Acute oral administration of low doses of methylphenidate targets calretinin neurons in the rat septal area

Abstract

Methylphenidate (MPD) is a commonly administered drug to treat children suffering from attention deficit hyperactivity disorder (ADHD). Alterations in septal driven hippocampal theta rhythm may underlie attention deficits observed in these patients. Amongst others, the septo-hippocampal connections have long been acknowledged to be important in preserving hippocampal function. Thus, we wanted to ascertain if MPD administration, which improves attention in patients, could affect septal areas connecting with hippocampus. We used low and orally administered MPD doses (1.3, 2.7 and 5 mg/Kg) to rats what mimics the dosage range in humans. In our model, we observed no effect when using 1.3 mg/Kg MPD; whereas 2.7 and 5 mg/Kg induced a significant increase in c-fos expression specifically in the medial septum (MS), an area intimately connected to the hippocampus. We analyzed dopaminergic areas such as nucleus accumbens and striatum, and found that only 5 mg/Kg induced c-fos levels increase. In these areas tyrosine hydroxylase correlated well with c-fos staining, whereas in the MS the sparse tyrosine hydroxylase fibers did not overlap with c-fos positive neurons. Double immunofluorescence of c-fos with neuronal markers in the septal area revealed that co-localization with choline acethyl transferase, parvalbumin, and calbindin with c-fos did not change with MPD treatment; whereas, calretinin and c-fos double labeled neurons increased after MPD administration. Altogether, these results suggest that low and acute doses of methylphenidate primary target specific populations of caltretinin medial septal neurons.

Keywords: ADHD; calcium binding proteins; catecholamines; methylphenidate; theta rhythm.

Figure 1

c-fos expression augments with MPD treatment in MS/VDB, but not horizontal diagonal band (HDB). Map showing the location of the analyzed areas vertical medial septum (MS/VDB) and HBD (A). Graphs showing the quantification of c-fos positive area expressed as the percentage of the total area analyzed and normalized to the average of control values in MS/VDB (B) and HDB (C). Results were analyzed with one-way ANOVA, followed by Bonferroni post hoc test **p < 0.01). Representative images of c-fos staining in the MS/VDB vehicle or MPD treated at the indicated dosages (D–G); representative images of c-fos neurons in the HBD from rats treated with vehicle or MPD at the indicated dosages (H–K). Calibration bar 100 µm.

Figure 2

MPD induces c-fos expression in Striatum but not nucleus accumbens. Map showing the location of the analyzed areas (A). Quantification of c-fos positive area represented as percentage of total area analyzed and normalized by average value of controls, accumbens shell (B) and striatum (C). Results analyzed with one-way ANOVA, followed by Bonferroni post hoc test *p < 0.05. Representative images of c-fos staining in accumbens shell (D–G) and striatum (H–K) in vehicle and MPD treated rats at the indicated concentrations. Calibration bar 100 µm.

Figure 3

Double immunocitochemistry TH-c-fos. Representative images illustrating double TH and c-fos staining in the dorsal part of LS (A–C); MS/VDB (D–F); nuccleus accumbens (G–I) and striatum (J–L) in vehicle and MPD treated rats at the indicated dosages. The vertical line (D–F) represents the middle line of septum. Calibration bar 100 µm (A–L).

Figure 4

Quantification of double immunofluorescence. The number of double labeled neurons in the MS/VBD was expressed as percentage of total CR, CB, PV or ChAT positive neurons. Five to ten photographs were taken from at least three different subjects of control and 5 MPD groups.

Figure 5

Confocal images of double immunofluorescence. Low magnification captures immunofluorescence of a representative case MPD 5 mg/Kg showing different areas occupied by ChAT (green) (A); PV (green) (D); CB (G); and CR (J). c-fos positive neurons (red) (B,E,H,K); and merged (C,F,I,L). Calibration bar 100 µm (A–I) and 50 µm (J–L). High magnification of the double immunofluorescences was used for quantification and are represented in the insets. Calibration bar 50 µm.

Figure 6

Proposed mechanism for MPD role in attention. Taking together the present data and others we propose that MPD may inhibit dopamine re-uptake leaving more dopamine in the extracellular space (1). This extra amount of dopamine will more effectively activate D1/5 receptors in the medial septal neurons. Specifically, the activation of CR neurons in this area will potentiate the effect on intrinsic connections inside the medial septum and descending projections over areas projecting to hippocampus (2). Then subcortical projections may enhance theta rhythm and consequently attention mechanisms (3).