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Effects of voluntary exercise on hippocampal long-term potentiation in morphine-dependent rats
Neuroscience 256 (2014) 83–90
EFFECTS OF VOLUNTARY EXERCISE ON HIPPOCAMPAL LONG-TERM POTENTIATION IN MORPHINE-DEPENDENT RATS H. MILADI-GORJI, a,b A. RASHIDY-POUR, b* Y. FATHOLLAHI, a S. SEMNANIAN a AND M. JADIDI b,c a
morphine may influence cognitive functions in opiate addicts. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
b Laboratory of Learning and Memory, Research Center and Department of Physiology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran
Key words: voluntary exercise, morphine exposure, long-term potentiation, hippocampus.
Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
c Department of Medical Physics, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran
INTRODUCTION Abstract—This study was designed to examine the effect of voluntary exercise on hippocampal long-term potentiation (LTP) in morphine-dependent rats. The rats were randomly distributed into the saline–sedentary (Sal/Sed), the dependent–sedentary, the saline–exercise (Sal/Exc), and the dependent–exercise (D/Exc) groups. The Sal/Exc and the D/Exc groups were allowed to freely exercise in a running wheel for 10 days. The Sal/Sed and the morphine–sedentary groups were kept sedentary for the same extent of time. Morphine (10 mg/kg) was injected bi-daily (12 h interval) during 10 days of voluntary exercise. On day 11, 2 h after the morphine injection, the in vivo LTP in the dentate gyrus of the hippocampus was examined. The theta frequency primed bursts were delivered to the perforant path for induction of LTP. Population spike (PS) amplitude and the field excitatory post-synaptic potentials (fEPSP) slope were measured as indices of increase in synaptic efficacy. Chronic morphine increased the mean basal EPSP, and augmented PS–LTP. Exercise significantly increased the mean baseline EPSP and PS responses, and augmented PS–LTP in both saline and morphine-treated groups. Moreover, the increase of PS–LTP in the morphine–exercise group was greater (22.5%), but not statistically significant, than that of the Sal/Exc group. These results may imply an additive effect between exercise and morphine on mechanisms of synaptic plasticity. Such an interaction between exercise and chronic
Hippocampal long-term potentiation (LTP) is a form of synaptic plasticity that proposed as a cellular substrate of learning and memory (Bliss and Collingridge, 1993). Previous studies, using in vivo and in vitro methods, have shown that chronic morphine can reduce LTP in rat hippocampal synapses (Pu et al., 2002; Salmanzadeh et al., 2003a; Bao et al., 2007). In contrast, other studies have shown that chronic morphine exposure augments in vivo LTP in Schaffer collateral–CA1 synapses (Mansouri et al., 1997, 1999), and in vivo LTP in the lateral perforant bath (PP)granule cell synapses of the rat dentate gyrus (DG) (Ito et al., 2001; Lu et al., 2010b). The conflicts between these studies could be due to the fact that, different experimental protocols such as the pattern of stimulation, the time points of synaptic responses recording after the morphine injection, and the site of stimulation might be involved in the modulation of synaptic responses. For example, Pu et al. (2002) showed that chronic exposure of rats to morphine markedly reduced the capacity of hippocampal CA1 LTP during the period of drug withdrawal (9–12 h after the termination of chronic treatment), while Mansouri et al. (1997, 1999) demonstrated the augmented LTP in the Schalleral-CA1 synapses of the hippocampal slices taken from dependent, but not withdrawn rats. Many studies have assumed that abused drugs can hijack synaptic machinery that are dedicated to plastic changes in the excitability of principal hippocampus circuits (Robbins and Everitt, 1999; Wolf, 2002; Bao et al., 2007; Kauer and Malenka, 2007), and may induce maladaptive plasticity in this structure (Eisch et al., 2000). Such maladaptive plasticity in the hippocampus and other brain structures may underline learning and memory impairment induced by chronic morphine (Miladi-Gorji et al., 2008, 2011; Lu et al., 2010a). Reversing or preventing these drug-induced synaptic modifications may prove beneficial in the treatment of relapse and other related disorders (Wolf, 2002; Lu et al., 2010a).
*Corresponding author. Address: Laboratory of Learning and Memory, Research Center and Department of Physiology, Semnan University of Medical Sciences, 15131-38111 Semnan, Iran. Tel: +98-09121140221. E-mail addresses: [email protected] (A. Rashidy-Pour), [email protected] (Y. Fathollahi). Co-corresponding author. Address: Department of Physiology, School of Medical Sciences, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran. Abbreviations: ANOVA, analysis of variance; AP, Anterior-Posterior; BDNF, brain-derived neurotrophic factor; D/Exc, dependent–exercise; DG, dentate gyrus; D/Sed, dependent–sedentary; DV, Dorssal-ventral; fEPSP, field excitatory post-synaptic potentials; I/O, input/output; LTP, long-term potentiation; ML, Medial-lateral; NMDA, N-methylD-aspartate; PBs, primed bursts; PP, perforant bath; PS, population spike; Sal/Exc, saline–exercise; Sal/Sed, saline–sedentary; TrkB, tyrosine kinase B.
0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.09.056 83
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Recent studies have shown that physical activity can have a wide variety of beneficial effects in humans and experimental animals (Cotman and Engesser-Cesar, 2002; Kramer et al., 2006). It is increasingly clear that physical exercise maintains brain health and the synaptic plasticity (Alaei et al., 2006; Pietropaolo et al., 2008). Voluntary or forced exercises delay the onset of Alzheimer’s disease in humans (Cotman and Berchtold, 2007) and promote neuro-genesis in the mice DG (van Praag et al., 1999b). Voluntary exercise can increase performance on spatial learning and memory tasks that need normal hippocampal functioning (van Praag et al., 1999a; Vaynman et al., 2004; Van der Borght et al., 2007) and it can also alter LTP in the hippocampus (Farmer et al., 2004). We have recently demonstrated that voluntary exercise could ameliorate the spatial memory deficits in morphine-dependent rats through brain-derived neurotrophic factor (BDNF) receptors (tyrosine kinase B (TrkB)) (Miladi-Gorji et al., 2011). BDNF infusion has been shown to enhance LTP in the rat DG (Korte et al., 1998; Ying et al., 2002), while transgenic mice have been used to demonstrate that BDNF plays an important functional role in the expression of LTP in the hippocampus (Korte et al., 1995). BDNF levels are elevated during voluntary exercise which plays an important role in mediating exercise-induced enhancement of learning and memory as well as LTP (Vaynman et al., 2004). One of the aims of the present study was to investigate whether chronic exposure to morphine could influence synaptic transmission and LTP in the DG of the hippocampus. Another aim was to examine whether voluntary exercise could alter hippocampal synaptic plasticity and LTP in morphine-dependent rats. Such studies can be helpful for understanding the neurophysiological substrate of cognitive deficits seen in opiate addicts.
EXPERIMENTAL PROCEDURES Animals Adult male Wistar rats (220 ± 10 g) were individually housed in cages (50 26 25 cm) in a 12-h light/dark cycle at 22–24 °C, with food and water ad libitum. The experimental protocol was approved by the Ethical Review Board of Tarbiat Modares University (Iran). All the experimental procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Additionally, care was taken to reduce number of rats in each experiment as far as possible. Induction of morphine dependence Morphine sulfate (Temad Company, Tehran, Iran) was dissolved in a physiological saline. On the basis of previous studies (Pu et al., 2002; Miladi-Gorji et al., 2011), morphine dependence was induced by chronic intermittent subcutaneous injections of morphine (10 mg/ kg, 1 ml/kg) twice daily (07:00 and 19:00) for 10 days in
the presence or absence of voluntary exercise (see below).The control rats were treated similarly, with injections of saline (1 ml/kg) replacing morphine. Voluntary exercise paradigm Each of the exercising rats was given all day/night access to a cage equipped with a running wheel (diameter = 34.5 cm, width = 9.5 cm) (Novidan.Tab, Iran) that was freely rotated against a resistance of 100 g. Each wheel was equipped with a magnetic switch that was connected to a separate counter, which was located outside the animal house and the number of revolutions per hour was monitored. The number of revolutions for each wheel was recorded every day at 6 a.m. The sedentary rats were confined to similar cages with no access to a wheel. The exercising groups were exposed to exercise during the development of dependence on morphine, which took 10 days before the start of the electrophysiology experiments (Akhavan et al., 2008; Miladi-Gorji et al., 2011). Exercising and sedentary rats were remained single housed throughout the entire experiment. Experimental groups Rats (n = 7–8 rats per group) were divided into the saline–exercise (Sal/Exc), the saline–sedentary (Sal/Sed), the dependent–exercise (D/Exc), and the dependent– sedentary (D/Sed) groups. Electrophysiology Surgery. The animals were anesthetized with urethane (1.5 g/kg, i.p.) 2 h after the last injection of morphine on day 11. For electrophysiological recording, a bipolar Teflon-coated stainless steel (125 lm diameter) in the medial PP (coordinates: AnteriorPosterior (AP), 8.1 mm; Medial-lateral (ML), 4.1 mm; Dorssal-ventral (DV), 3.3 mm, from skull surface) and a recording electrode (a bipolar Teflon-coated stainless steel) in the DG granule cell layer (coordinates: AP, 3.8; ML, 2; DV, 2.7–3.2 from skull surface) were implanted (Pu et al., 2002; Abrari et al., 2009). Stimulation and recording. Fifteen minutes after electrode placement, constant current rectangular stimulus pulses (200 ls, 0.1 Hz) were delivered during a 20 min period after electrode placement (stabilization period). To achieve this, it was at times necessary to reposition the stimulating and/or recording electrodes until the highest potential could be obtained. When the variation in the population spike (PS) amplitude was less than ±10% for 20 min, the baseline recording was considered stable. An input/output (I/O) profile was established by increasing the stimulus intensity and measuring the PS amplitude. The stimulus intensity that evoked a PS or field excitatory post-synaptic potentials (fEPSP) of 50–60% of the baseline maximum response was chosen for subsequent train stimuli. The evoked response was
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amplified, filtered (bandpass: 1 Hz–3 kHz) and sampled at a rate of 20 kHz and stored on the hard disk. All recording and stimulation was performed using an on-line computerized oscilloscope–stimulator and data analysis interface system by Data Acquisition D3111 set up and NeuroTrace Software (www.ScienceBeam.com). After baseline synaptic responses had been stable for at least 20 min, the theta pattern primed bursts (PBs) tetanic stimulation was used for LTP induction which was consisted of eight PBs at intervals of 10 s. Each PB consisted of a single priming pulse followed 170 ms later by a burst of 10 pulses delivered at 100 Hz. ThetaPB stimulation is an effective protocol for inducing robust and persistent LTP, which is based on the physiology of hippocampus (Diamond et al., 1988; Larson and Lynch, 1989) After tetanus delivery, responses were recorded 5, 15, 30, 45, 60, 75, 90, 105, and 120 min after train stimuli. The magnitude of potentiation was evaluated as the percentage change in the PS amplitude and the slope of fEPSP relative to the pre-tetanus test value. The acceptable level of a defined LTP was more than a 15% increase in the PS amplitude and/or EPSP slope (Pu et al., 2002; Berchtold et al., 2005). Statistical analysis The data were expressed as the mean ± standard error of the mean (S.E.M.). Data analysis consisted of one or two-way analysis of variance (ANOVA). Post-hoc analyses consisted of the Tukey’s test. Student’s t-test was used for comparison of two-independent groups. The general linear model for repeated measures was used for group comparisons of overall differences in LTP between groups. Moreover, the analyses were done on the average of the whole 120-min poststimulation period. The statistical differences were considered to be significant at P < 0.05.
RESULTS Running distances One-way ANOVA with repeated measures (days) for the average distance run (m) at 10 days of voluntary exercise revealed the absence of a significant effect of groups (F1,12 = 0.001, P = 0.97), a significant effect of days (F9,108 = 2.24, P = 0.025) and no significant interaction between both factors (F9,108 = 0.83, P = 0.497) (Fig. 1). In general, the running distance is increased significantly in both groups as exercise days progressed. Meanwhile, the average distance covered during running (m) after 10 days of voluntary exercise did not differ significantly between the exercising groups: the Sal/Exc group (10285.59 ± 387) and the D/Exc group (9612.283 ± 231) (t12 = 0.033, P = 0.974) (Fig. 1). This indicates that rats exposed chronically to morphine showed equivalent levels of activity compared with control rats. The mean EPSP slope and PS amplitude before LTP induction Baseline recordings were obtained in the DG by electrical stimulation of the medial PP. As shown in Fig. 2, rats that
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Fig. 1. The average of running distance (expressed in meter per day) at 10 days of voluntary exercise in the exercising groups. The data are expressed as the mean ± S.E.M.
had completed the exercise protocol displayed an enhancement in the EPSP slope and PS amplitude when compared with sedentary controls. A two-way ANOVA on the EPSP slope (Fig. 2A) showed significant effects of exercise (F1,22 = 16.51, P = 0.001) and of morphine treatment (F1,22 = 5.8, P = 0.025), but no interaction between both factors (F1,22 = 0.2, P = 0.65). Analysis of the PS amplitude (Fig. 2B) also showed significant effects of exercise (F1,22 = 17.22, P = 0.0001), no significant effects of morphine (F1,22 = 2.0.2, P = 0.65) and no interaction between both factors (F1,22 = 0.14, P = 0.71). The between-group comparisons indicated that both the EPSP slope and PS amplitude was significantly higher in the Sal/ Exc group than the Sal/Sed group (both, P = 0.01). The EPSP slope, but not PS amplitude was significantly higher in the D/Sed group than the Sal/Sed group (P = 0.043). The EPSP slope and PS amplitude of the D/Exc group was significantly higher than that of the D/Sed group (P = 0.004; P = 0.005, respectively). The EPSP slope, but not PS amplitude of the D/Sed group was significantly higher than that of the Sal/Sed group (P = 0.04). Finally, the EPSP slope, but not PS amplitude was significantly higher in the D/Exc than Sal/Exc (P = 0. 038).
I/O profile The medial PP was stimulated at 0.1 Hz with single shocks that each evoked single PS at the DG. There was no frequency potentiation at stimulation rate of 0.1 Hz. The amplitude of evoked field potentials was measured at the five different stimulation intensities and I/O curves were constructed for different groups. The PS amplitude of the DG for different groups was increased in proportion to the increase in stimulus intensity. The baseline data for all groups were shown in Table 1. The mean stimulus intensity (lA) that was required to evoke the test response and the maximum response did not show a significant difference between groups. Dependence to morphine caused a nonsignificant increase in the mean stimulus intensity (lA) that was required to evoke the maximum response than the Sal/Sed group. A significant increase in the test response and maximum response also was seen as a result of exercising in control rats.
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Fig. 2. Effects of voluntary exercise on baseline synaptic responsiveness in morphine-dependent rats. This figure shows data (mean ± S.E.M) of the EPSP slope (A) and PS amplitude (B) during 20 min of stable recordings before LTP induction. The exercise protocol displayed an enhancement in the EPSP slope and PS amplitude when compared with sedentary controls. Chronic morphine enhanced the EPSP slope. (A) ⁄P = 0.016 and ⁄⁄ P = 0.004 represent significant differences between the Sal/Sed and Sal/Exc, and the D/Sed and D/Exc groups, respectively. ^P = 0.043 represents significant difference between the Sal/Sed and D/Sed. (B) ⁄P = 0.016 and ⁄⁄P = 0.005 represent the significant differences between the Sal/Sed and Sal/Exc and the D/Sed and D/Exc, respectively.
Table 1. The field responses recorded in the dentate gyrus evoked upon test stimulus pulses (0.1 Hz) in the medial perforant pathway (lA)
Group
Itest
Sal/Sed Sal/Exc D/Sed D/Exc
2416 ± 247 2000 ± 218 3000 ± 144 2214 ± 150
stimulus
PSAtest
stimulus
2.66 ± 0.44 8.20 ± 1.85* 4.22 ± 0.56 9.07 ± 1.92
(mV)
Imax (lA)
PSAmax (mV)
2375 ± 257 2607 ± 210 4000 ± 144 2964 ± 101
4.50 ± 0.46 13.33 ± 1.26* 8.27 ± 0.55 16.59 ± 2.23
Imax is the stimulus intensity evoking the maximum response (population spike amplitude, PSA max). Itest stimulus is the stimulus intensity that yielded 50–60% of the PSA max (PSAtest stimulus) was used for LTP induction. Morphine in the D/Sed group caused a non-significant increase in the mean stimulus intensity (lA) that was required to evoke the maximum response than the Sal/Sed group. * Sal/Sed and Sal/Exc (P < 0.05).
Effect of exercise on LTP Fig. 3 shows a typical evoked response during baseline recording and after PBs among the experimental groups. A two-way ANOVA on the mean EPSP slope (Fig. 4A) revealed the absence of significant effects of exercise (F1,22 = 0.39, P = 0.54) and of morphine treatment (F1,22 = 0.72, P = 0.4), and no significant interaction between both factors (F1,22 = 0.54, P = 0.47). A two-way ANOVA on the overall comparison of the PS amplitude during 120-min recording (Fig. 4B) indicated significant effects of exercise (F1,22 = 19.4, P = 0.0001) and of morphine treatment (F1,22 = 8.16, P = 0.009) and no significant interaction between both factors (F1,22 = 0.032, P = 0.86). The between-group comparisons indicated that the PS amplitude of the Sal/Exc and D/Sed groups was significantly higher than the Sal/Sed group (P = 0.018; P = 0.05, respectively). The difference of PS amplitude between the Sal/Exc and D/Exc groups was not significant.
DISCUSSION Morphine dependence enhances hippocampal LTP We found that chronic morphine administration increased the EPSP basal responses and augmented PS–LTP, confirming previous studies showing the positive effects of chronic morphine on LTP. For example, using
hippocampal slices from morphine-dependent rats, it has been shown that the LTP induced by the PBs tetanic stimulation is augmented in the Schaffer collateral–CA1 pyramidal cell synapses (Mansouri et al., 1997, 1999). Another in vitro study has shown that chronic infusions of morphine significantly augmented LTP in the lateral PP–DG synapses (Ito et al., 2001). A more recent work has also demonstrated that the induction of LTP in the spinal dorsal horn by C-fiber stimulation is strongly facilitated after 7 days of treatment with opioid, and that both the induction and consolidation phases of the potentiation are facilitated (Haugan et al., 2008). Our findings are very similar with previous studies showing that a single injection (10 mg/kg, 30 min before LTP induction) of morphine at 12 h after the termination of chronic opiate treatment resulted in the enhancement of LTP in hippocampal CA1 region (Pu et al., 2002; Bao et al., 2007). We found that morphine-dependent rats showed LTP enhancement 2 h after the last morphine injection. This result is unlikely because of spontaneous withdrawal, given that hyper-locomotion induced by morphine lasts longer than 2 h (Handal et al., 2002) and that the halflife of morphine (Handal et al., 2002; Kalvass et al., 2007) requires a longer duration before spontaneous withdrawal precipitates. Indeed, in rats, the behavioral effects of spontaneous withdrawal were observed only 8 h post-morphine (Schulteis et al., 1998). Thus, the enhanced PS–LTP observed in the dependent rats
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Fig. 3. Representative response recorded from the DG of hippocampus during baseline recording and after 5–120 min period post-HFS among the groups. Horizontal scale bar = 5 ms, Vertical scale bar = 5 mV.
could potentially be interpreted as an effect of residual chronic morphine and not a withdrawal effect. Our results suggest that morphine dependence causes changes in hippocampal neuronal circuitry that induces synaptic plasticity, which are manifested during tetanic stimulation as augmented by LTP. Since PSs are regulated by both synaptic excitation and synaptic inhibition and since synaptic inhibition seems to be strongly affected by chronic morphine exposure, it seems that the effects of morphine exposure on PS–LTP are caused by alterations in the local inhibitory circuitry rather than changes in the LTP mechanisms itself (Salmanzadeh et al., 2003b). One possible mechanism for the enhanced LTP in morphine-dependent rats is increased hippocampal BDNF following chronic treatment of morphine. BDNF is a member of the neurotrophin family that is expressed in various brain regions that are involved in spatial learning and memory and in LTP, such as the hippocampus (Ying et al., 2002; Vaynman et al., 2004; Kramer et al., 2006). BDNF acts by binding to the TrkB receptor with a high-affinity. Recently, we have reported
that chronic exposure to morphine enhanced the BDNF protein levels in the hippocampus in the sedentary morphine-dependent rats (Miladi-Gorji et al., 2011). Voluntary exercise during morphine dependence development enhances LTP We have found that voluntary exercise enhances PS–LTP in medial PP to DG synapses. However, we have found no differences in the size of evoked EPSPs in animals that engaged in voluntary exercise, suggesting that new neurons and/or synapses, specifically geared to exhibit synaptic plasticity, may account for the observed enhancement of LTP magnitude. This finding is in agreement with previous studies showing that voluntary exercise enhanced the capacity of the DG to exhibit LTP in mice (van Praag et al., 1999a; Farmer et al., 2004; Vaynman et al., 2004). Although the mechanism that underlies this effect of exercise remains unknown, it has been suggested that stimulation of hippocampal neurogenesis by exercise is accompanied by facilitated induction and maintenance of LTP; precisely in the
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Fig. 4. Effects of voluntary exercise on LTP in morphine-dependent rats. The data are expressed as the mean ± S.E.M. (Insets) Representative superimposed fEPSPs (A-left) and PS amplitude (B-left) at baseline and during 120 min after PBs tetanus delivery. The overall comparison of the mean EPSP slope (A-right) during a 120-min recording revealed no significant effects of groups. But PS–LTP (B-right) was significantly increased in exercising groups relative to control ones. Exercise increased PS–LTP in the DG of hippocampus. Also, the PS amplitude of the D/Sed group was significantly greater than that of the Sal/Sed group. ⁄Sal/Sed and Sal/Exc (P = 0.018), ^D/Sed and D/Exc (P = 0.032), #Sal/Sed and D/Sed (P = 0.05).
same region of the hippocampus in which the new neurons are generated, raising the possibility that newborn granule cells play a role in increased DG–LTP (van Praag et al., 1999b). At the cellular levels, wheel running has been shown to enhance the firing rate of hippocampal cells and synaptic plasticity (Wu et al., 2008). It has been also shown that using the weak theta patterned stimulus (wTPS, bursts of 100 Hz stimuli compared to the bursts of 400 Hz stimuli) induces LTP in the exercising animals than in controls (Farmer et al., 2004), suggesting that voluntary exercise could facilitate the induction of LTP in the DG by lowering the induction threshold for LTP in exercising rats. Given the importance of BDNF in synaptic plasticity and in learning and memory, it has been proposed that the exercise-induced increase in hippocampal BDNF levels might underlie the ability of exercise in selective enhancements of hippocampal functions, such as plasticity and cognitive functions (Yamada et al., 2002; Vaynman et al., 2004; O’Callaghan et al., 2007). In a recent study, we have demonstrated that physical activity ameliorates the spatial memory deficits in morphine-dependent rats through a TrkB-mediated mechanism (Miladi-Gorji et al., 2011). Acute application of BDNF and other neurotrophins regulate neuronal connectivity and synaptic efficacy, probably by
potentiating excitatory synaptic transmission and neuronal excitability in CA3 and the DG or attenuating of synaptic fatigue, enhancing of N-methyl-D-aspartate (NMDA) receptor responses and inhibiting GABAergic transmission or decreasing inhibitory postsynaptic currents (Lu and Chow, 1999). We found that exercise was able to enhance LTP in both dependent and non-dependent groups. Additionally, a grater (22.5%) but not statistically significant increase in PS–LTP in the D/Exc group than the Sal/Exc group was observed (Fig. 4B). This finding may suggest an existence of some degree of additive affects between chronic morphine and exercise on LTP. Recently, we reported similar additive effects of exercise and chronic morphine on BDNF levels in the hippocampus (Miladi-Gorji et al., 2011). These findings suggest that BDNF plays an important role in LTP enhancement following both exercise and chronic morphine treatment. However, further studies using a specific neutralizing antibody against the hippocampal BDNF receptor during the exercise period may reveal the role of BDNF in LTPenhancement in morphinedependent rats. In addition to BDNF, glutamate, the most important neurotransmitter in the brain, plays an important role in LTP as well as hippocampal-dependent learning and
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memory paradigms (Riedel et al., 2003). Physical activity is associated with increased expression of NMDA receptors and glutamate activity (Farmer et al., 2004; Dietrich et al., 2005). On the other hand, chronic morphine alters NMDA-mediated synaptic transmission in the brain (Pourmotabbed et al., 1998; Martin et al., 1999). These findings may indicate a role for glutamate and its NMDA receptors in a mutually potentiating effect between exercise and morphine on mechanisms of synaptic plasticity. Further research is required to clarify this issue.
CONCLUSION In conclusion, our study demonstrates that both exercise and chronic exposure to morphine enhances LTP in the DG of the hippocampus. While the mean increase of PS–LTP in the morphine–exercise group was greater than that of the Sal/Exc group, this difference was not statistically significant. Hence, although there is some possibility of additive affects between exercise and morphine on mechanisms of synaptic plasticity, further studies are required to establish this relationship and to determine how such an effect could influence the cognitive functions in opiate addicts.
CONFLICT OF INTEREST STATEMENT We attest that we have herein disclosed any and all financial or other relationships that could be construed as a conflict of interest and that all sources of financial support for this study have been disclosed. Acknowledgments—This work was supported by grants from Iran National Science Foundation (91001448) and Tarbiat Modares University (Tehan, Iran).
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(Accepted 30 September 2013) (Available online 17 October 2013)
EFFECTS OF VOLUNTARY EXERCISE ON HIPPOCAMPAL LONG-TERM POTENTIATION IN MORPHINE-DEPENDENT RATS H. MILADI-GORJI, a,b A. RASHIDY-POUR, b* Y. FATHOLLAHI, a S. SEMNANIAN a AND M. JADIDI b,c a
morphine may influence cognitive functions in opiate addicts. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
b Laboratory of Learning and Memory, Research Center and Department of Physiology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran
Key words: voluntary exercise, morphine exposure, long-term potentiation, hippocampus.
Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
c Department of Medical Physics, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran
INTRODUCTION Abstract—This study was designed to examine the effect of voluntary exercise on hippocampal long-term potentiation (LTP) in morphine-dependent rats. The rats were randomly distributed into the saline–sedentary (Sal/Sed), the dependent–sedentary, the saline–exercise (Sal/Exc), and the dependent–exercise (D/Exc) groups. The Sal/Exc and the D/Exc groups were allowed to freely exercise in a running wheel for 10 days. The Sal/Sed and the morphine–sedentary groups were kept sedentary for the same extent of time. Morphine (10 mg/kg) was injected bi-daily (12 h interval) during 10 days of voluntary exercise. On day 11, 2 h after the morphine injection, the in vivo LTP in the dentate gyrus of the hippocampus was examined. The theta frequency primed bursts were delivered to the perforant path for induction of LTP. Population spike (PS) amplitude and the field excitatory post-synaptic potentials (fEPSP) slope were measured as indices of increase in synaptic efficacy. Chronic morphine increased the mean basal EPSP, and augmented PS–LTP. Exercise significantly increased the mean baseline EPSP and PS responses, and augmented PS–LTP in both saline and morphine-treated groups. Moreover, the increase of PS–LTP in the morphine–exercise group was greater (22.5%), but not statistically significant, than that of the Sal/Exc group. These results may imply an additive effect between exercise and morphine on mechanisms of synaptic plasticity. Such an interaction between exercise and chronic
Hippocampal long-term potentiation (LTP) is a form of synaptic plasticity that proposed as a cellular substrate of learning and memory (Bliss and Collingridge, 1993). Previous studies, using in vivo and in vitro methods, have shown that chronic morphine can reduce LTP in rat hippocampal synapses (Pu et al., 2002; Salmanzadeh et al., 2003a; Bao et al., 2007). In contrast, other studies have shown that chronic morphine exposure augments in vivo LTP in Schaffer collateral–CA1 synapses (Mansouri et al., 1997, 1999), and in vivo LTP in the lateral perforant bath (PP)granule cell synapses of the rat dentate gyrus (DG) (Ito et al., 2001; Lu et al., 2010b). The conflicts between these studies could be due to the fact that, different experimental protocols such as the pattern of stimulation, the time points of synaptic responses recording after the morphine injection, and the site of stimulation might be involved in the modulation of synaptic responses. For example, Pu et al. (2002) showed that chronic exposure of rats to morphine markedly reduced the capacity of hippocampal CA1 LTP during the period of drug withdrawal (9–12 h after the termination of chronic treatment), while Mansouri et al. (1997, 1999) demonstrated the augmented LTP in the Schalleral-CA1 synapses of the hippocampal slices taken from dependent, but not withdrawn rats. Many studies have assumed that abused drugs can hijack synaptic machinery that are dedicated to plastic changes in the excitability of principal hippocampus circuits (Robbins and Everitt, 1999; Wolf, 2002; Bao et al., 2007; Kauer and Malenka, 2007), and may induce maladaptive plasticity in this structure (Eisch et al., 2000). Such maladaptive plasticity in the hippocampus and other brain structures may underline learning and memory impairment induced by chronic morphine (Miladi-Gorji et al., 2008, 2011; Lu et al., 2010a). Reversing or preventing these drug-induced synaptic modifications may prove beneficial in the treatment of relapse and other related disorders (Wolf, 2002; Lu et al., 2010a).
*Corresponding author. Address: Laboratory of Learning and Memory, Research Center and Department of Physiology, Semnan University of Medical Sciences, 15131-38111 Semnan, Iran. Tel: +98-09121140221. E-mail addresses: [email protected] (A. Rashidy-Pour), [email protected] (Y. Fathollahi). Co-corresponding author. Address: Department of Physiology, School of Medical Sciences, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran. Abbreviations: ANOVA, analysis of variance; AP, Anterior-Posterior; BDNF, brain-derived neurotrophic factor; D/Exc, dependent–exercise; DG, dentate gyrus; D/Sed, dependent–sedentary; DV, Dorssal-ventral; fEPSP, field excitatory post-synaptic potentials; I/O, input/output; LTP, long-term potentiation; ML, Medial-lateral; NMDA, N-methylD-aspartate; PBs, primed bursts; PP, perforant bath; PS, population spike; Sal/Exc, saline–exercise; Sal/Sed, saline–sedentary; TrkB, tyrosine kinase B.
0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.09.056 83
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Recent studies have shown that physical activity can have a wide variety of beneficial effects in humans and experimental animals (Cotman and Engesser-Cesar, 2002; Kramer et al., 2006). It is increasingly clear that physical exercise maintains brain health and the synaptic plasticity (Alaei et al., 2006; Pietropaolo et al., 2008). Voluntary or forced exercises delay the onset of Alzheimer’s disease in humans (Cotman and Berchtold, 2007) and promote neuro-genesis in the mice DG (van Praag et al., 1999b). Voluntary exercise can increase performance on spatial learning and memory tasks that need normal hippocampal functioning (van Praag et al., 1999a; Vaynman et al., 2004; Van der Borght et al., 2007) and it can also alter LTP in the hippocampus (Farmer et al., 2004). We have recently demonstrated that voluntary exercise could ameliorate the spatial memory deficits in morphine-dependent rats through brain-derived neurotrophic factor (BDNF) receptors (tyrosine kinase B (TrkB)) (Miladi-Gorji et al., 2011). BDNF infusion has been shown to enhance LTP in the rat DG (Korte et al., 1998; Ying et al., 2002), while transgenic mice have been used to demonstrate that BDNF plays an important functional role in the expression of LTP in the hippocampus (Korte et al., 1995). BDNF levels are elevated during voluntary exercise which plays an important role in mediating exercise-induced enhancement of learning and memory as well as LTP (Vaynman et al., 2004). One of the aims of the present study was to investigate whether chronic exposure to morphine could influence synaptic transmission and LTP in the DG of the hippocampus. Another aim was to examine whether voluntary exercise could alter hippocampal synaptic plasticity and LTP in morphine-dependent rats. Such studies can be helpful for understanding the neurophysiological substrate of cognitive deficits seen in opiate addicts.
EXPERIMENTAL PROCEDURES Animals Adult male Wistar rats (220 ± 10 g) were individually housed in cages (50 26 25 cm) in a 12-h light/dark cycle at 22–24 °C, with food and water ad libitum. The experimental protocol was approved by the Ethical Review Board of Tarbiat Modares University (Iran). All the experimental procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Additionally, care was taken to reduce number of rats in each experiment as far as possible. Induction of morphine dependence Morphine sulfate (Temad Company, Tehran, Iran) was dissolved in a physiological saline. On the basis of previous studies (Pu et al., 2002; Miladi-Gorji et al., 2011), morphine dependence was induced by chronic intermittent subcutaneous injections of morphine (10 mg/ kg, 1 ml/kg) twice daily (07:00 and 19:00) for 10 days in
the presence or absence of voluntary exercise (see below).The control rats were treated similarly, with injections of saline (1 ml/kg) replacing morphine. Voluntary exercise paradigm Each of the exercising rats was given all day/night access to a cage equipped with a running wheel (diameter = 34.5 cm, width = 9.5 cm) (Novidan.Tab, Iran) that was freely rotated against a resistance of 100 g. Each wheel was equipped with a magnetic switch that was connected to a separate counter, which was located outside the animal house and the number of revolutions per hour was monitored. The number of revolutions for each wheel was recorded every day at 6 a.m. The sedentary rats were confined to similar cages with no access to a wheel. The exercising groups were exposed to exercise during the development of dependence on morphine, which took 10 days before the start of the electrophysiology experiments (Akhavan et al., 2008; Miladi-Gorji et al., 2011). Exercising and sedentary rats were remained single housed throughout the entire experiment. Experimental groups Rats (n = 7–8 rats per group) were divided into the saline–exercise (Sal/Exc), the saline–sedentary (Sal/Sed), the dependent–exercise (D/Exc), and the dependent– sedentary (D/Sed) groups. Electrophysiology Surgery. The animals were anesthetized with urethane (1.5 g/kg, i.p.) 2 h after the last injection of morphine on day 11. For electrophysiological recording, a bipolar Teflon-coated stainless steel (125 lm diameter) in the medial PP (coordinates: AnteriorPosterior (AP), 8.1 mm; Medial-lateral (ML), 4.1 mm; Dorssal-ventral (DV), 3.3 mm, from skull surface) and a recording electrode (a bipolar Teflon-coated stainless steel) in the DG granule cell layer (coordinates: AP, 3.8; ML, 2; DV, 2.7–3.2 from skull surface) were implanted (Pu et al., 2002; Abrari et al., 2009). Stimulation and recording. Fifteen minutes after electrode placement, constant current rectangular stimulus pulses (200 ls, 0.1 Hz) were delivered during a 20 min period after electrode placement (stabilization period). To achieve this, it was at times necessary to reposition the stimulating and/or recording electrodes until the highest potential could be obtained. When the variation in the population spike (PS) amplitude was less than ±10% for 20 min, the baseline recording was considered stable. An input/output (I/O) profile was established by increasing the stimulus intensity and measuring the PS amplitude. The stimulus intensity that evoked a PS or field excitatory post-synaptic potentials (fEPSP) of 50–60% of the baseline maximum response was chosen for subsequent train stimuli. The evoked response was
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amplified, filtered (bandpass: 1 Hz–3 kHz) and sampled at a rate of 20 kHz and stored on the hard disk. All recording and stimulation was performed using an on-line computerized oscilloscope–stimulator and data analysis interface system by Data Acquisition D3111 set up and NeuroTrace Software (www.ScienceBeam.com). After baseline synaptic responses had been stable for at least 20 min, the theta pattern primed bursts (PBs) tetanic stimulation was used for LTP induction which was consisted of eight PBs at intervals of 10 s. Each PB consisted of a single priming pulse followed 170 ms later by a burst of 10 pulses delivered at 100 Hz. ThetaPB stimulation is an effective protocol for inducing robust and persistent LTP, which is based on the physiology of hippocampus (Diamond et al., 1988; Larson and Lynch, 1989) After tetanus delivery, responses were recorded 5, 15, 30, 45, 60, 75, 90, 105, and 120 min after train stimuli. The magnitude of potentiation was evaluated as the percentage change in the PS amplitude and the slope of fEPSP relative to the pre-tetanus test value. The acceptable level of a defined LTP was more than a 15% increase in the PS amplitude and/or EPSP slope (Pu et al., 2002; Berchtold et al., 2005). Statistical analysis The data were expressed as the mean ± standard error of the mean (S.E.M.). Data analysis consisted of one or two-way analysis of variance (ANOVA). Post-hoc analyses consisted of the Tukey’s test. Student’s t-test was used for comparison of two-independent groups. The general linear model for repeated measures was used for group comparisons of overall differences in LTP between groups. Moreover, the analyses were done on the average of the whole 120-min poststimulation period. The statistical differences were considered to be significant at P < 0.05.
RESULTS Running distances One-way ANOVA with repeated measures (days) for the average distance run (m) at 10 days of voluntary exercise revealed the absence of a significant effect of groups (F1,12 = 0.001, P = 0.97), a significant effect of days (F9,108 = 2.24, P = 0.025) and no significant interaction between both factors (F9,108 = 0.83, P = 0.497) (Fig. 1). In general, the running distance is increased significantly in both groups as exercise days progressed. Meanwhile, the average distance covered during running (m) after 10 days of voluntary exercise did not differ significantly between the exercising groups: the Sal/Exc group (10285.59 ± 387) and the D/Exc group (9612.283 ± 231) (t12 = 0.033, P = 0.974) (Fig. 1). This indicates that rats exposed chronically to morphine showed equivalent levels of activity compared with control rats. The mean EPSP slope and PS amplitude before LTP induction Baseline recordings were obtained in the DG by electrical stimulation of the medial PP. As shown in Fig. 2, rats that
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Fig. 1. The average of running distance (expressed in meter per day) at 10 days of voluntary exercise in the exercising groups. The data are expressed as the mean ± S.E.M.
had completed the exercise protocol displayed an enhancement in the EPSP slope and PS amplitude when compared with sedentary controls. A two-way ANOVA on the EPSP slope (Fig. 2A) showed significant effects of exercise (F1,22 = 16.51, P = 0.001) and of morphine treatment (F1,22 = 5.8, P = 0.025), but no interaction between both factors (F1,22 = 0.2, P = 0.65). Analysis of the PS amplitude (Fig. 2B) also showed significant effects of exercise (F1,22 = 17.22, P = 0.0001), no significant effects of morphine (F1,22 = 2.0.2, P = 0.65) and no interaction between both factors (F1,22 = 0.14, P = 0.71). The between-group comparisons indicated that both the EPSP slope and PS amplitude was significantly higher in the Sal/ Exc group than the Sal/Sed group (both, P = 0.01). The EPSP slope, but not PS amplitude was significantly higher in the D/Sed group than the Sal/Sed group (P = 0.043). The EPSP slope and PS amplitude of the D/Exc group was significantly higher than that of the D/Sed group (P = 0.004; P = 0.005, respectively). The EPSP slope, but not PS amplitude of the D/Sed group was significantly higher than that of the Sal/Sed group (P = 0.04). Finally, the EPSP slope, but not PS amplitude was significantly higher in the D/Exc than Sal/Exc (P = 0. 038).
I/O profile The medial PP was stimulated at 0.1 Hz with single shocks that each evoked single PS at the DG. There was no frequency potentiation at stimulation rate of 0.1 Hz. The amplitude of evoked field potentials was measured at the five different stimulation intensities and I/O curves were constructed for different groups. The PS amplitude of the DG for different groups was increased in proportion to the increase in stimulus intensity. The baseline data for all groups were shown in Table 1. The mean stimulus intensity (lA) that was required to evoke the test response and the maximum response did not show a significant difference between groups. Dependence to morphine caused a nonsignificant increase in the mean stimulus intensity (lA) that was required to evoke the maximum response than the Sal/Sed group. A significant increase in the test response and maximum response also was seen as a result of exercising in control rats.
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Fig. 2. Effects of voluntary exercise on baseline synaptic responsiveness in morphine-dependent rats. This figure shows data (mean ± S.E.M) of the EPSP slope (A) and PS amplitude (B) during 20 min of stable recordings before LTP induction. The exercise protocol displayed an enhancement in the EPSP slope and PS amplitude when compared with sedentary controls. Chronic morphine enhanced the EPSP slope. (A) ⁄P = 0.016 and ⁄⁄ P = 0.004 represent significant differences between the Sal/Sed and Sal/Exc, and the D/Sed and D/Exc groups, respectively. ^P = 0.043 represents significant difference between the Sal/Sed and D/Sed. (B) ⁄P = 0.016 and ⁄⁄P = 0.005 represent the significant differences between the Sal/Sed and Sal/Exc and the D/Sed and D/Exc, respectively.
Table 1. The field responses recorded in the dentate gyrus evoked upon test stimulus pulses (0.1 Hz) in the medial perforant pathway (lA)
Group
Itest
Sal/Sed Sal/Exc D/Sed D/Exc
2416 ± 247 2000 ± 218 3000 ± 144 2214 ± 150
stimulus
PSAtest
stimulus
2.66 ± 0.44 8.20 ± 1.85* 4.22 ± 0.56 9.07 ± 1.92
(mV)
Imax (lA)
PSAmax (mV)
2375 ± 257 2607 ± 210 4000 ± 144 2964 ± 101
4.50 ± 0.46 13.33 ± 1.26* 8.27 ± 0.55 16.59 ± 2.23
Imax is the stimulus intensity evoking the maximum response (population spike amplitude, PSA max). Itest stimulus is the stimulus intensity that yielded 50–60% of the PSA max (PSAtest stimulus) was used for LTP induction. Morphine in the D/Sed group caused a non-significant increase in the mean stimulus intensity (lA) that was required to evoke the maximum response than the Sal/Sed group. * Sal/Sed and Sal/Exc (P < 0.05).
Effect of exercise on LTP Fig. 3 shows a typical evoked response during baseline recording and after PBs among the experimental groups. A two-way ANOVA on the mean EPSP slope (Fig. 4A) revealed the absence of significant effects of exercise (F1,22 = 0.39, P = 0.54) and of morphine treatment (F1,22 = 0.72, P = 0.4), and no significant interaction between both factors (F1,22 = 0.54, P = 0.47). A two-way ANOVA on the overall comparison of the PS amplitude during 120-min recording (Fig. 4B) indicated significant effects of exercise (F1,22 = 19.4, P = 0.0001) and of morphine treatment (F1,22 = 8.16, P = 0.009) and no significant interaction between both factors (F1,22 = 0.032, P = 0.86). The between-group comparisons indicated that the PS amplitude of the Sal/Exc and D/Sed groups was significantly higher than the Sal/Sed group (P = 0.018; P = 0.05, respectively). The difference of PS amplitude between the Sal/Exc and D/Exc groups was not significant.
DISCUSSION Morphine dependence enhances hippocampal LTP We found that chronic morphine administration increased the EPSP basal responses and augmented PS–LTP, confirming previous studies showing the positive effects of chronic morphine on LTP. For example, using
hippocampal slices from morphine-dependent rats, it has been shown that the LTP induced by the PBs tetanic stimulation is augmented in the Schaffer collateral–CA1 pyramidal cell synapses (Mansouri et al., 1997, 1999). Another in vitro study has shown that chronic infusions of morphine significantly augmented LTP in the lateral PP–DG synapses (Ito et al., 2001). A more recent work has also demonstrated that the induction of LTP in the spinal dorsal horn by C-fiber stimulation is strongly facilitated after 7 days of treatment with opioid, and that both the induction and consolidation phases of the potentiation are facilitated (Haugan et al., 2008). Our findings are very similar with previous studies showing that a single injection (10 mg/kg, 30 min before LTP induction) of morphine at 12 h after the termination of chronic opiate treatment resulted in the enhancement of LTP in hippocampal CA1 region (Pu et al., 2002; Bao et al., 2007). We found that morphine-dependent rats showed LTP enhancement 2 h after the last morphine injection. This result is unlikely because of spontaneous withdrawal, given that hyper-locomotion induced by morphine lasts longer than 2 h (Handal et al., 2002) and that the halflife of morphine (Handal et al., 2002; Kalvass et al., 2007) requires a longer duration before spontaneous withdrawal precipitates. Indeed, in rats, the behavioral effects of spontaneous withdrawal were observed only 8 h post-morphine (Schulteis et al., 1998). Thus, the enhanced PS–LTP observed in the dependent rats
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Fig. 3. Representative response recorded from the DG of hippocampus during baseline recording and after 5–120 min period post-HFS among the groups. Horizontal scale bar = 5 ms, Vertical scale bar = 5 mV.
could potentially be interpreted as an effect of residual chronic morphine and not a withdrawal effect. Our results suggest that morphine dependence causes changes in hippocampal neuronal circuitry that induces synaptic plasticity, which are manifested during tetanic stimulation as augmented by LTP. Since PSs are regulated by both synaptic excitation and synaptic inhibition and since synaptic inhibition seems to be strongly affected by chronic morphine exposure, it seems that the effects of morphine exposure on PS–LTP are caused by alterations in the local inhibitory circuitry rather than changes in the LTP mechanisms itself (Salmanzadeh et al., 2003b). One possible mechanism for the enhanced LTP in morphine-dependent rats is increased hippocampal BDNF following chronic treatment of morphine. BDNF is a member of the neurotrophin family that is expressed in various brain regions that are involved in spatial learning and memory and in LTP, such as the hippocampus (Ying et al., 2002; Vaynman et al., 2004; Kramer et al., 2006). BDNF acts by binding to the TrkB receptor with a high-affinity. Recently, we have reported
that chronic exposure to morphine enhanced the BDNF protein levels in the hippocampus in the sedentary morphine-dependent rats (Miladi-Gorji et al., 2011). Voluntary exercise during morphine dependence development enhances LTP We have found that voluntary exercise enhances PS–LTP in medial PP to DG synapses. However, we have found no differences in the size of evoked EPSPs in animals that engaged in voluntary exercise, suggesting that new neurons and/or synapses, specifically geared to exhibit synaptic plasticity, may account for the observed enhancement of LTP magnitude. This finding is in agreement with previous studies showing that voluntary exercise enhanced the capacity of the DG to exhibit LTP in mice (van Praag et al., 1999a; Farmer et al., 2004; Vaynman et al., 2004). Although the mechanism that underlies this effect of exercise remains unknown, it has been suggested that stimulation of hippocampal neurogenesis by exercise is accompanied by facilitated induction and maintenance of LTP; precisely in the
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Fig. 4. Effects of voluntary exercise on LTP in morphine-dependent rats. The data are expressed as the mean ± S.E.M. (Insets) Representative superimposed fEPSPs (A-left) and PS amplitude (B-left) at baseline and during 120 min after PBs tetanus delivery. The overall comparison of the mean EPSP slope (A-right) during a 120-min recording revealed no significant effects of groups. But PS–LTP (B-right) was significantly increased in exercising groups relative to control ones. Exercise increased PS–LTP in the DG of hippocampus. Also, the PS amplitude of the D/Sed group was significantly greater than that of the Sal/Sed group. ⁄Sal/Sed and Sal/Exc (P = 0.018), ^D/Sed and D/Exc (P = 0.032), #Sal/Sed and D/Sed (P = 0.05).
same region of the hippocampus in which the new neurons are generated, raising the possibility that newborn granule cells play a role in increased DG–LTP (van Praag et al., 1999b). At the cellular levels, wheel running has been shown to enhance the firing rate of hippocampal cells and synaptic plasticity (Wu et al., 2008). It has been also shown that using the weak theta patterned stimulus (wTPS, bursts of 100 Hz stimuli compared to the bursts of 400 Hz stimuli) induces LTP in the exercising animals than in controls (Farmer et al., 2004), suggesting that voluntary exercise could facilitate the induction of LTP in the DG by lowering the induction threshold for LTP in exercising rats. Given the importance of BDNF in synaptic plasticity and in learning and memory, it has been proposed that the exercise-induced increase in hippocampal BDNF levels might underlie the ability of exercise in selective enhancements of hippocampal functions, such as plasticity and cognitive functions (Yamada et al., 2002; Vaynman et al., 2004; O’Callaghan et al., 2007). In a recent study, we have demonstrated that physical activity ameliorates the spatial memory deficits in morphine-dependent rats through a TrkB-mediated mechanism (Miladi-Gorji et al., 2011). Acute application of BDNF and other neurotrophins regulate neuronal connectivity and synaptic efficacy, probably by
potentiating excitatory synaptic transmission and neuronal excitability in CA3 and the DG or attenuating of synaptic fatigue, enhancing of N-methyl-D-aspartate (NMDA) receptor responses and inhibiting GABAergic transmission or decreasing inhibitory postsynaptic currents (Lu and Chow, 1999). We found that exercise was able to enhance LTP in both dependent and non-dependent groups. Additionally, a grater (22.5%) but not statistically significant increase in PS–LTP in the D/Exc group than the Sal/Exc group was observed (Fig. 4B). This finding may suggest an existence of some degree of additive affects between chronic morphine and exercise on LTP. Recently, we reported similar additive effects of exercise and chronic morphine on BDNF levels in the hippocampus (Miladi-Gorji et al., 2011). These findings suggest that BDNF plays an important role in LTP enhancement following both exercise and chronic morphine treatment. However, further studies using a specific neutralizing antibody against the hippocampal BDNF receptor during the exercise period may reveal the role of BDNF in LTPenhancement in morphinedependent rats. In addition to BDNF, glutamate, the most important neurotransmitter in the brain, plays an important role in LTP as well as hippocampal-dependent learning and
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memory paradigms (Riedel et al., 2003). Physical activity is associated with increased expression of NMDA receptors and glutamate activity (Farmer et al., 2004; Dietrich et al., 2005). On the other hand, chronic morphine alters NMDA-mediated synaptic transmission in the brain (Pourmotabbed et al., 1998; Martin et al., 1999). These findings may indicate a role for glutamate and its NMDA receptors in a mutually potentiating effect between exercise and morphine on mechanisms of synaptic plasticity. Further research is required to clarify this issue.
CONCLUSION In conclusion, our study demonstrates that both exercise and chronic exposure to morphine enhances LTP in the DG of the hippocampus. While the mean increase of PS–LTP in the morphine–exercise group was greater than that of the Sal/Exc group, this difference was not statistically significant. Hence, although there is some possibility of additive affects between exercise and morphine on mechanisms of synaptic plasticity, further studies are required to establish this relationship and to determine how such an effect could influence the cognitive functions in opiate addicts.
CONFLICT OF INTEREST STATEMENT We attest that we have herein disclosed any and all financial or other relationships that could be construed as a conflict of interest and that all sources of financial support for this study have been disclosed. Acknowledgments—This work was supported by grants from Iran National Science Foundation (91001448) and Tarbiat Modares University (Tehan, Iran).
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(Accepted 30 September 2013) (Available online 17 October 2013)