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).
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.
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.
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.
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].
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).
Figure 7.
MPH enhancement of NMDA-R current is mediated via intracellular Ca2+ dependent PLC/PKC signaling pathway.
MPH produced no effect on NMDA-induced current when the PLC inhibitor U73122, the PKC inhibitor chelerythrine, the Ca2+ chelating reagent BAPTA, or the IP3 inhibitor 2-APB was administered, but still enhanced the current when the PKA inhibitor fragment 5-24 (PKI5-24) or L-type Ca2+ channel blocker nefedipine was applied. NMDA-R currents were recorded in the presence of CNQX, TTX and BMI, with holding potential of -40 mV. Shown in figure are the normalized histograms for MPH effects in the presence of U73122, chelerythrine, PKI5-24, BAPTA, nefedipine and 2-APB. *P<0.05 vs. control. **P<0.01 vs. control, paired t-test. Numbers mean the cell recorded.
Figure 8.
PRE-084 enhances NMDA-induced current.
The specific σ1 receptor agonist PRE-084 (5 µM) enhanced NMDA-induced current, as MPH did. Recordings of NMDA-R current were performed in the presence of CNQX (20 µM), TTX (1 µM) and BMI (20 µM), with holding potential of −40 mV. **P<0.01 vs. control, n = 6, paired t-test.
Figure 9.
A novel mechanism for the MPH enhancement of NMDA-R activity.
As a blocker of DA/NE transporters, MPH increases the DA/NE level in the synaptic cleft. In addition, MPH at higher dose could potentiate NMDA-R mediated exciatatory synaptic transmission, via a catecholamine-independent mechanism. Through action at postsynaptic σ1 receptor, MPH activates PLC, which subsequently cleaves PIP2 into DG and IP3. Under IP3-induced intracellular Ca2+ release, PKC activated by DG may result in the phosphorylation of NMDA receptors, and enhancement of NMDA-R activity.