Molecular and Neurochemical Effects of Methylphenidate on the Developing Brain
Attention deficit hyperactivity disorder (ADHD) affects 2.6-4.5% of the worlds population, with affected children often performing poorly at school. The catecholamine hypothesis of ADHD proposes that the disorder results from a hypofunction of the fronto-striato-cerebellar catecholaminergic (i.e. dopaminergic and noradrenergic) system. However, abnormalities in the glutamatergic and GABAergic systems have also been implicated in the symptoms of ADHD. In addition, previous reports indicate that a deviation from the common pattern of cerebral lateralisation also contribute to some of the symptoms of ADHD, with under-activation of the right prefrontal and right parietal cortices observed in ADHD patients. The psychostimulant, methylphenidate (MPH) is the preferred drug for the treatment of the disorder, however the long-term consequences of the drug are not yet established. Here, we have investigated the effects of acute and chronic MPH treatment on monoaminergic protein markers, genes and their corresponding proteins involved in synaptic plasticity, as well as the drug’s effect on metabolic pathways. The investigations were conducted on brain regions implicated in the symptoms of ADHD in adolescent rats. Our data shows in Chapter 3 that MPH increases whole tissue and extracellular dopamine levels in the frontal cortex and striatum. Interestingly, this effect was more prominent in the left sides of these brain areas. Chronic MPH treatment also upregulated dopamine (DAT) and noradrenaline (NET) transporters in the striatum and the nucleus accumbens, an effect that could limit the long-term effectiveness of the drug, as larger doses may be required to reach a functional blockade of the upregulated transporters and achieve clinical efficacy. In addition, chronic MPH but not acute treatment increased protein expression of the: vesicular monoamine transporter (VMAT2), dopamine D1 receptor, and the catecholamine rate-limiting enzyme tyrosine hydroxylase (TH), in most of the examined brain areas. By using proton nuclear magnetic resonance (1H-NMR) spectroscopy, this thesis also shows that acute MPH enhances the levels of the large neutral amino acids (LNAAs), tyrosine and phenylalanine in the cerebrum (Chapter 4). In Chapter 5 the thesis further demonstrates that chronic but not acute MPH treatment increases the levels of tyrosine and phenylalanine in the frontal cortex, while also decreasing the levels of these LNAAs in the plasma, suggesting that the drug increases the transport of the these two LNAAs from the plasma into parts of the brain. Given that these two LNAAs are precursors of dopamine synthesis, their enhanced levels following MPH treatment in the frontal cortex but not the striatum (no significant increase of tyrosine or phenylalanine in striatum) this finding might contribute to the elevated cortical levels of dopamine as shown in Chapter 3. This novel finding has resulted in our hypothesis that MPH may alleviate ADHD symptoms in two ways: (1) by a blockade of DAT and NET, and (2) by enhancing the brain pool of tyrosine and phenylalanine. Interestingly, MPH had a similar effect on the energy-related metabolites, lactate and acetate, favouring their transport from the plasma into the frontal cortex and hippocampal areas. In addition, glutamate and GABA levels were also increased by MPH in the frontal cortex and hippocampus. Together, these findings provide support for the contribution of dopaminergic, glutamatergic and energy-related metabolites in the mechanism of action of the anti-ADHD drug, MPH. Contrasting findings were observed following acute and chronic MPH treatment in the cerebellum (Chapter 6), which were both dose and treatment duration-dependent. Acute treatment decreased cerebellar glutamate, N-acetylaspartate and VMAT2, while chronic treatment increased glutamate, tyrosine and VMAT2. However, the chronic MPH-induced increase of tyrosine and VMAT2 did not correlate with increased cerebellar dopamine, which would suggest that in the cerebellum, the increased tyrosine may elevate the synthesis of noradrenaline rather than dopamine. In chapter 7, the thesis shows that chronic MPH treatment increases mRNA and protein expression of markers mediating neuroplasticity. Here, chronic MPH increased Arc, IRSp53/58, Cdc42, and Arp2 levels in the: striatum, nucleus accumbens and hippocampus. The MPH-induced increase of these synaptic plasticity-associated markers in these brain regions may induce changes in dendritic spine morphology, as well as in the consolidation of memory strength and drug-seeking behaviour. In conclusion, the present thesis demonstrates, by applying a wide range of bioanalytical techniques, that the anti-ADHD drug, MPH, has major effects on: catecholaminergic protein markers, metabolic markers, and proteins mediating neuronal plasticity in the adolescent brain. The factors influencing some of these effects were dependent on regional differences, cerebral lateralisation, drug dosage and treatment duration.
- PhD