Methylphenidate (MPH), commercially called Ritalin or Concerta, has been widely used as a drug for Attention Deficit Hyperactivity Disorder (ADHD). Noteworthily, growing numbers of young people using prescribed MPH improperly for pleasurable enhancement, take high risk of addiction. Thus, understanding the mechanism underlying high level of MPH action in the brain becomes an important goal nowadays. As a blocker of catecholamine transporters, its therapeutic effect is explained as being due to proper modulation of D1 and α2A receptor. Here we showed that higher dose of MPH facilitates NMDA-receptor mediated synaptic transmission via a catecholamine-independent mechanism, in layer V∼VI pyramidal cells of the rat medial prefrontal cortex (PFC). To indicate its postsynaptic action, we next found that MPH facilitates NMDA-induced current and such facilitation could be blocked by σ1 but not D1/5 and α2 receptor antagonists. And this MPH eliciting enhancement of NMDA-receptor activity involves PLC, PKC and IP3 receptor mediated intracellular Ca 2+ increase, but does not require PKA and extracellular Ca 2+ influx. Our additional pharmacological studies confirmed that higher dose of MPH increases locomotor activity via interacting with σ1 receptor. Together, the present study demonstrates for the first time that MPH facilitates NMDA-receptor mediated synaptic transmission via σ1 receptor, and such facilitation requires PLC/IP3/PKC signaling pathway. This novel mechanism possibly explains the underlying mechanism for MPH induced addictive potential and other psychiatric side effects.

Funding: This work was supported by grants from the Ministry of Science and Technology of China (2006CB500807; 2011CBA00406) and the National Natural Science Foundation of China (30700218, 30821002 and 30990263). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Considering MPH’s important role in regulating PFC function and ADHD medication, our study attempted to characterize the pharmacological and cellular mechanism of MPH in layer V∼VI pyramidal cells in the medial prefrontal cortex of rats. We found that MPH facilitates NMDA-receptor mediated excitatory synaptic transmission through σ1 receptors via PLC/PKC signaling pathway, revealing a novel mechanism for MPH action.

Interestingly, recent studies have indicated that sigma-1 (σ1) receptor is a new target for those stimulants used for ADHD, like cocaine [30] , [31] , amphetamine [32] – [34] , and 3,4-methylenedioxymethamphetamine (MDMA) [35] . σ receptor was first described as a subtype of opioid receptor [36] or phencyclidine (PCP) receptor [37] . As σ receptor is not the high-affinity binding site of naltrexone or thienylcyclohexylpiperidine (TCP), it was re-classified as a non-opioid receptor [38] or non-PCP receptor [39] , [40] . Two subtypes of σ receptor have been described: σ1 and σ2 receptors [41] . By measuring the mRNA level in the brain, sigma-1 receptor protein is highly distributed in the PFC, striatum and hippocampus, etc [42] . On the cellular level, σ1 receptor showing a post-synaptic distribution, is enriched in the endoplasmic reticulum and on the plasma membrane through the dynamic translocation [42] . Activation of the σ1 receptor would modulate Ca 2+ entry through plasma membrane (i.e., via K + channel, NMDA receptor, voltage-sensitive Ca 2+ channel), and intracellular Ca 2+ mobilization (i.e., via IP3 receptor) [43] , [44] .

On the other hand, escalating or higher doses of MPH induce behavioral sensitization, like locomotor hyperactivity in animals [21] , [22] , which is associated with a robust DA release in the PFC, caudate-putamen, and nucleus accumbens, etc [1] , [14] , [23] . Among these brain areas, a series of studies by Dafny et al indicated that PFC is still an important area for acute or chronic administration of MPH induced sensitization in free-moving animals [24] – [26] . Moreover, higher doses or long-term medication of MPH may lead some psychiatric adverse effects like depressive symptoms both in animals and patients [27] – [29] . Although higher doses of MPH actions may be due to excessive stimulation on D1, α1 and/or β1 receptors [1] , its explicit receptor mechanism underlying the side effects like addiction and other psychiatric effects remains mostly unexplored.

Through strengthening DA/NE transmission in PFC, low to moderate doses of MPH have been shown to improve working memory in animals [13] , [17] , [18] . Importantly, recent electrophysiological studies explored more on the receptor mechanisms for MPH actions. For example, in vivo acutely administered MPH exerts excitatory actions on PFC neurons by indirectly activating α2-adrenoceptors and D1 receptors [1] , [17] – [19] . And in vitro, MPH could enhance excitability of pyramidal PFC neurons by activating α2 receptors located in interneurons [20] .

In the ADHD patients, the symptoms are mostly consistent with the dysfunction of the PFC [8] , [9] , where is a high-function area guiding and organizing attention, thought and affection [10] . As a blocker of dopamine (DA) and norepinephrine (NE) transporters [11] , [12] , low to moderate levels of MPH increase both extracellular DA and NE in PFC [13] , and DA in the striatum [14] . Interestingly, a recent animal study showed that low dose of MPH infusion into PFC facilitates working memory performance, while MPH into striatum does not affect this PFC-dependent cognition task [15] . Thus, these evidence support the notion that PFC is a main site involving in MPH’s therapeutic actions [1] , [16] .

Methylphenidate (MPH, known as Ritalin or Concerta), is a commonly used stimulant medication for Attention-deficit/hyperactivity disorder (ADHD) [1] , [2] . As acutely administered MPH has a good safety profile, and improves executive function performance in both diagnosed ADHD patients and general healthy population [3] – [6] , its prescription has been strikingly increased nowadays. However, these young people using prescribed MPH improperly for pleasurable enhancement, have high risk of being addicted [7] .

Results

Whole-cell patch clamp recordings were conducted in layer V∼VI pyramidal cells in slices of rat mPFC. Pyramidal cells were identified by their morphological and electrophysiological features. These neurons have pyramidal-shaped cell bodies and long apical dendrites extending toward superficial layers, as revealed by IR-DIC. They had a resting membrane potential more negative than −60 mV and an action potential larger than 70 mV, with no spontaneous discharge. They exhibited a spike frequency adaptation in response to a depolarizing current pulse and could be characterized as “regular spiking” pyramidal cells [45].

MPH Enhances NMDA- and Non-NMDA-R Mediated Synaptic Transmission We first tested if treatment with methylphenidate hydrochloride (MPH) could affect excitatory synaptic transmission in pyramidal cells. Recordings of eEPSC were conducted in the continuous presence of the GABAergic antagonist bicuculline (BMI). Under voltage-clamp at a holding potential of −70 mV, eEPSCs were evoked at a stimulation rate of 0.033 Hz. These eEPSCs could be completely inhibited by co-application of AP-5 (50 µM) and CNQX (20 µM) (data not shown), indicating that the currents were mediated by ionotropic glutamate receptors. As shown in Figure 1B and 1C, bath application of MPH (10, 50 µM) significantly enhanced eEPSC. While MPH with 1 µM produced no effect (Figure 1A, 105.2±8.9% of the baseline eEPSC, n = 8, paired t-test, P>0.05), MPH with 10 µM significantly enhanced eEPSC (129.5±8.6% of the baseline eEPSC, n = 8, P<0.01), and such enhancement was more evident when MPH dose was 50 µM (142.0±9.3% of the baseline eEPSC, n = 12, P<0.001). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. MPH enhances both non-NMDA- and NMDA-R mediated eEPSC. (A) MPH with 1 µM had no effect on the amplitude of eEPSC. P>0.05 for MPH vs. control, n = 8, paired t-test. (B) MPH with 10 µM significantly enhanced the amplitude of eEPSC. **P<0.01 vs. control, n = 8, paired t-test. (C) MPH with 50 µM significantly enhanced the amplitude of eEPSC. ***P<0.001 vs. control, n = 12, paired t-test. (D) MPH (50 µM) enhanced non-NMDA-R mediated eEPSC. Recordings of eEPSC were carried out in the presence of AP-5 (50 µM; NMDA receptor antagonist), with holding potential of −70 mV. **P<0.01 vs. control, n = 8, paired t-test. (E) MPH (50 µM) enhanced NMDA-R mediated eEPSC. Recordings of eEPSCs were performed in the presence of CNQX (20 µM; non-NMDA receptor antagonist), with holding potential of −40 mV to relieve the voltage-dependent Mg2+ blockade of NMDA receptor. **P<0.01 vs. control, n = 8, paired t-test. All traces of the synaptic currents are the average of 10 consecutive eEPSC responses. Recordings of eEPSCs were conducted in the continuous presence of BMI, with holding potential of −70 mV (A–D) or −40 mV (E). https://doi.org/10.1371/journal.pone.0051910.g001 To characterize if MPH enhancement came from a facilitation of NMDA-receptor (NMDA-R) or non-NMDA-receptor (non-NMDA-R) components, or both, we examined the effects of MPH on NMDA-R and non-NMDA-R mediated eEPSC, respectively. When non-NMDA-R current was recorded, we held the membrane potential at −70 mV and applied AP-5 (50 µM) to block NMDA-R. The non-NMDA-R eEPSC could be blocked wholly by the non-NMDA-R antagonist CNQX (20 µM) (data not shown). When NMDA-R current was recorded, we held the membrane potential at −40 mV (to relieve the voltage-dependent Mg2+ blockade of NMDA-R channel) and applied CNQX (20 µM) to block non-NMDA-R. NMDA-R mediated eEPSC could be blocked completely by the NMDA-R antagonist AP-5 (50 µM) (data not shown). As shown in Figure 1D and 1E, MPH (50 µM) significantly enhanced both non-NMDA- and NMDA-R mediated eEPSC (non-NMDA-R eEPSC: 131.0±5.2% of baseline, n = 8, P<0.01; NMDA-R eEPSC: 151.6±11.9% of baseline, n = 8, P<0.01, paired t-test).

MPH Facilitates Excitatory Synaptic Transmission via Pre- and Postsynaptic Mechanisms As a blocker of dopamine (DA) and norepinephrine (NE) transporters, MPH could increase the concentrations of DA and NE in synaptic cleft [14]. Thus, MPH enhancement of eEPSC may be a result of enhanced synaptic transmission mediated by catecholamine. If so, the facilitation effect of MPH should not exist after catecholamine is depleted. In this experiment, we used reserpine, an inhibitor of the vesicular monoamine transporter, to deplete catecholamine (see the methods). As shown in Figure 2A, NE and DA were almost completely depleted in reserpine-treated slices (6.0±2.1% of baseline for NE, n = 6 slices; and 16.1±3.4% for DA, n = 3 slices). In such slices, MPH produced no effect on non-NMDA-R eEPSC (97.1±9.8% of the baseline eEPSC, n = 7, P>0.05) (Figure 2B), but still enhanced NMDA-R eEPSC (135.5±16.2% of the baseline eEPSC, n = 6, P<0.05) (Figure 2C). Thus, MPH enhances non-NMDA-R eEPSC via a catecholamine-dependent mechanism (presynaptic mechanism), whereas it facilitates NMDA-R eEPSC through a catecholamine-independent way. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. MPH enhances NMDA-R mediated eEPSC under depletion of catecholamine. (A) The levels of NE and DA were almost completely depleted in slices treated with reserpine. n = 6 slices for NE, and n = 3 slices for DA. (B) In reserpine-treated slices, MPH (50 µM) produced no effect on non-NMDA-R mediated eEPSC. Recordings of eEPSCs were performed in the presence of AP-5 (50 µM), with holding potential of −70 mV. P>0.05 for MPH vs. control, n = 7, paired t-test. (C) In reserpine-treated slices, MPH (50 µM) still enhanced NMDA-R mediated eEPSC. Recording of eEPSCs were performed in the presence of CNQX (20 µM), with holding potential of −40 mV. *P<0.05 vs. control, n = 6, paired t-test. https://doi.org/10.1371/journal.pone.0051910.g002 To future confirm this notion, we pharmacologically isolated the patched cells by bath applying TTX and BMI, and puff administered glutamate to induce non-NMDA-R current or NMDA to induce NMDA-R current. The non-NMDA- and NMDA-R currents could be eliminated by CNQX and AP-5, respectively. As shown in Figure 3A, MPH had no effect on non-NMDA-R current (98.4±6.5% of the baseline, n = 7, P>0.05, paired t-test), but significantly enhanced NMDA-R current (129.6±6.2% of the baseline, n = 10, P<0.01) (Figure 3B), indicating that there exists a post-synaptic mechanism mediating MPH facilitation of NMDA-R current. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. MPH has no effect on non-NMDA-R current, but enhances NMDA-R current in pharmacologically-isolated cells. (A) MPH (50 µM) produced no effect on non-NMDA-R current. Recordings of non-NMDA-R currents were performed in the presence of AP-5 (50 µM), TTX (1 µM) and BMI (20 µM), with holding potential of −70 mV. As seen, pressure-application of glutamate (100 µM) induced an inward non-NMDA-R current (left), and this current was unchanged when MPH was administered (right). P>0.05 for MPH vs. control, n = 7, paired t-test. (B) MPH (50 µM) enhanced NMDA-R current. Recordings of NMDA-R currents were performed in the presence of CNQX (20 µM), TTX (1 µM) and BMI (20 µM), with holding potential of −40 mV. As shown, pressure-application of NMDA (100 µM) induced an inward NMDA-R current (left), and this current was enhanced when MPH was applied (right). **P<0.01 vs. control, n = 10, paired t-test. https://doi.org/10.1371/journal.pone.0051910.g003

MPH Enhances NMDA-R Response through σ1 but not D1/5 and α2 Receptors It is important to know the receptor mechanism underlying MPH facilitation of NMDA-R mediated synaptic transmission. Behavioral pharmacological studies have shown that MPH improves prefrontal cortical cognitive functions through actions at NE α2 and DA D1 receptors [1], [17]–[19]. It has been reported that MPH increases the excitability of PFC pyramidal neurons via activation of α2 receptors [20]. Moreover, stimulation on D1 receptors has been shown to potentiate NMDA-R current in rat PFC [46]. Thus, we tested if D1 and/or α2 receptors involve in MPH enhancement of NMDA-R current. As shown in Figure 4, MPH still enhanced NMDA-R current when the D1/5 antagonist SCH39166 and the α2 antagonist yohimbine were co-administered (119.4±6.0% of the baseline, n = 11, P<0.01) (Figure 4A) or applied separately (data not shown). Thus, MPH enhancement of NMDA-R response was not directly mediated by D1 and α2 receptors. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. MPH enhancement of NMDA-R current is mediated by σ1, but not D1/5 and α2 receptors. (A) MPH (50 µM) still enhanced NMDA-induced current when the D1/5 receptor antagonist SCH39166 (1 µM) and the α2 receptor antagonist yohimbine (1 µM) were co-administered. **P<0.01 vs. control, n = 11, paired t-test. (B) MPH (50 µM) did not enhance NMDA-induced current in the presence of the potent σ1 receptor antagonist haloperidol (1 µM). P>0.05 for MPH vs. control, n = 8, paired t-test. (C) MPH (50 µM) did not enhance NMDA-induced current in the presence of the selective σ1 receptor antagonist AC915 (1 µM). P>0.05 for MPH vs. control, n = 9, paired t-test. NMDA-R currents were recorded in the presence of CNQX (20 µM), TTX (1 µM) and BMI (20 µM), with holding potential of −40 mV. https://doi.org/10.1371/journal.pone.0051910.g004 Previous studies have shown that stimulation of σ1 receptor regulates NMDA-R mediated intracellular calcium elevation, NMDA-R current and NMDA-R mediated synaptic transmission [44], [47], [48]. Thus, we speculated that MPH enhancement of NMDA-R current might have something to do with σ1 receptor. To address this speculation, we investigated MPH effect in the presence of haloperidol (1 µM), a potent σ1 receptor antagonist [49], and found that MPH facilitation of NMDA-R current did not appear when haloperidol was bath applied (104.0±5.3% of the baseline, n = 8, P>0.05) (Figure 4B). Since haloperidol is also a D2 receptor antagonist, we then examined the effect of AC915, a selective σ1 receptor antagonist, to further confirm the role of σ1 receptor. As shown in Figure 4C, MPH enhancement of NMDA-R response was blocked in the presence of AC915 (1 µM) (93.7±3.4% of the baseline, n = 9, P>0.05). Taken together, these results indicate that MPH could act at σ1 receptor to enhance NMDA-R response.

Competitive Binding Assays Reveal that MPH could Bind with σ1 Receptor It has been shown that σ1 receptor becomes a new binding target for some psychostimulants like cocaine, methamphetamine, and MDMA (3,4-methylenedioxymethamphetamine). Recently, several studies have reported that the σ1 receptor ligand pharmacophore possesses a common N-substituted trace amines [50], [51]. Like methamphetamine and MDMA, MPH also has a similar N-substituted trace amines (Figure 5A), suggesting that MPH could bind with σ1 receptor. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Binding assay of MPH with σ1 receptor. (A) MPH has a N-substituted trace amines similar to those of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). (B) The amount of σ1 receptor in the liver tissue was nearly 8 times of that in the mPFC. (C) Competitive binding curves of haloperidol, NE-100, and MPH against [3H]-(+)-pentazocine. Haloperidol (10 µM) was used to define non-specific binding. (D) Affinities of haloperidol, NE-100 and MPH with σ1 receptor. IC50 was calculated by nonlinear regression using a sigmoidal function (PRISM, Graphpad, San Diego, CA). Inhibition constants (Ki) were calculated using the equation Ki = IC50/(1+ C/Kd), where Kd was the equilibrium dissociation constant of σ1 receptor for [3H]-(+)-pentazocine (3 nM) in rat liver [54]. https://doi.org/10.1371/journal.pone.0051910.g005 To address this, we conducted competition binding assays. σ1 receptors were labeled in rat liver homogenates, using the radioactive σ1 receptor ligand [3H]-(+)-pentazocine (5 nM). Previous study showed that the Bmax (maximal number of binding sites) of [3H]-(+)-pentazocine for σ1 receptor in the liver (2929 fmol/mg) is nearly 10 times higher than in the brain (280 fmol/mg) [52], [53]. Our western blot experiment also showed the amount of σ1 receptor in the liver is nearly 8 times of that in the mPFC (Ratio of gray density for σ1 receptor/GAPDH in the liver: 1.61±0.08; in the mPFC: 0.24±0.04) (Figure 5B). Thus, we selected liver tissue instead of mPFC tissue to prepare σ1 receptor for binding assays. Both NE-100 and haloperidol, which are high-affinity σ1 receptor ligands, were used to confirm the reliability of our binding assay system. The competitive binding curves of NE-100, haloperidol and MPH against [3H]-(+)-pentazocine were shown in Figure 5C. The inhibition constant (Ki) of haloperidol for σ1 receptor was similar with that reported by Klouz et al [54]. Our data showed that MPH could bind with σ1 receptor in a competitive manner (Figure 6C). The Ki of MPH for σ1 receptor was 14.91±4.22 µM (Figure 5D).

σ1 Receptor Involves in MPH-induced Locomotive Hyperactivity Therefore after, we adapted behavioral pharmacological experiments [35], to test if σ1 receptor involves in MPH-induced locomotor hyperactivity in mice. As described by previous studies, higher doses of MPH lead locmotor hyperactivity in rodents [21], [55], [56]. Indeed as shown in Figure 6A, MPH (2.5, 5, 10 mg/kg, i.p.) produced a stimulatory effect on the locomotor activity of the mice in a dose-dependent manner: saline group (n = 7), 1 mg/kg (P>0.05 vs saline, n = 7, post-hoc Dunnett’s tests), 2.5 mg/kg (P<0.05 vs saline, n = 7), 5 mg/kg (P<0.05 vs saline, n = 7), and 10 mg/kg (P<0.001 vs saline, n = 7). And post-hoc LSD multiple comparisons confirmed that 10 mg/kg group led more evident effect than other groups (F[4], [30] = 11.62, P<0.0001). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. MPH induces locomotor hyperactivity via interaction with σ1 receptor. (A) Swiss Webster mice were injected (i.p.) with saline and MPH (1, 2.5, 5 and 10 mg/kg). 30 min later, MPH produced a significant stimulatory effect on locomotor activity in a dose-dependent manner. The horizontal activity was analyzed for 30 min in the open field. *P<0.05 and ***P<0.001 vs. saline, n = 7 for each group, post-hoc Dunnett’s tests. (B) BD1063 (10, 20 and 30 mg/kg) itself did not affect basal locomotion of the mice, compared with saline group. n = 7 for each group. No significance. (C) Pretreatment with BD1063 (10, 20, and 30 mg/kg) effectively blocked 10 mg/kg MPH-induced locomotor hyperactivity. n = 7 for saline, and n = 6 for other groups. ***P<0.001 vs. saline and other groups, post-hoc LSD multiple comparisons. (D) Pretreatment with BD1063 (10 mg/kg) shifted the MPH’s dose-response curves to the right. The mice in the left curve were pretreated with saline, then injected with MPH (0–15 mg/kg). Other group in the right curve was pretreated with BD 1063 (10 mg/kg), then injected with MPH (5–30 mg/kg). MPH with 5 mg/kg and 10 mg/kg groups, *P<0.05 in the absence of BD1063 vs. in the presence of BD1063, n = 7 for each group, post-hoc Student-Newman-Keuls. (E) Locomotor activity trace of MPH (10 mg/kg) stimulatory mice pretreated with saline (middle), were significantly different from saline control (left). Pretreated with 10 mg/kg BD1063 (right) effectively blocked MPH’s effect (middle). https://doi.org/10.1371/journal.pone.0051910.g006 In the next antagonism experiments, a selective σ1 receptor antagonist BD1063 [57], was challenged to alter the stimulatory effect of MPH. As indicated by Figure 6B, BD1063 (10, 20, 30 mg/kg) itself did not alter basal locomotor activity of the mice, compared to saline group (n = 7 for each group, no significance). Importantly, we found that pretreatment with BD1063 could effectively block the MPH-induced hyperactivity (Figure 6C and 6E). 10 mg/kg MPH group pretreated with saline (0 mg/kg BD1063, n = 7) was significantly different from saline control (n = 7) and other BD1063 pretreatment groups (F[4], [27] = 10.261, P<0.0001, n = 6 for each group, post-hoc LSD multiple comparisons). Moreover, there was no significance between saline control and MPH pretreated with BD1063 (10, 20, or 30 mg/kg). Because BD1063 has a higher affinity for σ1 receptor compared with MPH [57], pretreated with BD1063 would pre-occupy σ1 receptors in vivo, and shift the MPH-induced dose-response curves. Indeed, we demonstrated that BD1063 (10 mg/kg) pretreatment elicited an obvious shift to the right in the MPH’s dose-response curves (Figure 6D, see the methods for details). And the locomotor activities of MPH (5 and 10 mg/kg) groups with saline were statistically higher than those pretreated with BD1063 (n = 7 for each group, P<0.05, Student-Newman-Keuls). Taken together, the behavioral pharmacological results provided evidence that higher dose of MPH induces the locomotor hyperactivity via interaction with σ1 receptors.