A Clinically-Relevant Dose of Methylphenidate Enhances Synaptic Inhibition in the Juvenile Rat Prefrontal Cortex

Abstract

Methylphenidate (MPH) is perhaps the most commonly prescribed psychoactive substance for young children and adolescents; however, its effects on the immature brain are not well understood. MPH is increasingly abused by adolescents and prescriptions are being issued to increasingly younger children without rigorous psychological testing, raising the potential for misdiagnosis; it is therefore crucial to understand how this drug might impact a healthy, developing brain. Recently, we have shown that a clinically-relevant dose of MPH depresses the activity of pyramidal neurons in the prefrontal cortex of normal juvenile rats, but its effects on inhibitory synaptic transmission remain to be explored. We therefore recorded spontaneous (s), miniature (m), and evoked (e) inhibitory postsynaptic currents (IPSCs) in layer 5 pyramidal neurons in juvenile rat prefrontal cortex. We found a dose-dependent effect of MPH on sIPSC frequency but not amplitude, where 0.3 mg/kg significantly decreased frequency, but 1 mg/kg significantly increased frequency. Moreover, mIPSCs were not affected by either dose of MPH, whereas the amplitudes, as well as paired-pulse ratios and coefficient of variations of evoked IPSCs were significantly increased after MPH treatment, indicating a presynaptic action. Tonic GABA current was also not affected by MPH treatment. Taken together, these results suggest that MPH administration to a healthy juvenile may enhance excitation of GABAergic interneurons; thus shifting the excitation-inhibition balance in the prefrontal cortex towards inhibition, and depressing overall prefrontal cortical activity. Our findings also indicate that the adolescent brain is more sensitive to MPH than previously thought, and dose ranges need to be reconsidered for age as well as size.

Keywords: ADHD; GABA; Interneuron; Juvenile; Prefrontal cortex; Psychostimulant.

Figure 1

Acute treatment with MPH dose-dependently affects the frequency, but not amplitude, of sIPSCs. A) Representative traces of sIPSC recordings from saline-treated neurons, 0.3 mg/kg MPH-treated neurons, and 1.0 mg/kg MPH-treated neurons. B) The frequency of sIPSCs was significantly decreased following 0.3 mg/kg MPH (n = 12, p = 0.0002), but significantly increased following 1.0 mg/kg MPH (n = 12, p = 0.0006). C) The amplitudes of sIPSCs following both 0.3 mg/kg MPH (n = 12, p = 0.16) and 1.0 mg/kg MPH (n = 12, p = 0.3) were not significantly affected compared with saline control (n = 10). D) The sIPSC decay times were significantly increased following either dose of MPH, although to a greater degree following 0.3 mg/kg treatment (p = 0.0009) than 1.0 mg/kg treatment (p = 0.044).

Figure 2

The amplitude of evoked IPSCs increased following 0.3 mg/kg and 1 mg/kg MPH. A) Representative traces of eIPSCs from saline, 0.3 mg/kg or 1 mg/kg MPH-treated neurons. B) Graphical representation of eIPSC amplitudes. The amplitudes of evoked IPSCs were significantly increased following MPH [F (2, 27), = 81.79, p = 3.5 × 10⁻¹²]. C) Stimulus intensity was not significantly different across the three treatment groups (p > 0.05). D) Paired-pulse ratios were significantly increased following 1 mg/kg MPH treatment [F (2, 27) = 24.87; p = 1.41 × 10⁻⁶] but not 0.3 mg/kg; further analysis revealed that each paired-pulse ratio was significantly increased following the 1 mg/kg MPH when compared to the equivalent saline paired-pulse ratio. Following 0.3 mg/kg MPH treatment, only the amplitudes to pulses 3 (p = 0.026) and 9 (p = 0.017) were increased. E) Coefficient of variances were increased by 0.3 mg/kg MPH (p = 0.021) and 1 mg/kg MPH (p = 0.0006).

Figure 3

Miniature IPSCs are unchanged by MPH treatment. A) Representative traces from saline and 1 mg/kg MPH-treated neurons reveal similar amplitude and frequency. B) Frequency was unchanged by MPH treatment (p = 0.35). C) Amplitude of mIPSCs was unchanged by MPH treatment (p = 0.35), and D) Decay time was not altered (p = 0.12).

Figure 4

Tonic GABAergic current is not affected by MPH treatment. A) Representative traces showing recordings from saline control and 1 mg/kg MPH-treated animals (n = 8 saline, 7 MPH). B) The baseline current recorded in the first five minutes in the presence of DNQX and APV is not affected by MPH treatment (−79 + 9.3pA for saline vs. −96 + 26pA for MPH; p = 0.277). C) Tonic GABA current, measured as the current shift induced by the application of gabazine, is not affected by MPH treatment (1.6 + 4.45pA for saline vs. 5.06 + 8.72pA for MPH; p = 0.366).