Methylphenidate exerts dose-dependent effects on glutamate receptors and behaviors

Abstract

Background: Methylphenidate (MPH), a psychostimulant drug used to treat attention-deficit/hyperactivity disorder, produces the effects of increasing alertness and improving attention. However, misuse of MPH has been associated with an increased risk of aggression and psychosis. We sought to determine the molecular mechanism underlying the complex actions of MPH.

Methods: Adolescent (4-week-old) rats were given one injection of MPH at different doses. The impact of MPH on glutamatergic signaling in pyramidal neurons of prefrontal cortex was measured. Behavioral changes induced by MPH were also examined in parallel.

Results: Administration of low-dose (.5 mg/kg) MPH selectively potentiated N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs) via adrenergic receptor activation, whereas high-dose (10 mg/kg) MPH suppressed both NMDAR-mediated and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated EPSCs. The dual effects of MPH on EPSCs were associated with bidirectional changes in the surface level of glutamate receptor subunits. Behavioral tests also indicated that low-dose MPH facilitated prefrontal cortex-mediated temporal order recognition memory and attention. Animals injected with high-dose MPH exhibited significantly elevated locomotive activity. Inhibiting the function of synaptosomal-associated protein 25, a key SNARE protein involved in NMDAR exocytosis, blocked the increase of NMDAR-mediated EPSCs by low-dose MPH. In animals exposed to repeated stress, administration of low-dose MPH effectively restored NMDAR function and temporal order recognition memory via a mechanism dependent on synaptosomal-associated protein 25.

Conclusions: These results provide a potential mechanism underlying the cognitive-enhancing effects of low-dose MPH as well as the psychosis-inducing effects of high-dose MPH.

Keywords: AMPA receptors; NMDA receptors; SNAP-25; methylphenidate; prefrontal cortex; stress.

Conflict of interest statement

All authors report no biomedical financial interests or potential conflicts of interest.

Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Low-dose MPH selectively enhances NMDAR-EPSC, while high-dose MPH reduces both NMDAR- and AMPAR-EPSC

(A, B) Input-output curves of NMDAR-EPSC (A) or AMPAR-EPSC (B) evoked by a series of stimulation intensities in PFC pyramidal neurons from rats with a single injection (i.p.) of saline, low-dose MPH (0.5 mg/kg) or high-dose MPH (10 mg/kg). *: p < 0.05, **: p < 0.01. Inset: representative EPSC traces. Scale bars: 50 pA, 100 ms (A); 50 pA, 20 ms (B). (C, D) Bar graph showing the paired-pulse ratio (PPR) of NMDAR-EPSC (interstimulus interval: 100ms) (C) or decay time constant of NMDAR-EPSC (D) in PFC pyramidal neurons taken from animals injected with saline, low-dose MPH or high-dose MPH. Inset: representative NMDAR-EPSC traces evoked by paired pulses. #: p < 0.001. Scale bar: 50 pA, 100 ms.

Figure 2. Low-dose MPH increases the surface level of NMDAR subunits, while high-dose MPH decreases the surface NMDAR and AMPAR expression

(A) Immunoblots and quantification analysis of the surface and total NMDAR and AMPAR subunits in PFC slices from rats injected with saline or MPH (0.5 mg/kg, or 2.5 mg/kg, i.p.). *: p < 0.05, #: p < 0.001. (B) Immunoblots and quantification analysis of the surface and total NMDAR and AMPAR subunits from the rats treated with saline or high-dose MPH (10 mg/kg, i.p.). *: p < 0.05.

Figure 3. Low-dose MPH enhances the temporal order recognition memory and attentional set shifting, while high-dose MPH elevates locomotor activity

(A, C) Bar graph (mean ± SEM) showing the discrimination ratio (DR) of temporal order recognition memory (TORM) tasks in animals treated with saline vs. MPH (A, 0.5 mg/kg, i.p.; C, 10mg/kg, i.p.) *: p < 0.05. (B) Bar graph showing the number of trials to criterion (6 consecutive correct trials) for each discrimination stage of the attentional set-shifting task in animals treated with saline or a low-dose MPH (0.5 mg/kg, i.p.). **: p < 0.01. (D) Bar graph showing the number of midline crossing in locomotion apparatus for animals injected with saline vs. MPH (low or high dose). #: p < 0.001.

Figure 4. Low-dose MPH potentiates NMDAR-EPSC via norepinephrine reuptake inhibition and adrenergic receptor activation

(A, B) Summarized input-output curves of NMDAR-EPSC in PFC pyramidal neurons from rats treated with saline, Maprotiline (A, 20 mg/kg, i.p.), or GBR-12909 (B, 5 mg/kg, i.p.). Inset: representative traces of NMDAR-EPSC. Scale bar: 50 pA, 200 ms. **: p < 0.01. (C) Summarized input-output curves of NMDAR-EPSC in saline- vs. MPH- (0.5 mg/kg, i.p.) injected rats pre-treated with prazosin and yohimbine (Prazosin, 1 mg/kg, Yohimbine, 5 mg/kg, i.p., injected 0.5 hr before MPH injection). Inset: representative NMDAR-EPSC traces. Scale bar: 50 pA, 100 ms. (D) Summarized input-output curves of NMDAR-EPSC in saline- vs. MPH-injected rats pre-treated with SCH23390 and sulpiride (SCH23390, 1 mg/kg, sulpiride, 50 mg/kg, i.p., injected 0.5 hr before MPH injection). Inset: representative traces. Scale bar: 50 pA, 100 ms. **: p < 0.01.

Figure 5. SNAP-25 participates in the potentiation of NMDAR-EPSC and cognitive functions by low-dose MPH

(A) Summarized input-output curves of NMDAR-EPSC in saline- vs. MPH- (0.5 mg/kg, i.p.) injected rats pre-treated with SNAP-25 blocking peptide (SNAP-25 pep, 0.6 pmol/g, i.v.) or a scrambled peptide (sc pep, 0.6 pmol/g, i.v.). Inset: representative EPSC traces. Scale bar: 50 pA, 200 ms. *: p < 0.05. (B, C) Immunoblots (B) and quantification analysis (C) of the surface and total NMDAR subunits in rat PFC slices from saline- vs. MPH-injected rats pre-treated with SNAP-25 blocking peptide or a scrambled peptide. **: p < 0.01. (D, E) Bar graphs showing the discrimination ratio (DR) of temporal order recognition memory (TORM) tasks (D) or number of trials to criterion at each discrimination stage of the attentional set-shifting task (E) in MPH (0.5 mg/kg, i.p.)-injected animals pre-treated with a scrambled peptide or SNAP-25 blocking peptide. *: p < 0.05.

Figure 6. PFC infusion of SNAP-25 blocking peptide abolished the low-dose MPH-induced enhancement of NMDAR-EPSC and temporal order recognition memory

(A, B) Summarized input-output curves of NMDAR-EPSC (A) or bar graph (mean ± SEM) showing the discrimination ratio (DR) of temporal order recognition memory (TORM) tasks (B) in saline vs. MPH (0.5 mg/kg, i.p.)-injected animals with PFC infusion of a scrambled peptide or SNAP-25 blocking peptide (3 pmol/site). Inset: representative EPSC traces. Scale bar: 50 pA, 200 ms. *: p < 0.05, **: p<0.01.

Figure 7. Low-dose MPH restores the impaired NMDAR function and recognition memory in animals exposed to repeated stress

A, summarized input-output curves of NMDAR-EPSC in control or repeatedly stressed (RS) rats treated with saline or MPH (0.5 mg/kg, i.p.) without or with SNAP-25 peptide (S26p, 0.6 pmol/g, i.v.) pretreatment. *: p < 0.05, #: p < 0.001. Inset: representative NMDAR-EPSC traces. Scale bar: 50 pA, 200 ms. B, C, Bar graphs (mean ± SEM) showing the DR (B) and total exploration time (C) of TORM tasks in repeatedly stressed animals injected with saline or MPH without or with SNAP-25 peptide pretreatment. **: p < 0.01.