The Impact of Psychostimulant Administration During Development on Adult Brain Functions Controlling Motivation, Impulsivity and Cognition
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The impact of psychostimulant administration during development on adult brain functions controlling motivation, impulsivity and cognition. Mathieu Di Miceli Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy. July 2016 Thesis conducted under the supervision of Dr. Benjamin S. Gronier, first supervisor and Dr. Tyra S. Zetterström, second supervisor. Department of Pharmacology, School of Pharmacy, De Montfort University, Leicester, LE1 9BH, United Kingdom Abstract ADHD pharmacotherapy uses methylphenidate (MPH), D-amphetamine (D- amph), two psychostimulants targeting dopamine transporters, or atomoxetine (ATX), specifically targeting norepinephrine transporters. We have assessed the pharmacological mechanisms of these three drugs on the in vitro efflux of neurotransmitters in rat prefrontal cortex (PFC) and striatal slices as well as on the in vivo electrical activities of PFC pyramidal neurons, striatal medium spiny neurons, ventral tegmental area dopamine neurons or dorsal raphe nucleus serotonin neurons, using single cell extracellular electrophysiological recording techniques. We have also tested whether chronic methylphenidate treatment, during either adolescence or adulthood, could have long-lasting consequences on body growth, depression and neuronal functions. Release experiments showed that all ADHD drugs induce dose-dependent dopamine efflux in both the PFC and striatum, with different efficacies, while only D- amph induced cortical norepinephrine efflux. Atomoxetine induced an unexpected massive dopamine outflow in striatal regions, by mechanisms that depend on physiological parameters. Our electrophysiological studies indicate that all three drugs equally stimulate the excitability of PFC pyramidal neurons, in basal and NMDA-evoked conditions, when administered acutely (3 mg/kg). While the electrophysiological effects elicited by psychostimulants may be dependent on D1 receptor activation, those induced by atomoxetine relied on different mechanisms. In the ventral tegmental area (VTA), methylphenidate (2 mg/kg), but not atomoxetine, induced firing and burst activity reductions, through dopamine D2 autoreceptor activation. Reversal of such effects (eticlopride 0.2 mg/kg) revealed an excitatory effect of methylphenidate on midbrain dopamine neurons that appear to be dependent on glutamate pathways and the combination of D1 and alpha-1 receptors. Finally, acute intraperitoneal psychostimulant i injections increased vertical locomotor activity as well as NMDA2B protein expression in the striatum. Some animals chronically treated with intraperitoneal administrations (methylphenidate 4 mg/kg/day or saline 1.2 ml/kg/day) showed decreased body weight gain. Voluntary oral methylphenidate intake induces desensitisation to subsequent intravenous methylphenidate challenges, without altering dopamine D2 receptor plasticity. Significant decreases in striatal NMDA2B protein expression were observed in animals chronically treated. After adolescent MPH treatment, midbrain dopaminergic neurons do not display either desensitisation or sensitisation to intravenous methylphenidate re-challenges. However, partial dopamine D2 receptor desensitisation was observed in midbrain dopamine neurons. Using behavioural experiments, cross-sensitisation between adolescent methylphenidate exposure and later-life D-amphetamine challenge was observed. Significant decreases in striatal NMDA2B protein expression were observed in animals chronically treated, while striatal medium spiny neurons showed decreased sensitivities to locally applied NMDA and dopamine. While caffeine is devoid of action on baseline spike generation and burst activity of dopamine neurons, nicotine induces either firing rate enhancement, firing rate reduction, or has no consequences. Adolescent methylphenidate treatment leads to decreased neuronal sensitivities to the combination of nicotine, MPH and eticlopride, compared to controls. Finally, nicotine partially prevented D-amphetamine-induced increase of rearing activities. Our results show that increases in the excitability of PFC neurons in basal conditions and via NMDA receptor activation may be involved in the therapeutic response to ADHD drugs. Long-term consequences were observed after psychostimulant exposure. Such novel findings strengthen the mixed hypothesis in ADHD, whereby both dopamine and glutamate neurotransmissions are dysregulated. Therefore, ADHD therapy may now focus on adequate balancing between glutamate and dopamine. ii Acknowledgements First I would like to thank De Montfort University for allowing me to follow this Ph-D program, for paying my tuition fees and giving me financial support in the form of a stipend. For the last 3 years, it has been a real pleasure to come to the University and work with such amazing colleagues, to all of whom I express my heartfelt gratitude. I would also like to thank all the members of staff involved in the radioactivity work: Liz O’Brien, for all the help she provided regarding isotope regulations, radioactivity handling as well as laboratory health and safety; Nazmin Juma and Dr. Ketan Ruparelia for their patience in collecting the radioactive waste and pointing out some mistakes they found in my log book entries while doing so. This work could never have been done without technical support from the B.S.U. team, including Anita O’Donoghue, Stephen Bowen (†) and Mike Storer. Their assistance in maintaining high standards within the animal unit has allowed us to save time and effort, but has also had an impact on the quality of work that we were able to perform. I would like to thank all the technicians in room 2.17 for their immense technical support, in particular David Reeder, Amrat Khorana, Claire West, Jo Tonkin and Leonie Hough. By providing gallons of purified water as well as many consumables, this work could not have been done without their almost daily assistance. On more than one occasion, I have found myself thinking about all the animals used during the last 3 years. I would like to emphasis the help they provide by increasing our knowledge in biology and Science. Memento Mori. I would also like to express my gratitude to my second supervisor, Dr. Tyra Zetterström for her support and ideas from start to end of this Ph-D. Last but of great importance, I personally thank my supervisor, Dr. Benjamin Gronier, for his three years of support, his extensive scientific knowledge and for increasing my research skills. Pour tout cela et tout le reste, merci beaucoup ! À ma famille, sans laquelle je n’aurais jamais pu arriver jusqu’au doctorat. iii Abbreviations ADHD Attention Deficit and Hyperactivity Disorder AMPA 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid ATX Atomoxetine BAPTA-AM Bis(aminophenoxy)ethane-N-tetraacetic acid-acetoxymethyl ester CNVs Copy number variation(s) D-amph Dextro-amphetamine oC Degree Celsius DAT Dopamine transporter EC50 Dose inducing 50% of maximum response EPSCs Excitatory post-synaptic currents fMRI Functional magnetic resonance imaging g - kg Gram - kilogram GABA γ-amino-butyric acid GlyT1 Glycine reuptake transporter 1 IC50 Dose inducing 50% of maximum inhibition i.e. id est IPSCs Inhibitory post-synaptic currents KATP ATP-gated potassium channel Ki Inhibitory constant, binding affinity KIR Inwardly rectifying potassium channel Kv1 Voltage-gated potassium channel, family 1 l Litre LTD Long-term depression LTP Long-term potentiation mEq Milliequivalent min Minute ml Millilitre mm Millimetre mM Millimolar MΩ Megaohm iv MPH Methylphenidate ms Millisecond MSN Medium spiny neurons nA Nanoampere NET Norepinephrine transporter nM Nanomolar NMDA N-methyl-D-aspartate % Percentage PFC Prefrontal cortex rpm Rotations per minute s - sec Second SERT Serotonin transporter SHR Spontaneously hypertensive rat SNPs Single nucleotide polymorphism(s) µm Micrometre µM Micromolar VMAT2 Vesicular monoamine transporter 2 VTA Ventral tegmental area v/v Volume to volume ratio w/v Weight to volume ratio v Table of content The impact of psychostimulant administration during development on adult brain functions controlling motivation, impulsivity and cognition. ...................................... i Abstract ............................................................................................................................ i Acknowledgements ........................................................................................................ iii Abbreviations ................................................................................................................. iv Table of content ............................................................................................................. vi Table of figures .............................................................................................................. xi List of tables ................................................................................................................ xvii Introduction .................................................................................................................... 1 ADHD: statistics and symptoms ................................................................................... 1 Aetiology of ADHD ...................................................................................................... 2 Neurotransmitters and brain regions implicated in ADHD .......................................... 4 ADHD drugs pharmacology ........................................................................................