Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons

BRES : 43671

pp:  1211ðcol:fig: : NILÞ

Model7 brain research ] (]]]]) ]]]–]]]

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 Q1 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons Shuzhuo Zhang, Yuelei Jin, Xiaoyan Liu, Lujia Yang, Zhi juan Ge, Hui Wang, Jin Lin, Jianquan Zhengnn Beijing Institute of Pharmacology and Toxicology, 27 Taiping Street, Beijing 100850

art i cle i nfo

ab st rac t

Article history:

Methamphetamine (METH) is a psychostimulant drug. Abuse of METH produces long-term

Accepted 24 July 2014

behavioral changes including behavioral, sensitization, tolerance, and dependence. It induces neurotoxic effects in several areas of the brain via enhancing dopamine (DA) level abnormally,

Keywords:

which may cause a secondary release of glutamate (GLU). However, repeated administration

Methamphetamine

of METH still increases release of GLU even when dopamine content in tissue is significantly

Hippocampal neurons

depleted. It implies that some other mechanisms are likely to involve in METH-induced GLU

Excitatory postsynaptic currents

release. The goal of this study was to observe METH affected glutamatergic synaptic

Whole-cell recording

transmission in rat primary cultured hippocampal neurons and to explore the mechanism of METH modulated GLU release. Using whole-cell patch-clamp recordings, we found that METH (0.1–50.0 μM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). However, METH decreased the frequency of sEPSCs and mEPSCs at high concentration of 100 μM. The postsynaptic NMDA receptor currents and P/Q-type calcium channel were not affected by the use of METH (10,100 μM). METH did not present visible effect on N-type Ca2þ channel current at the concentration lower than 50.0 μM, but it was inhibited by use of METH at a 100 μM. The effect of METH on glutamatergic synaptic transmission was not revered by pretreated with DA receptor antagonist SCH23390. These results suggest that METH directly modulated presynaptic GLU release at a different concentration, while dopaminergic system was not involved in METH modulated release of GLU in rat primary cultured hippocampal neurons. & 2014 Published by Elsevier B.V.

1.

Introduction

Methamphetamine (METH) is a psychostimulant drug. METH abuse throughout the world has markedly increased during

recent years (Lezcano and Bergson, 2002; Chirwa et al., 2005) and represents a significant public health concern (Reiner et al., 2009). Drug abuse, including METH, can cause longlasting changes in neuronal systems (Cadet et al., 2005;

n

Corresponding author. Corresponding author. Fax: þ86 10 68211656. E-mail addresses: [email protected] (J. Li), [email protected] (J. Zheng).

nn

http://dx.doi.org/10.1016/j.brainres.2014.07.040 0006-8993/& 2014 Published by Elsevier B.V.

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671

2

121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

brain research ] (]]]]) ]]]–]]]

Nestler, 2001) and alter synaptic plasticity (Kauer and Malenka, 2007; Thompson et al., 2002). Sub-acute METH administration increased the expression of the plasmalemmal glial-glutamate transporter EAAT3 in the hippocampal (Smith et al., 2007). METH increases extracellular concentrations of DA and glutamate in the striatum (Ernst and Chang, 2008), while the combined effects of DA and GLU releases are thought to produce oxidative stress and glutamate-mediated excitotoxicity to DA nerve terminals. Although there are reports addressing the effects of METH on glutamate synaptic transmission in the striatum and hippocampus (He et al., 2014), the specific nature and pharmacology of METH-evoked changes in hippocampal glutamate-mediated excitatory postsynaptic transmission is not clearly understood. METHinduced increases in corticostriatal GLU release occur via an indirect polysynaptic mechanism that is mediated by D1 DA receptors in the substantia nigra (Mark et al., 2004). Our study shows that extracellular concentrations of GLU remain elevated at 24 h after METH, while METH-induced DA release is no longer present 4 hours after METH (Mark et al., 2007). These results suggest that the METH-facilitated release of GLU may involve not only the dopaminergic systems, but also other unknown mechanisms. The hippocampus is a limbic structure that is involved in learning and memory (Kensinger et al., 2001), and is a target for psychostimulants (Vorel et al., 2001). It represents a critical structure in methamphetamine modulation of the reward circuit (Hori et al., 2010; Keleta and Martinez, 2012; Onaivi et al., 2002; Ricoy and Martinez, 2009). Since hippocampus is implicated in the reinstatement of psychostimulant self-administration, and may even contribute to the rewarding properties of METH, it is important to study and understand the potential effects of METH in this brain region. A recent discovery shows that rats will self-administer METH directly into their hippocampus (Ricoy and Martinez, 2009). Chronic stress resulted in an upregulation of glutamate function and an enhanced glutamate response to METH may have implications for glutamate responsiveness in chronically stressed animals exposed to other challenges or stressors (Raudensky and Yamamoto, 2007). To observe a possible direct effect of METH on GLU release, the cultured hippocampal neurons are used in the present study as the hippocampus has been increasingly recognized as the brain region that is highly vulnerable to METH-induced changes in neuronal signaling and is rich in glutamatergic neurons (Maze et al., 2011). The acute effects of METH on glutamate baseline synaptic transmission were examined by recording EPSCs. EPSCs are recorded by the whole-cell recording technique as the main outcome measurement for glutamate neurotransmitter release in hippocampal neurons. It is found that METH increases the frequency and the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) at the concentration ranging from 0.1 to 50.0 μM. In the presence of tetrodotoxin (TTX), miniature spontaneous excitatory postsynaptic currents (mEPSCs) can be recorded, while METH still enhances the frequency of mEPSCs, revealing METHenhanced release of glutamate. Our study also addressed whether dopamine involved in METH affects the glutamatemediated excitatory postsynaptic transmission in rat primary cultured hippocampal neurons. We found that less than 0.2%

TH-positive cell (dopaminergic neuron) lie in primary cultured hippocampal neurons from postnatal rats 10–12 hours. This is likely due to the fact that cultured neurons from the rats we applied were postnatal within 12 h, and the THþ cells could not survive. This finding is consistent with a previous report (Shimodu, 1992) where it was shown that D1/D5 dopamine receptors are expressed in rat primary cultured hippocampal neurons (Lezcano and Bergson, 2002). We further found that the effect of METH on GLU release is not affected by the use of dopamine D1/D5 receptor antagonist SCH23390. These results suggest that dopamine was most likely not involved in METH-mediated increase in the release of glutamate in rat primary cultured hippocampal neurons. METH did not display remarkable effects on NMDA and AMPA receptor-mediated currents and the voltage-gated calcium channels. Our results suggest that METH may modulate GLU release directly through a given presynaptic mechanism.

2.

Results

2.1. METH affected the frequency and the amplitude of sEPSCs After 10–14 days, the cultured hippocampal neurons were recorded. After the whole-cell recording configuration was established, the sEPSCs could be recorded in most neurons. The sEPSCs were blocked by a combined AMPA receptor antagonist CNQX (20 μM), NMDA receptor antagonist (L-AP5 50 μM), and KA receptor antagonist kynurenic acid (10 μM) (data not shown) in the presence of GABAA antagonist bicuculline, which revealed that the sEPSCs were mediated by neurotransmitter GLU. It was found that METH (10 μM) significantly increased the frequency of sEPSCs (K-S statistic, n ¼10, po0.05), which was reversed by washing out for about 5 min (Fig. 1A and B). The frequency of sEPSCs was 1. 4370.52 Hz for control, 2.7370.32 Hz in the presence of METH, 1.7070.31 Hz after washout (n¼10, po0.05). The amplitude of sEPSCs was enhanced by the use of METH (10 μM) (K-S statistic, n¼ 10, po0.05, Fig. 1C). The median amplitude of sEPSCs was 72.4378.78 pA for control, 89.457 9.90 pA in the application of METH (10 μM), 77.9378.90 pA after washout (n¼10, po0.05). At concentrations 0.1, 1.0, 5.0, 10.0, and 50.0 μM, METH increased the frequency of sEPSCs for 223740%, 318746%, 246762%, 225739%, and 176741%, respectively (n¼10, po0.05). However, METH, at concentration of 100 μM, decreased the frequency of sEPSCs by 87713% (n¼10, po0.05, Fig. 1D). METH enhanced the amplitude of sEPSCs for 126.379.80%, 145.6712.80%, 135.577.15%, 132.876.92%, 1267 4.70% at concentrations of 0.1, 1.0, 5.0, 10.0, and 50.0 μM, respectively (n¼10, po0.05, Fig. 1E). METH decreased the amplitude of sEPSCs by 5977.39% at concentration of 100 μM.

2.2. METH regulated the frequency and amplitude of mEPSCs We further examined the METH-mediated alteration of presynaptic GLU release by observing the effect of METH on mEPSCs, which were action potential-independent spontaneous GLU release from presynaptic terminals. The bath solution included

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671 brain research ] (]]]]) ]]]–]]]

181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

3

Fig. 1 – METH affects the frequency and the amplitude of sEPSCs in rat primary hippocampal neurons. A, Samples of sEPSCs recordings are shown. sEPSCs were recorded before (labeled as Control), after 10 min of 10 μi METH treatment, and after 15 min of washout. B, Cumulative plot of inter-event interval from A is shown. METH shortened the inter-event interval, which caused a significant increase of the frequency of sEPSCs from 1. 4370.51 Hz in control to 2.7370.32 Hz in METH on average (n ¼ 10, po0.05). C, Cumulative distribution of the sEPSC amplitude from the same neuron represented in A is plotted in the absence (Control) and presence of 10 μa METH. The increased sEPSC frequency occurred with the enhancement of the sEPSCs amplitude. On average, the amplitude of sEPSCs increased from control 72.4378.78 pA to 89.4579.90 pA in10 μA METH (n ¼10, po0.05). D, Histogram plot represents the change of the sEPSC frequency in the presence of different concentrations of METH (n indicates significant difference by t-test at the level of po0.05, while nn indicates level of po0.01, n ¼10). E, Histogram plot shows the change of the sEPSCs amplitude in the presence of different concentrations of METH (* indicates significant difference by t-test at the level of po0.05, n ¼10).

bicuculline (100 μM) and TTX (1 μM) to block the GABAA receptor chloride channels and voltage-gated sodium channel, respectively. METH (10 μM and 100 μM) was applied in the following experiments. It was found that the frequency of mEPSCs was increased significantly in the presence of METH (10 μM) (K-S statistic, n¼8, po0.05, Fig. 2A and B), which suggested that METH enhanced presynaptic GLU release. K-S test showed that the amplitude of mEPSCs was not changed significantly by the application of METH (10 μM) (n¼8, p40.05, Fig. 2C). mEPSCs frequency was 1.0170.19 Hz for control, 1.8670.21 Hz in the presence of METH, 1.0270.21 Hz after 5 min of washout (n¼8, po0.05, Fig. 2D). mEPSCs amplitude was 21.7870.64 pA for control, 21.4670.53 pA in the presence of METH, 21.517 0.61 pA after 5 min of washout (n¼8, p40.05, Fig. 2E). On the other hand, the frequency and the amplitude of mEPSCs were both decreased significantly by the application of METH (100 μM) (n¼8, each, po0.05, Fig. 2F–J).

2.3. METH had no significant effect on NMDA and AMPA receptor-mediated currents To examine the possible involvement of GLU-mediated postsynaptic NMDA and AMPA receptors in METH-induced changes in glutamatergic synaptic transmission, AMPA and NMDA receptor-mediated currents were studied. To record NMDA receptor-mediated currents, exogenous NMDA (100 μM) and glycine (20 μM) were co-applied to hippocampal neurons at a holding potential of  50 mV. After 5 min pretreatment of METH (10 or 100 μM, in this and subsequent experiments, a total of METH superfusion time depended on when the agonist-induced current returned to original level, usually 6–7 min), the peak amplitudes of NMDA receptor-mediated currents before and after METH treatment were compared. On average, the peak NMDA receptor-mediated current was 441745 pA for the control group and 446756 pA and

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671

4

241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

brain research ] (]]]]) ]]]–]]]

Fig. 2 – Effects of METH on mEPSCs in rat primary cultured hippocampal neurons. A. Raw data traces of mEPSCs were recorded in control, after treatment of 10 lM METH and Washout. B. Cumulative plot for inter-event interval from figure A is shown and METH increased the frequency of mEPSCs (K-S statistic, n ¼8, po0.05). C, Cumulative distribution of the mEPSCs amplitude from the same neuron represented in A is shown and the amplitude of mEPSC was not affected by use of METH (K-S statistic, n¼ 8, p40.05). D, Histogram plot represents the change of the mEPSC frequency in the presence of METH (10 lM) (n indicates significant difference by t-test at the level of po0.05). E, Histogram plot shows amplitude of mEPSCs was not altered in the presence of METH (10 lM). (n¼ 8 p40.05). F, Raw data traces of mEPSCs were recorded in control, after treatment of 100 lM METH and Washout. G, Cumulative plot for inter-event interval from figure F is shown and METH decreased the frequency of mEPSCs (K-S statistic, n ¼8, po0.05). H, Cumulative distribution of mEPSCs amplitude from the same neuron represented in F is shown in control and in the presence of METH (100 μM). Decreased mEPSCs frequency occurred with the reduction of the mEPSC amplitude (K-S statistic, n¼ 8, po0.05). I, Histogram plot represents the change of the mEPSC frequency in the presence of METH (100 lM) (* indicates significant difference by t-test at the level of po0.05, n ¼8 ). J, Histogram plot shows amplitude of mEPSCs was decreased by use of METH (10 lM) (n ¼8, po0.05 ). 443738 pA for 10 μM and 100 μM METH-treated groups, respectively (n¼ 5, p40.05). This result showed that NMDA receptor-mediated currents were not affected directly by the application of METH (Fig. 3A–C). Exogenous application of AMPA (5 μM) induced an inward current at a holding potential of  70 mV. The mean amplitude of AMPA-mediated current was 251714 pA (Fig. 3D) for control and 260716 pA or 2577 15 pA in the presence of METH, 10 or 100 μM (n¼ 5, p40.05,

Fig. 3E–G), suggesting AMPA receptor was not directly affected by METH.

2.4. METH blocked N-type calcium channel currents, but not P/Q-type calcium channel currents Both N-type and P/Q-type calcium channels are tightly coupled to GLU release (Inchauspe et al., 2007). Previous experiments

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671 brain research ] (]]]]) ]]]–]]]

301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

5

Fig. 3 – NMDA and AMPA receptor-mediated currents were not affected by application of METH. A, The NMDA receptor current was induced by NMDA (100 lM)þglycine (20 lM) (Control). B and C show the currents induced by 100 lM NMDAþ20 lM glycine in the presence of METH (10 lM) and (100 lM), respectively. The amplitude of NMDA receptor-mediated currents was not affected by the application of METH at both tested concentrations (n ¼5, p40.05). D, AMPA receptor-mediated current was recorded by AMPA (5 lM) (control). E and F show the amplitude of AMPA receptor-mediated currents was not affected significantly in the presence of METH at 10 and 100 lM concentration, respectively (n ¼5, p40.05).

have shown that at least four distinct types of voltage-gated calcium channel (L-, N-, P/Q-, and R-type) are expressed in cultured hippocampal neurons and sensitive to different blockers (Takahashi and Momiyama, 1993). To observe the effect of METH on different kinds of voltage-gated calcium channels, three distinct blockers (verapamil for L-, MVIIA for N-, and ω-agatoxin IVA for P/Q-type calcium channels) were applied in the present experiment to define L-,N-, and P/Q-type currents, respectively (Wen et al., 2005). The voltage-gated P/Q-type Ca2þ channel currents were elicited by 300 ms step depolarization to þ60 mV from a holding potential of  80 mV in the presence of verapamil (10 μM) and MVIIA (3 μM). METH (10 μM, 100 μM) did not show any visible effect on the kinetic of P/Q-type Ca2þ channel currents (n¼8, p40.05, Fig. 4A). The I-V curves showed that the amplitude was not affected by the application of METH either (Fig. 4B). This result indicated that METH had no significant effect on P/Q-type Ca2þ channel currents. The voltagegated N-type calcium channel currents were recorded by 300 ms step depolarization to þ60 mV from a holding potential of  80 mV in the presence of verapamil (10 μM) and ω-agatoxin IVA (0.5 μM). METH, at 10 μM, did not show any visible effect on N-type Ca2þ channel currents (Fig. 4C). However, N-type Ca2þ channel currents were depressed when a high concentration of METH (100 μM) was applied to the neurons. The inhibitory rate reached 6276% (n¼ 8, po0.05) at a holding potential of 0 mV (Fig. 4D).

antagonist SCH23390 was used. The frequency of mEPSCs was increased by the application of METH (10 μM), which was not altered in the presence of SCH23390 (10 μM) (n¼ 8, po0.05, Fig. 5A and B). The frequency of mEPSCs was 0. 9370.34 Hz for control, 1.9370.41 Hz in the presence of METH and SCH23390, and 0.8970.32 Hz after washout (n¼ 8, po0.05 ). The amplitude of mEPSCs was not affected in the presence of METH (10 μM) and SCH23390 (10 μM) (n ¼8, p40.05, Fig. 8C). METH, at 100 μM, decreased the frequency and the amplitude of mEPSCs. Both changes were not affected by pre-application of SCH23390 (10 μM) (n ¼8, po0.05, respectively, Fig. 5D–F).

2.6. Tyrosine hydroxylase (TH)-positive neuron was almost not found in rat primary cultured hippocampal neurons To further study whether dopamine was involved in METHinduced increase of glutamate release, the TH was used as a selective marker to identify dopaminergic cells in our cultured hippocampal neurons (Fish et al., 2013). We found no TH-positive cells in most rat primary cultured hippocampal neurons (Fig. 6A and B). However, TH-positive cells were shown in rat cultured mesencephalic neurons (Fig. 6C and D), which was used as a TH staining positive control.

3. 2.5. The effect of METH on mEPSCs was not affected by pre-application of DA receptor antagonist SCH23390 To study whether DA was involved in METH-induced change of GLU synaptic transmission, D1/D5-selective DA receptor

Discussion

Experiments in our manuscript provide evidence of significant effects of METH on excitatory synaptic transmission in primary cultured hippocampal neurons. METH (0.1–50 μM) increased the frequency and amplitude of sEPSCs, while the

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671

6

361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

brain research ] (]]]]) ]]]–]]]

Fig. 4 – Effects of METH on P/Q-type and N-type calcium channel currents. A, P/Q-type calcium channel currents were recorded in control (top) and after 10 min of METH application (100 μM, bottom). B, I-V curve relationship shows no significant effect of METH (10 or 100 μM ) on P/Q-type calcium channel currents (n ¼ 5, p40.05). C, The N-type calcium channel currents were recorded in control (top) and the presence of METH (100 μM). D, I-V curve relationship shows that METH (100 μM) inhibited the N-type calcium channel currents (n ¼ 5, po0.05), while METH (10 μM) did not significantly affect the N-type calcium channel currents (n ¼5, p40.05).

concentration of 100 μM decreased it. Furthermore, frequency of mEPSCs was enhanced by the application of METH and suggested that METH increases presynaptic membrane GLU release, which is not altered by use of D1/D5 receptor antagonist SCH233990, NMDA, AMPA, and P/Q-type calcium channel. Furthermore, N-type calcium channel is not involved in METH-mediated increase in GLU release. The current findings illustrate that the other unknown mechanism most likely involves METH-mediated increases in presynaptic GLU release in rat primary cultured hippocampal neurons. According to prior reports, METH concentrations of 5–200 μM had no significant effects on cell toxicity, while 500 μM METH showed significant toxic effect to human neurons and astrocytes (about 50% cell death compared with control) following 24 h exposure (Abdul Muneer et al., 2011). Therefore, in our experiments we used METH concentrations between 0.1 and 100 μM. The clinical studies showed that a dose of METH 30 mg i.v. is estimated to result in a concentration of 14 μM in the human brain. METH abusers commonly inject dosages of tens to hundreds of milligrams (Fowler et al., 2008). The concentrations of METH used in these experiments (0.1–100 μM) fall within clinically relevant range.

Unlike METH-induced striatal DA release, which occurs directly via reverse transport (Panenka WJ, 2013), METH- Q2 induced striatal GLU release is considered to be initiated indirectly by the activation of dopamine pathways (Sonsalla et al., 1989). However, the extracellular concentration of striatal GLU remains at elevated level for at least 24 h after METH, while METH-induced DA release is no longer present 4 h after METH consumption (Mark et al., 2007; Ricoy and Martinez, 2009). This study suggests that the other mechanism are likely involved in METH-mediated release of GLU. Therefore, we explored the mechanism whether dopamine system was involved in METH-mediated increase of GLU release in rat primary cultured hippocampal neurons. In the present experiment, TH-positive cells (dopaminergic neurons) less than 0.2% were found in rat primary cultured hippocampal neurons from postnatal rats 10–12 h. This finding was also supported by a previous report where the survival rate of dopaminergic neurons was 90% in primary cultured ventral mesencephalon of the E14 rat fetus, while the survival rate of dopaminergic neurons was 0.1–0.25% at cultured neurons of postnatal rats 10–12 h (Shimoda et al., 1992). This could represent the reason why the release of GLU

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671 brain research ] (]]]]) ]]]–]]]

421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

7

Fig. 5 – METH affected the frequency and the amplitude of mEPSCs, which was not changed by pre-application of dopamine receptor antagonist SCH23390. A, The experiment was carried out in the presence of SCH23390 (10 μM) and the raw currents were recorded in the absence, in the presence, and washout of10 μM METH. B, Cumulative plot for inter-event interval from A is shown in the absence (Control) and presence of 10 μM METH. METH still significantly increased the frequency of mEPSCs in the presence of SCH23390 (n ¼8, po0.05). C, Cumulative distribution of the mEPSC amplitude from the same neuron represented in A was shown and the amplitude of mEPSCs was not affected by use of METH in the presence of SCH23390 (n ¼8, p40.05). D, The experiment was carried out in the presence of SCH23390 (100 μM) and mEPSCs were recorded in the absence, in the presence, and washout of METH (10 μM).E, Cumulative plot for inter-event interval from D is shown and METH (100 μM) decreased the frequency of mEPSCs in the presence of SCH23390 (n ¼8, po0.05). F, Cumulative distribution of the mEPSCs amplitude from the same neuron represented in D is shown and the amplitude of mEPSCs was decreased by use of METH (100 μM) in the presence of SCH23390 (n ¼8, po0.05).

was enhanced by the application of METH in rat primary cultured hippocampal neurons, which was not altered by the application of dopamine D1/D5 receptor antagonist. We provide the evidence that METH facilitates GLU release in rat primary cultured hippocampal neurons and the effect is independent on dopaminergic systems. The D1 and D5 receptors are members of the D1-like family of DA receptors and

are expressed in rat hippocampal neurons (Kalueff et al., 2007). Both of them are involved in METH-induced psychostimulation and neurotoxicity (Friend and Keefe, 2013). Preclinical and clinical evidence recently accumulated suggest that GLU plays a pivotal role in drug self-administration, reward-related processes, drug-induced conditioned associations, and relapse (Santos-Vera et al., 2013). Dissecting

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671

8

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

brain research ] (]]]]) ]]]–]]]

Fig. 6 – Tyrosine hydroxylase (TH)-positive neuron was not found in rat primary cultured hippocampal neurons. A, TH-positive neuron was not found in rat primary cultured hippocampal neurons. B, Nucleus was staining with DAPI and merge with A. C, TH-positive neuron (red) was found in rat primary cultured mesencephalic neurons. D, Nucleus was staining with DAPI and merge with C. A,B,C,D (all with 40  objective).

the mechanism of how METH influences the GLU-mediated synaptic transmission in neurons will help us understand the complexity of its neurotoxicity. It is known that synaptic interactions between cultured hippocampal neurons can be very strong, especially in low density cultured neurons (Wang et al., 2009; Segal and Furshpan, 1990). To electrophysiological recording, density of cultured neuron was low in our preparation. In the region of the hippocampus, the two main neural populations, pyramidal neurons and interneurons establish intricate synaptic connections (Freund and Buzsaki, 1996). Due to superimposed polysynaptic circuitry, METH increases the glutamate release and enhances the excitability of pyramidal neuron or interneuron. Although the primary cultured hippocampal neurons do not keep the same neuron circuitry when compared to hippocampal slice, the use of cultured cells to study connectivity is controversial because the endogenous three-dimensional structure of hippocampus is completely dissolved and networks regrown in a two-dimensional sheet. On the other hand, major phenomena observed in culture, such as LTP and homeostatic plasticity, have also been observed in slice and in vivo, justifying the use of cultured cells as a model system for investigating the synapse transmission (Schultz, 2002; Huang and Kandel, 1995). In a local circuitry, it could be assumed that METH enhances presynaptic pyramidal neuron glutamate release, which mediates mEPSCs and sEPSCs and their reordering in pyramidal or interneuron in our preparation. Elevating GLU synaptic transmission in local circuitry and changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction (Ernst et al., 2000). The hippocampus is a

particularly suitable structure for studying the role of psychostimulants in light of its exceptional monoaminergic innervation and its support of ICSS (Ursin et al., 1966; German and Bowden, 1974). The hippocampus has a significant site for drug reward/reinforcement, most likely due to its anatomical circuit to the VTA via the NAc and VP (Lisman and Grace, 2005). Thus, glutamate synaptic transmission enhanced by application of METH in hippocampus is most likely involved in reward circuitry in vivo. To observe the effects of METH on glutamatergic synaptic transmission in primary cultured hippocampal neurons, N-type calcium channel and P/Q-type calcium channel currents were recorded. It is established that synaptic transmission at most CNS synapses is mediated by joint actions of multiple subfamilies of the voltage-gated calcium channels, most prominently, P/Q- and N-type calcium channel (Takahashi, 2005; Takahashi and Momiyama, 1993). Presynaptic voltagegated Ca2þ channels are activated on action potential (AP) invasion to allow for rapid influx of extracellular Ca2þ, which, in turn, triggers synaptic vesicle fusion and neurotransmitter release (Adams et al., 2003; Northrop et al., 2011). In the present study, METH had no significant effect on P/Q type Ca2þ channels. On the other hand, METH (10 μM) did not present visible effects on N-type Ca2þ channel currents, but had an inhibitory effect at a high concentration of 100 μM with an inhibitory rate of 6275.9%. Therefore, METH (100 μM) decreased the release of GLU, most likely through the inhibition of the frequency and amplitude of mEPSCs. In clinic, the user of METH at high doses produces the symptoms of hallucination, dizziness, and anxiety (Fowler et al., 2008). These symptoms are produced by blockade of N-type calcium

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671 brain research ] (]]]]) ]]]–]]]

541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600

channels, since N-type calcium channels inhibitors also produce the similar symptoms in clinic (Pope and Deer, 2013). Furthermore, in our study the high concentration (100 μM) of METH decreased the frequency of mEPSCs, which can likely be attributed to the effects on neuronal viability. This opinion was supported by a previous report where the high concentration of METH affected neuronal survival and direct toxicity of METH for neurons (Abdul Muneer et al., 2011). However, the detailed mechanisms are still being explored. As METH increases the frequency of mEPSCs, we believe that presynaptic mechanism involves the METHinduced phenomenon. Our findings indicate that N-type calcium and P/Q-type calcium channels are not involved in METH-enhanced release of glutamate. The previous report showed that external Ca2þ triggers spontaneous glutamate release more weakly than evoked release in mouse neocortical neurons. Blockade of voltage-activated calcium channels has no effect on the spontaneous release rate or its dependence on [Ca 2þ] (Vyleta and Smith, 2011). This is the most likely reason why N-type calcium channel currents and P/Qtype calcium channel are not involved in METH-induced release of glutamate. sEPSCs and mEPSCs represent the GLU receptor postsynaptic response to presynaptic action potential dependent and independent release of the neurotransmitter vesicles at functional synapses, respectively. Both of them provide an index of synaptic efficacy and connectivity at a quantitative level (Liu et al., 2004). While the frequency of mEPSCs reflects presynaptic release quantitatively, the amplitude of sEPSCs and mEPSCs is affected by the amount of neurotransmitter release and sensitivity of postsynaptic receptors together. As METH enhances the amplitude of sEPSCs, we studied the direct effect of METH on NMDA and AMPA receptor-mediated currents. METH had no direct effect on NMDA and AMPA receptor-mediated currents in hippocampal neurons. Therefore, it was more likely that the increased amplitude of sEPSCs might be produced by an enhanced amount of GLU release, which induced a temporal and spatial summation of sEPSCs. The study showed that serotonergic mechanism responsible for increase in baseline synaptic transmission is possible, since serotonin can reduce inhibition of excitatory synaptic transmission in the CA1 region of the hippocampus via activation of 5-HT1A receptors, while METH is a powerful releaser of 5HT. (Steketee and Kalivas, 2011). METH is a substrate for the dopamine transporter and profoundly increases the concentration of extracellular monoamines dopamine (DA), serotonin (5-HT), and norepinephrine (NE) by redistributing these neurotransmitters from synaptic vesicles to the cytosol, in addition to inducing reverse transport and competing for transmitter uptake at their cognate transporters (Swant et al., 2010). However, the reported effects of 5-HT in the hippocampus are divergent, as some have documented inhibitory (Kimura et al., 2004; Pugliese et al., 1998; Jahnsen, 1980), while others have shown excitatory effects, or no effect on excitatory synaptic transmission (Jahnsen, 1980; Kimura et al.,2004). Therefore, the further exploration is needed in order to answer whether serotonergic changes are involved in the METH-induced effects on glutamatemediated excitatory postsynaptic currents in our experiment.

9

Future studies will unequivocally discern whether serotonergic modulation is required for METH to increase glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Conclusively, the question of the exact mechanism on how METH induces GLU release remains unclear and required further investigation.

4.

Conclusion

The effect of METH on glutamatergic synaptic transmission was not reversed by the pretreatment of DA receptor antagonist SCH23390. These results suggest that METH modulates presynaptic GLU release without the involvement of the dopaminergic system in rat primary cultured hippocampal neurons.

5.

Materials and methods

5.1.

Cultures

Wistar rats at postnatal younger than 12 h were anesthetized with ether and decapitated, in accordance with the guideline of the Beijing Institute of Biological Science Animal Research Advisory Committee (SCXK-JUN-2007-004). The experimental protocol was approved by Beijing Institute of Biological Science Animal Research Advisory Committee. Primary culture of hippocampal cells was established using a previously reported protocol (Zhang et al., 2008).

5.2.

Electrophysiology

Hippocampal cultures at 10–14 days were transferred to a recording chamber placed on the stage of an inverted Olympus IX70 microscope. In most neurons, the excitatory postsynaptic currents could be recorded in whole-cell voltage clamp mode with a clamping membrane potential of  70 mV. The recording electrodes (borosilicate glass; 4–5 MΩ) were filled with an internal solution containing (in mM): Cs gluconate 130, MgCl2 2, HEPES 10, and Na2ATP 2 (pH was adjusted to 7.35 with CsOH, osmolarity was approximately 280 mOsm). For the mEPSCs experiment, TTX (1 μM) and bicuculline (100 μM) were added to the recording medium. For Ca 2þ current recording, 1 μM TTX, 5 mM TEA-Cl, and 2 mM 4-AP were added to the extracellular solution to block Naþ and Kþ channels, respectively. The intracellular pipette solution contained (in mM): CsCl 140, HEPES 10, EGTA 10, TEA-Cl 5, and Na2ATP 2 (pH was adjusted to 7.35 with CsOH, osmolarity was about 280 mOsm) (Xia and Arai, 2011; Finnegan et al., 2005). Signals were amplified with Axopatch-1D (Axon Instruments, USA) and recorded with pClamp8.2 software (Axon Instruments, USA). Data were analyzed with the software Clampfit10.2 (Axon Instruments, USA) and Origin7.5 (OriginLab Corporation, USA).

5.3.

Immunohistochemistry

The rat primary hippocampal neurons cultured on top of coverslips were washed with phosphate buffered saline (PBS) buffer and fixed with iced methanol. Cells were stained with

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671

10

601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660

brain research ] (]]]]) ]]]–]]]

rabbit anti-TH antibody 1:1000 (ab41528, Abcam) overnight. After that, they were washed with PBS buffer three times for total of 30 min. The cells were then immersed in PBS buffer containing Cy3-conjugated donkey anti-rabbit antibody (1:300; Jackson ImmunoResearch Laboratories Inc.) for 60 min. Thereafter, cells were washed three times with PBS for total of 60 min. Coverslips were then mounted on slides and pictures were captured with fluorescence microscope (Olympus, Japan) and analyzed using Adobe Photoshop version 7.

5.4.

Drugs

TTX, bicuculline, CNQX, verapamil, MVIIA, and ω-agatoxin IVA were purchased from Sigma (St Louis, MO, USA). TTX was dissolved in aqueous sodium acetate buffer (pH 4.5). Bicuculline and CNQX were dissolved in DMSO. Verapamil, MVIIA, and ω-agatoxin IVA were dissolved in dH2O. All drugs were stocked at a high concentration and stored at –20 1C. They were dissolved in the extracellular solution for appropriate concentration just before administration.

5.5.

Statistical analysis

Original current traces were analyzed using the Clampfit 10.2 software (Molecular Devices, UK). All curve fittings were carried out using Origin 7.5 (OriginLab Corporation, USA). All data were reported as mean7SEM. The nonparametric Kolmogorov–Smirnov test was used to compare amplitudes and frequencies of mEPSCs and sEPSCs between two experimental groups. The level of significance was set at po0.05.

Disclosure The project was supported by the foundation of National Key Basic Research Program (No. 2007CB512307), National Natural Science Foundation of China (No. 81100995) and National Key Technology R&D Program of China (2012BAI01B07).

Conflict of Interest The authors declare no conflict of interest.

r e f e r e n c e

Abdul Muneer, P.M., Alikunju, S., Szlachetka, A.M., Haorah, J., 2011. Methamphetamine inhibits the glucose uptake by human neurons and astrocytes: stabilization by acetyl-Lcarnitine. PloS one 6, e19258. Adams, D.J., Smith, A.B., Schroeder, C.I., Yasuda, T., Lewis, R.J., 2003. Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals. J. Biol. Chem. 278, 4057–4062. Cadet, J.L., Jayanthi, S., Deng, X., 2005. Methamphetamineinduced neuronal apoptosis involves the activation of multiple death pathwaysRev. Neurotox. Res. 8, 199–206. Chirwa, S., Aduonum, A., Pizarro, J., Reasor, J., Kawai, Y., Gonzalez, M., McAdory, B.S., Onaivi, E., Barea-Rodriguez, E.J., 2005. Dopaminergic DA1 signaling couples growth-associated

protein-43 and long-term potentiation in guinea pig hippocampus. Brain Res. Bull. 64, 433–440. Ernst, T., Chang, L., 2008. Adaptation of brain glutamate plus glutamine during abstinence from chronic methamphetamine use. J. Neuroimmune Pharmacol. 3, 165–172. Ernst, T., Chang, L., Leonido-Yee, M., Speck, O., 2000. Evidence for long-term neurotoxicity associated with methamphetamine abuse: A 1H MRS study. Neurology 54, 1344–1349. Finnegan, T.F., Chen, S.R., Pan, H.L., 2005. Effect of the {mu} opioid on excitatory and inhibitory synaptic inputs to periaqueductal gray-projecting neurons in the amygdala. J. Pharm. Exp. Ther.312, 441–448. Fish, E.W., Krouse, M.C., Stringfield, S.J., Diberto, J.F., Robinson, J.E., Malanga, C.J., 2013. Changes in sensitivity of reward and motor behavior to dopaminergic, glutamatergic, and cholinergic drugs in a mouse model of fragile x syndrome. PloS one 8, e77896. Fowler, J.S., Volkow, N.D., Logan, J., Alexoff, D., Telang, F., Wang, G.J., Wong, C., Ma, Y., Kriplani, A., Pradhan, K., Schlyer, D., Jayne, M., Hubbard, B., Carter, P., Warner, D., King, P., Shea, C., Xu, Y., Muench, L., Apelskog, K., 2008. Fast uptake and long-lasting binding of methamphetamine in the human brain: comparison with cocaine. NeuroImage 43, 756–763. Freund, T.F., Buzsaki, G., 1996. Interneurons of the hippocampus. Hippocampus 6, 347–470. Friend, D.M., Keefe, K.A., 2013. A role for D1 dopamine receptors in striatal methamphetamine-induced neurotoxicity. Neurosci. Lett.555, 243–247. German, D.C., Bowden, D.M., 1974. Catecholamine systems as the neural substrate for intracranial self-stimulation: a hypothesis. Brain Res. 73, 381–419. He, Z., Chen, Y., Dong, H., Su, R., Gong, Z., Yan, L., 2014. Inhibition of vesicular glutamate transporters contributes to attenuate methamphetamine-induced conditioned place preference in rats. Beh. Brain Res. 267, 1–5. Huang, Y.Y., Kandel, E.R., 1995. D1/D5 receptor agonists induce a protein synthesis-dependent late potentiation in the CA1 region of the hippocampus. Proc. Natl. Acad. Sci. U.S.A 92, 2446–2450. Inchauspe, C.G., Forsythe, I.D., Uchitel, O.D., 2007. Changes in synaptic transmission properties due to the expression of N-type calcium channels at the calyx of Held synapse of mice lacking P/Q-type calcium channels. J. Physiol.584, 835–851. Jahnsen, H., 1980. The action of 5-hydroxytryptamine on neuronal membranes and synaptic transmission in area CA1 of the hippocampus in vitro. Brain Res. 197, 83–94. Kalueff, A.V., Ren-Patterson, R.F., Murphy, D.L., 2007. The developing use of heterozygous mutant mouse models in brain monoamine transporter research. Trends pharmacol. Sci. 28, 122–127. Kauer, J.A., Malenka, R.C., 2007. Synaptic plasticity and addiction. Nature Rev. Neurosci. 8, 844–858. Kimura, T., Ishihara, K., Ozawa, K., Sasa, M., 2004. Augmentation of serotonin-induced inhibition of neuronal activity in the hippocampus following repeated treatment with methamphetamine. Ann. N. Y. Acad. Sci. 1025, 242–247. Lezcano, N., Bergson, C., 2002. D1/D5 dopamine receptors stimulate intracellular calcium release in primary cultures of neocortical and hippocampal neurons. J. Neurophysiol. 87, 2167–2175. Lisman, J.E., Grace, A.A., 2005. The hippocampal-VTA loop: controlling the entry of information into long-term memory. Neuron 46, 703–713. Liu, J., Yu, B., Neugebauer, V., Grigoriadis, D.E., Rivier, J., Vale, W.W., Shinnick-Gallagher, P., Gallagher, J.P., 2004. Corticotropinreleasing factor and Urocortin I modulate excitatory glutamatergic synaptic transmission. J. Neurosci. 24, 4020–4029.

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040

BRES : 43671 brain research ] (]]]]) ]]]–]]]

661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703

Mark, K.A., Quinton, M.S., Russek, S.J., Yamamoto, B.K., 2007. Dynamic changes in vesicular glutamate transporter 1 function and expression related to methamphetamineinduced glutamate release. J. Neurosci. 27, 6823–6831. Mark, K.A., Soghomonian, J.J., Yamamoto, B.K., 2004. High-dose methamphetamine acutely activates the striatonigral pathway to increase striatal glutamate and mediate long-term dopamine toxicity. J. Neurosci. 24, 11449–11456. Maze, I., Feng, J., Wilkinson, M.B., Sun, H., Shen, L., Nestler, E.J., 2011. Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens. Proc. Natl. Acad. Sci. U.S.A 108, 3035–3040. Nestler, E.J., 2001. Molecular basis of long-term plasticity underlying addiction. Nat. Rev. Neurosci. 2, 119–128. Northrop, N.A., Smith, L.P., Yamamoto, B.K., Eyerman, D.J., 2011. Regulation of glutamate release by alpha7 nicotinic receptors: differential role in methamphetamine-induced damage to dopaminergic and serotonergic terminals. J. Pharmacol. Exp. Ther.336, 900–907. Pope, J.E., Deer, T.R., 2013. Ziconotide: a clinical update and pharmacologic review. Expert Opin. Pharmacother.14, 957–966. Pugliese, A.M., Passani, M.B., Corradetti, R., 1998. Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices. Br. J. Pharmacol.124, 93–100. Raudensky, J., Yamamoto, B.K., 2007. Effects of chronic unpredictable stress and methamphetamine on hippocampal glutamate function. Brain Res. 1135, 129–135. Reiner, B.C., Keblesh, J.P., Xiong, H., 2009. Methamphetamine abuse, HIV infection, and neurotoxicity. Int. J. Physiol. Pathophysiol. Pharmacol.1, 162–179. Ricoy, U.M., Martinez Jr., J.L., 2009. Local hippocampal methamphetamine-induced reinforcement. Front Beh. Neurosci. 3, 47. Santos-Vera, B., Vazquez-Torres, R., Marrero, H.G., Acevedo, J.M., Arencibia-Albite, F., Velez-Hernandez, M.E., Miranda, J.D., Jimenez-Rivera, C.A., 2013. Cocaine sensitization increases I h current channel subunit 2 (HCN(2)) protein expression in structures of the mesocorticolimbic system. J. Mol. Neurosci. 50, 234–245. Schultz, W., 2002. Getting formal with dopamine and reward. Neuron 36, 241–263. Segal, M.M., Furshpan, E.J., 1990. Epileptiform activity in microcultures containing small numbers of hippocampal neurons. J. Neurophysiol. 64, 1390–1399.

11

Shimoda, K., Sauve, Y., Marini, A., Schwartz, J.P., Commissiong, J.W., 1992. A high percentage yield of tyrosine hydroxylase-positive cells from rat E14 mesencephalic cell culture. Brain Res. 586, 319–331. Smith, K.J., Self, R.L., Butler, T.R., Mullins, M.M., Ghayoumi, L., Holley, R.C., Littleton, J.M., Prendergast, M.A., 2007. Methamphetamine exposure antagonizes N-methyl-Daspartate receptor-mediated neurotoxicity in organotypic hippocampal slice cultures. Brain Res. 1157, 74–80. Sonsalla, P.K., Nicklas, W.J., Heikkila, R.E., 1989. Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science 243, 398–400. Steketee, J.D., Kalivas, P.W., 2011. Drug wanting: behavioral sensitization and relapse to drug-seeking behavior. Pharmacol. Rev. 63, 348–365. Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H., 2010. Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus. PloS one 5, e11382. Takahashi, T., 2005. Dynamic aspects of presynaptic calcium currents mediating synaptic transmission. Cell Calcium37, 507–511. Takahashi, T., Momiyama, A., 1993. Different types of calcium channels mediate central synaptic transmission. Nature 366, 156–158. Thompson, A.M., Gosnell, B.A., Wagner, J.J., 2002. Enhancement of long-term potentiation in the rat hippocampus following cocaine exposure. Neuropharmacol. 42, 1039–1042. Ursin, R., Ursin, H., Olds, J., 1966. Self-stimulation of hippocampus in rats. J. Comp. Physiol. Psychol. 61, 353–359. Vyleta, N.P., Smith, S.M., 2011. Spontaneous glutamate release is independent of calcium influx and tonically activated by the calcium-sensing receptor. J. Neurosci. 31, 4593–4606. Wang, J., Hasan, M.T., Seung, H.S., 2009. Laser-evoked synaptic transmission in cultured hippocampal neurons expressing channelrhodopsin-2 delivered by adeno-associated virus. J. Neurosc Methods183, 165–175. Wen, L., Yang, S., Qiao, H., Liu, Z., Zhou, W., Zhang, Y., Huang, P., 2005. SO-3, a new O-superfamily conopeptide derived from Conus striatus, selectively inhibits N-type calcium currents in cultured hippocampal neurons. Br. J. Pharmacol.145, 728–739. Xia, Y.F., Arai, A.C., 2011. Prolactin-releasing peptide enhances synaptic transmission in rat thalamus. Neuroscience 172, 1–11. Zhang, S., Su, R., Zhang, C., Liu, X., Li, J., Zheng, J., 2008. C101, a novel 4-amino-piperidine derivative selectively blocks N-type calcium channels. Eu. J. Pharmacol.587, 42–47.

Please cite this article as: Zhang, S., et al., Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.07.040