groupswere the control–rest group, the control–exercise group, the diabetes–restgroup and the diabetes–exercise group (n=6 for each experimental group). The exerciseprogram was moderate exercise consisting of treadmill running at 17 m/min and 0-degreeinclination for 40min/day, 7 days/week, for 12 weeks. The paired pulse paradigmwas used tostimulate the perforant pathway and field excitatory post-synaptic potentials (fEPSP) wererecorded in dentate gyrus (DG). In the diabetic–rest group paired pulse facilitation wassignificantly increased comparing to the control–rest group. However, there were nodifferences between responses of the control–exercise and diabetes–exercise groupscompared to the control–rest group. The present results suggest that the pre-synapticcomponent of synaptic plasticity in the dentate gyrus is affectedunder diabetic conditions andthat treadmill running prevents this effect. The data support the possibility that alterations intransmissionmay account, in part, for learning andmemory deficits induced in diabetes, andthat treadmill running is helpful in alleviating the neural complications of diabetes mellitus.

© 2008 Elsevier B.V. All rights reserved.

Keywords:HippocampusShort-term memoryPaired pulse facilitationDentate gyrusDiabetesTreadmill running

.Reisi).e; AP, anterior posterior; Ca, calcium; CNS, central nervous system; DG, dentate gyrus; DV,xcitatory post-synaptic potential; fEPSP, field excitatory post-synaptic potential; Fig, figure;population spike; GABA, gamma-aminobutyric acid; g/kg, gram per kilogram; g, gram; Hz,eritoneal; IPSP, inhibitory post-synaptic potential; KHz, kilo hertz; µA, microampere; mg/dl,r kilogram; ML, medial lateral; mm, millimeter; ms, millisecond; n, number; °C, degree of, paired pulse depression; PPI, paired pulse index; PP, paired pulse; PS, population spike; s,Streptozotocin

er B.V. All rights reserved.

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Fig. 1 – Input–output curves (mean±SEM) of A: the PSamplitude and B: the EPSP slope in dentate gyrus.

1. Introduction

Diabetes mellitus (DM), a deficiency in insulin secretion orresistance to insulin action or both (Gaven et al., 1997), is acommon and serious metabolic disorder with numeroussecondary complications. Behavioral and electrophysiologicalexperiments have shown that diabetes induces cognitiveimpairments by affecting the hippocampus (Biessels et al.,1996, 2002). However, the mechanism of these impairments indiabetes has not been well understood.

Involvement of synaptic plasticity in the hippocampus inlearningandmemoryhasbeenclearly identified, and increasingevidencehas shown that diabetes impairs synaptic plasticity, orlearning and memory gradually from 8 weeks after theinduction of diabetes (Biessels et al., 1996; Kamal et al., 2000)to a maximum of after 12 weeks (Biessels et al., 1996; Chabotet al., 1997; Kamal et al., 1999). At the cellular level, diabetesaffects synaptic transmission, by influencing both pre-synaptic(Bitar et al., 1985; Chu et al., 1986; Lackovic et al., 1990; Yamatoet al., 2004) and post-synaptic (Chabot et al., 1997; Gardoni et al.,2002) components. The streptozotocin-induced diabetes altersthe synaptic terminal structure in the hippocampus, includingthe rearrangement of vesicles (Magarinos et al., 1997), depletionof synaptic vesicles and retraction and simplification of apicaldendrites of hippocampal neurons (Magarinos and McEwen,2000). However, it is still not clear if the defects in synapticplasticity in the hippocampus in diabetes are pre- or post-synaptic or both (Kamal et al., 2006). In addition, these deficitswerepartially reversedby theuseof insulin (Biesselsetal., 1998).

Previous studies have shown that exercise can contribute toimprovement of the central neural complications of diabetesmellitus (Kim et al., 2002). Regular physical exercise has beenshown to have beneficial effects on neural health and function,andalso, it canprotectneurons fromvariousbrain insults (Carroet al., 2001; Tillerson et al., 2003). It has been demonstrated thatexercise can increase the speedof learningandestablishmentofmemory and improve cognitive performance (Anderson et al.,2000; Ang et al., 2006; Faber et al., 2002; Van praag et al., 1999).Subsequent to voluntary exercise synaptic efficiency was en-hanced in the dentate gyrus (DG) of the hippocampus in adultrat andmice (Farmer et al., 2004; Van praag et al., 1999). Physicalactivity affects synaptic transmission by influencing the releaseofmost neurotransmitters (Meeusen and DeMeirleir, 1995) andthe receptor content at post-synaptic sites (Dietrich et al., 2005).

The phenomena of paired pulse facilitation (PPF) and depres-sion (PPD),well knownas short-term formsof synaptic plasticity,are generally accepted as a model for evaluation of the pre-

Table 1 – Mean blood glucose concentrations and bodyweightmg/dl±SEMand g±SEM, respectively, in each group

Control–rest

Control–exercise

Diabetes–rest

Diabetes–exercise

Blood glucose(mg/dl)

88±5.9 89.25±12.1 More than600⁎ (N600)

474.86±27.6⁎

Body weight(g)

262±3.4 250.6±7 178.4±10.6⁎ 187±11.1⁎

⁎Significantly different (p<0.05) from the control–rest group.

synaptic component of the synapse (Commins et al., 1998; Chenet al., 1996; Gottschalk et al., 1998). PPF is an enhancement in theamplitude of the EPSP evoked by a second stimulus that followsthe first one in the paired pulse paradigm with a short inter-stimulus interval (Lashgari et al., in press). Facilitation is theresult of an increase in probability of neurotransmitter releasethat is mainly attributed to residual Ca2+ in the nerve terminalsafter the first stimulus (Carter et al., 2002; Katz andMiledi, 1968).Short-term synaptic plasticity lasting from a fewmilliseconds toa few minutes serves as a flexible mechanism for temporalinformation processing in higher cortical integration. The pur-pose of this electrophysiological study was to investigate, usingpaired pulse stimulation and field potential recordings method,the effects of treadmill running on short-term pre-synaptic plas-ticity in thedentate gyrusof streptozotocin-induceddiabetic rats.

2. Results

2.1. Body weights and blood glucose

The body weights and blood glucose concentrations at the endof experiments are shown in Table 1. The body weights andblood glucose concentrations of controls are both significantlydifferent from that of diabetes–rest and diabetes–exercisegroups (univariate ANOVA, p<0.05). Table 1 shows a decreaseof blood glucose levels in diabetes–exercise group compared tothe diabetes–rest group (p<0.05).

2.2. Input/output (I/O) function

As it is shown in Fig. 1, a repeated measure ANOVA indicatedthat the stimulus–response curves in the DG measured as PS

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amplitude (1A) and EPSP slope (1B) before induction of pairedpulse in order to evaluate synaptic potency, had no significantdifference between the groups.

2.3. Paired pulse (PP)

As illustrated in Fig. 2, the effects of diabetes and exercise onpaired pulse indices in dentate gyrus were determined. ArepeatedmeasureANOVArevealed thatpairedpulse facilitationwas increased, asmeasured by the population spike ratio, at allinter-stimulus intervals in the diabetic-rest group compared tothe control–rest group (Figs. 2A, C). However, these increases

Fig. 2 – The effect of diabetes and exercise on recurrentinhibition/facilitation in dentate gyrus of the hippocampus atA: the population spike amplitude ratio, (percentage of meanPS2/PS1±SEM), and B: EPSP slope ratio (percentage of meanEPSP2/EPSP1±SEM) (B). C: single traces recorded atinter-stimulus interval 30 ms were shown in. (*p<0.05 withrespect to the control group. n=6 for each experimentalgroup).

were significant (p<0.05) at inter-stimulus intervals of 15, 20, 30and 50 ms. Although, there is an increase at inter-stimulusintervals of 15, 20 and 30 ms and a decrease at inter-stimulusintervals of 50, 70, 100 and 120 ms in PPF (as measured by thepopulation spike ratio) in the control–exercise group comparedto the control–rest group (Figs. 2A, C), there were no significantdifferences between the groups other than diabetic–rest groupcompared to control–rest group. Paired pulse facilitation wassignificantly greater in the diabetic–rest group compared to thediabetic–exercise group at inter-stimulus intervals of 10, 15, 20,30 (p<0.01) and 50 ms (p<0.05).

Paired pulse facilitation induced in the EPSP slope ratiowere significant at inter-stimulus intervals of 15, 20 and 30ms(p<0.05) in the diabetic–rest group compared to the control–rest group; and at inter-stimulus intervals of 15, 20 and 30 ms(p<0.05) in the diabetic–rest group compared to the diabetic–exercise group (Figs. 2B, C).

3. Discussion

In this study, we report that diabetes impairs paired pulsefacilitation in the DG of the hippocampus after the electricalstimulation of the perforant pathway, and, that regular tread-mill running prevents PPF impairments in diabetic rats, whilehaving no significant effect in healthy rats.

Our present results demonstrated that diabetes may sig-nificantly impair pre-synaptic components of synaptic plasti-city in the DG. This conclusion is supported by increased pairedpulse indices. These results are consistentwith previous reportsthat have shown that diabetes impairs the release of neuro-transmitters in various parts of CNS, especially in hippocampus(Bitar et al., 1985; Chu et al., 1986; Lackovic et al., 1990; Leunget al., 2006; Uda et al., 2006). Biessels et al. (1996) did not observeany pre-synaptic changes in the CA1 area of hippocampus.However, it should be considered that there are differences inGABAergic modulation in CA1 and DG synapses in thehippocampus (Song et al., 2001; Suzuki and Okada, 2007).

In the central nervous system, GABAergic neurons regulateneuronal excitability, post-synaptic actionpotential firing, anddendritic and synaptic integration (Jensen and Mody, 2001;Miles et al., 1996). In the hippocampus, recurrent inhibition,mediated by GABAergic interneurons, regulates excitability ofgranular and pyramidal cells by both feed-back and feed-forwardmechanisms (Steffensen and Henriksen, 1991). Recur-rent inhibition in the hippocampus can be assessed byemploying paired pulse stimulation. Although residual Ca2+

in the nerve terminals after the first stimulus facilitatesprobability of neurotransmitter release (Carter et al., 2002;Katz and Miledi, 1968), resulting in PPF; at inter-stimulusintervals of 10–40 ms, recurrent inhibition caused by hippo-campal interneurons produces a decline in the EPSP indices ofthe second wave relative to the first. However, at inter-stimulus intervals of 50–150 ms, there is facilitation and anincrease in EPSP indices of the secondwave relative to the first.Inhibition is due to activation of post-synaptic GABAA recep-tors (Ceri et al., 1991; Lashgari et al., 2007; Wang et al., 2007).The PPF at inter-stimulus intervals of 15, 20 and 30 ms in ourstudy (Fig. 2) demonstrates that recurrent inhibition is im-paired in diabetes. This finding is consistent with studies that

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have been shown that diabetes alters GABA homeostasis andchanges cognitive responses (Gomez et al., 2003). It has beendemonstrated that extracellular GABA levels and glutamatedecarboxylase (GAD) and GABA transaminase (GABA-T) activ-ity in the brain are decreased during streptozotocin-induceddiabetes (Guyot et al., 2001; Honda et al., 1998). Reduction ofGABA release or blockade of GABAergic transmission couldcontribute to unblocking of the release of excitatory aminoacids (Levi, 1984) and cause a post-synaptic depolarization ofneurons (Abel and McCandless, 1992), resulting in PPF (Lash-gari et al., 2007).

As a secondary observation, our study demonstrates thattreadmill running alone does not significantly change the pre-synaptic component of synaptic plasticity in the DG (Fig. 2) andthis is consistent with previous reports (Dietrich et al., 2005;Farmer et al., 2004). However, others have observed that physicalexercise can influence release and metabolism of neurotrans-mitters (Leung et al., 2006; Meeusen and De Meirleir, 1995;Molteni et al., 2002). These differences are probably related todifferences in the exercise protocol (voluntary versus forced), incombination with the intensity (in forced exercise models) anddurationof exercise exposure (Cotmanet al., 2007). In thepresentstudy, the exercise protocol was forced treadmill running whichis associated with certain level of stress (Arida et al., 2004). Thestress causedby the forcedparadigmmight impact thebeneficialeffects of exercise, such that results might be different fromthose using voluntary running. However, extensive researchdemonstrates that treadmill exercisehasneuroprotectiveeffects(Sim et al., 2004). These effects have been best defined withrespect to reducing brain injury, and to delaying the onset of anddecline in several neurodegenerativediseases (Colino et al., 2002;Saviane et al., 2002; Tsodyks and Markram, 1997).

Our results show that there is a significant differencebetween diabetes–rest and diabetes–exercise groups in recur-rent inhibition in the DG (Fig. 2), and that exercise prevents thedestructive changes in interneurons in diabetes. A possibleexplanation for the results reported here is that increases inGABA as a result of the running are due to changes in theneuron–astrocyte network (Araque et al., 1999; Del Arco et al.,2003). Indeed, other studies have shown that cell proliferationin the DG is suppressed during diabetic conditions (Kim et al.,2002) and that these changes may be experimentally modifiedby treadmill running (Uda et al., 2006). However, severalstudies have shown that exercise in brain injured subjectspromoted functional recovery without any effect on tissuemass (Johansson and Ohlsson, 1996; Ohlsson and Johansson,1995). During running activity, excitatory amino acid carrier 1(EAAC1), a glutamate transporter, was up-regulated in the rathippocampus (Molteni et al., 2002). EAAC1 is one of the fiveisoforms of the glutamate transporter responsible for theremoval of extracellular glutamate from the synaptic cleft(Gadea and Lopez-Colome, 2001). Because extracellular levelsof glutamate are high in the DM (Guyot et al., 2001), the in-creasedexpressionof EAAC1may represent a protectivemech-anism activated by exercise in diabetes. In addition, physicalexercise is known to down-regulate the expression of somesubtype of glutamate receptors (Guezennec et al., 1998). Al-though exercise seems to have both preventative and ther-apeutic effects on the learning defects in diabetes, theunderlying mechanisms are poorly understood and most

studies have focused on the hippocampus, where exercise-induces neurogenesis (Kim et al., 2002, 2003; Uda et al., 2006).

In the present results, we have shown that the pre-synapticcomponent of synaptic plasticity in the DG is affected underdiabetic conditions and that treadmill running had protectiveeffects on synaptic transmission in the DG against the destruc-tive effects of diabetes. However, treadmill running dose notshow any such effects in normal subjects. The data correspondto the possibility that alterations in transmission may account,in part, for learning and memory deficits in diabetes, and thattreadmill running is helpful in alleviating the neural complica-tions of diabetes mellitus.

4. Experimental procedures

4.1. Subjects

Male Wistar rats (starting weight 150–180 g) were housed fourper cage andmaintained on a 12h–12h light–dark cycle in an airconditioned constant temperature (23±1 °C) room, with foodand water made available ad libitum. The Ethic Committee forAnimal Experiments at Tabriz University approved the studyand all experiments were conducted in accordance with theEuropean Communities Council Directive of 24 November 1986(86/609/EEC).

Animals were divided into four groups: the control–restgroup, the control–exercise group, the diabetes–rest group andthe diabetes–exercise group (n=6 for each experimental group).To induce diabetes, streptozotocin (Sigma Chemical Co, St Luis,MO, USA) was dissolved in saline and a single intra-peritonealinjection of STZ (60mg/kg)was given to eachanimal (Tuzcu andBaydas, 2006). To confirm the induction of diabetes, 3 days afterthe STZ injection, blood glucose levels were determined fromblood samples obtained by tail prick using a strip-operatedblood glucose monitoring system (Healthy Living, Samsung,Korea), and the animals with blood glucose levels higher than300 mg/dl were selected.

4.2. Treadmill running

Rats in the control–exercise and diabetes–exercise groups weresubjected to run at speed of 17m/min for 40min daily (7 days aweek), for 12 weeks at 0° of inclination. To familiarize, animalswith the experimental set up, animalswere left on the treadmillfor 40 min once a day for 2 consecutive days without operationof the treadmill, then from the third day onward, the treadmillwas switched on and the speed increased from 5 to 17 m/minand the duration increased from 10 to 40min over the course of5 days. Electric shocks were used sparingly to motivate theanimal to run. Fromweek 2 onwards, after warm-up, speed andduration were kept constant at 17 m/min and 40 min per run.The non-runners groups were put on the treadmill withoutrunning for the same duration as the runners.

4.3. Hippocampal electrophysiology

4.3.1. Surgical procedureAfter 12 weeks of diabetes induction and exercise, rats wereanesthetized with urethane (1.8 g/kg, i.p.) and their heads were

Fig. 3 – Schematic diagram of population spike and EPSPslope analysis. The population spike (PS) parametersanalyzed as: [(VB−VC)+(VD−VC)] /2, and EPSP slopeanalyzed as: CD slope.

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fixed in a stereotaxic head-holder. A heating pad was used tomaintain body temperature at 36.5±0.5 °C. The skullwas exposedand two small holes were drilled at the positions of thestimulating and recording electrodes. The exposed cortex waskept moist by the application of paraffin oil. A concentric bipolarstimulatingelectrode (stainlesssteel, 0.125mmdiameter,Advent,UK) was placed in the perforant pathway (AP=−8.1 mm;ML=4.3 mm; DV=3.2 mm), and a stainless steel recordingelectrode was lowered into the DG until the maximal responsewas observed (AP=−3.8 mm; ML=2.3 mm; DV=2.7–3.2 mm)(Paxinos andWatson, 2005). In order tominimize trauma to braintissue, the electrodes were lowered very slowly (2 mm/min).

4.3.2. Electrophysiological recordings and PP inductionExtracellular evoked responses were obtained from the dentategranule cell population following stimulation of the perforantpathway. Extracellular field potentials were amplified (×100)and filtered (1Hz to 3KHzbandpass) using aDAM80differentialamplifier (WPI, USA). Signals were passed through an analogueto digital interface (Powerlab/4SP, ADInstruments, Australia) toa computer and data were analyzed using custom software.Stimulation intensity was adjusted to evoke about 40% of themaximal response of the population spike (PS) and fieldexcitatory post-synaptic potential (fEPSP) (Lashgari et al., 2007).As shown in Fig. 3, the PS amplitude was measured as [thedifference in voltage between the peak of the first positive waveand the peak of the first negative deflection (VB−VC)+thedifference in voltage between the peak of the second positivewave and the peak of the first negative deflection (VD−VC)]/2,and the fEPSP slope was measured as the maximum slope (10–90%) between the peak of the first negative deflection and thesecond positive wave (CD slope). PS and EPSPs were evoked intheDGregionusing0.1Hzstimulation.Baseline recordingsweretaken at least 30 min prior to each experiment.

Paired pulse depression/facilitation was measured by deli-vering five consecutive evoked responses of paired pulses at 10,15, 20, 30, 50, 70, 100 and 120 ms inter-stimulus intervals to theperforant pathway at a frequency of 0.1 Hz (10 s interval). Thepopulation spike amplitude ratio [second population spikeamplitude/first population spike amplitude at percent; PS2/PS1%, paired pulse index (PPI)] and the fEPSP slope ratio [secondfEPSP slope/first fEPSP slope at percent; fEPSP2/fEPSP1%] weremeasured at different inter-stimulus intervals and compared tothe control group (Lashgari et al., 2007).

4.3.3. Input/output functionsStimulus–response or input/output (I/O) functions were ac-quired by systematic variation of the stimulus current (100–1000 µA) in order to evaluate synaptic potency before inductionof pairedpulse. Stimuluspulsesweredeliveredat 0.1Hzand fiveresponses at each current level were averaged.

4.3.4. Physiological and histological verificationsImplantation of electrodes in the correct location was deter-mined by physiological and histological verification. For phy-siological verification, first stimulating electrodewas implantedin theperforantpath, then,placementof the recording electrodein DG, a second electrode was lowered very slowly from skulllevel until DV=−2.7. At this depth, therewas a low voltage EPSPwith deep PS. Then the recording electrodewas lowered to DV=−3.2, where is a high voltage EPSP with small PS and reversedthan previous EPSP that is characteristic of the DG. Forhistological verifications at the end of each experiment, ratswere perfused transcardially with a 10% formalin solution andthe brain removed and fixed in 10% formalin for at least 3 days.Subsequently, transverse sections through the brain were cutusing a freezing microtome to locate electrode tracts. Thesection were examined under a microscope and compared tothe rat brain atlas (Paxinos and Watson, 2005).

4.4. Statistical analysis

Results are given as mean±S.E.M. Data were analyzedstatistically using repeated measure of ANOVA (three-factormixed ANOVA). Transforming data (square root) were con-sidered when differences between the variances of the groupswere significant. Probabilities less than 0.05 were consideredsignificantly different.

Acknowledgments

This research was supported by the Drug Applied ResearchCenter of Tabriz University of Medical Sciences, Tabriz, Iran.Also, the authors wish to thank Dr S.M. Noorbakhsh for helpwith the electrophysiological set up and Dr. Crista Barberini atColumbia University for revising as a native English speakerand for his excellent comments.

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