Early postnatal exposure to methylphenidate alters stress reactivity and increases hippocampal ectopic granule cells in adult rats

Abstract

To mimic clinical treatment with methylphenidate (MPH; Ritalin) for attention deficit/hyperactivity disorder (ADHD), rat pups were injected with MPH (5 mg/kg, i.p.) or placebo twice daily during their nocturnal active phase from postnatal day (PND) 7-35. Thirty-nine days after the last MPH administration (PND 76), four litters of rats experienced stressful conditions during the 2003 New York City blackout. MPH-treated rats that endured the blackout lost more weight and regained it at a slower pace than controls (p<0.05; N=7-11 per group). Furthermore, MPH-treated rats had elevated systolic arterial blood pressure (from 115.6+/-1.2 to 126+/-1.8 mmHg; p<0.05), assessed on PND 130 by tail cuff plethysmography. Immunocytochemical studies of transmitter systems in the brain demonstrated rearrangements of catecholamine and neuropeptide Y fibers in select brain regions at PND 135, which did not differ between blackout and control groups. However, MPH-treated rats that endured the blackout had more ectopic granule cells in the hilus of the dorsal hippocampal dentate gyrus compared to controls at PND 135 (p<0.05; N=6 per group). These findings indicate that early postnatal exposure to high therapeutic doses of MPH can have long lasting effects on the plasticity of select brain regions and can induce changes in the reactivity to stress that persist into adulthood.

Fig. 1. Following the New York City blackout body weight was more affected in rats that had received early postnatal MPH

Six weeks after cessation of treatment, male rats that received MPH and experienced the New York City blackout lost weight instead of continuing to gaining weight as compared to control rats that also endured the New York City blackout. Inset shows changes in the weight gain at PND 79 relative to days preceding the black out (PND 74–78). p < 0.05; N= 7–11 per group; bars represent average and SEM

Fig. 2. Three months after cessation of administration, rats that received MPH and also endured the New York City blackout had significantly higher ABP

Three months after cessation of treatment (approximately PND130), MPH administration during development (PND7 - 35) significantly influenced ABP in male rats that had endured the New York City blackout. * = p< 0.05 (post hoc); bars represent averages and SEM. Numbers inside bars represent animals/group.

Fig. 3. Distribution of catecholamine fibers or neuropeptide Y was not different 3 months later (PND135) after MPH administration in animals that endured the New York City blackout

A. Density of TH-immunoreactive fibers in the medial prefrontal cortex (PFC) of MPH-administered rats (A₂) compared to controls (A₁). Atlas level 9, AP = +2.80 from Bregma. cc: corpus callosum. B. TH-immunoreactivity in coronal sections of striatum (atlas level 16, AP = +0.10 from Bregma) in control (B₁) and MPH administered rats (B₂). C. Controls (C₁) and MPH-administered rats (C₂) had similar densities of TH-labeled fibers in the central hilus (ch). Gcl: granule cell layer, sgz: subgranular zone. Atlas level 32, AP = −3.90 from Bregma. D. NPY-immunoreactive fibers in the PVN in MPH-treated rats (D₂) compared to controls (D₁). Atlas levels 26, AP = −1.78 from Bregma. All atlas references from [44]. Pictures were adjusted for brightness and contrast. Bar A and C: 50 μm; B: 200 μm; D: 250 μm

Fig. 4. More ectopic granule cells were observed in adult rats that had received MPH during development and had endured the New York City blackout

Photomicrographs of the dorsal hippocampal dentate gyrus (atlas level 32, AP = −3.90 from Bregma [44]) labeled with PROX in a control (A) and MPH-administered rat (B) that had endured the New York City blackout. In both conditions, PROX-labeled nuclei were most prominent in the granule cell layer (GCL). However, more PROX-labeled nuclei (arrows) were found in the hilus (hil), especially the subgranular zone of the MPH-administered rats. Bar, 100 μm. C. A significant increase (*) in the number of PROX-labeled nuclei in the hilus was seen in MPH administered rats compared to controls. * = p< 0.05 (independent sample t-test), N = 6/condition. Pictures were adjusted for brightness and contrast. Bar, 50 μm.