Tonic GABAergic inhibition as a new way to regulate mesolimbic dopamine system Elena Vashchinkina Institute of Biomedicine, Pharmacology University of Helsinki Academic Dissertation To be presented with the permission of the Medical Faculty of the University of Helsinki, for public examination in lecture hall 3, Biomedicum Helsinki 1, Haartmaninkatu 8, on December 13th 2013 at 12 noon Helsinki 2013 Supervisor Professor Esa R. Korpi, MD PhD Institute of Biomedicine, Pharmacology Faculty of Medicine University of Helsinki, Finland Reviewers Docent Mikko Airavaara, PhD Institute of Biotechnology University of Helsinki, Finland Docent Tarja Stenberg, MD PhD Institute of Biomedicine, Physiology University of Helsinki, Finland Dissertation Opponent Professor Kimmo Jensen, MD PhD Department of Biomedicine Aarhus University, Denmark ISBN 978-952-10-9636-5 (paperback) ISBN 978-952-10-9637-2 (PDF) http://ethesis.helsinki.fi Unigrafia OY Helsinki 2013 TABLE OF CONTENTS Abstract ......................................................................................................................... 1 Original publications ................................................................................................... 2 Abbreviations ............................................................................................................... 3 Glossary of terms ......................................................................................................... 4 1 INTRODUCTION ..................................................................................................... 5 2 REVIEW OF THE LITERATURE ........................................................................ 7 2.1 The GABAA receptor system ........................................................................... 7 2.1.1 Diversity of inhibition through GABAA receptors ....................................... 8 2.1.2 GABAA receptors underlying tonic currents ............................................... 10 2.1.3 Origin of tonic currents ............................................................................... 11 2.1.4 The mechanism of tonic inhibition ............................................................. 12 2.1.5 Modulators of extrasynaptic GABAA receptors ......................................... 13 2.1.6 Extrasynaptic GABAA receptor as a potential drug target .......................... 17 2.2 The dopamine reward system ....................................................................... 20 2.2.1 Neurophysiology of reward ........................................................................ 21 2.2.2 Dopamine reward circuits ........................................................................... 22 2.2.3 Afferent control of VTA neurons ............................................................... 22 2.2.4 Diversity of VTA neurons .......................................................................... 24 2.2.5 Synaptic plasticity in VTA DA neurons ..................................................... 26 2.2.6 Drug-induced synaptic plasticity in VTA DA neurons .............................. 27 2.2.7 Modulation of dopamine system via GABAergic agents ........................... 29 3 AIMS OF THE STUDY ......................................................................................... 31 4 MATERIALS AND METHODS .......................................................................... 33 4.1 Experimental animals .................................................................................... 33 4.2 Electrophysiological studies .......................................................................... 33 4.2.1 Acute midbrain VTA-slice preparation ...................................................... 34 4.2.2 Identification of the targeted neurons ......................................................... 34 4.2.3 Electrophysiological recordings and data analysis ..................................... 34 4.3 Behavioral studies .......................................................................................... 34 4.3.1 Locomotor activity of mice ......................................................................... 35 4.3.2 Light-dark choice test ................................................................................. 35 4.3.2 Individual exploratory behavior .................................................................. 35 4.3.3 Place-conditioning for preference/aversion ................................................ 35 4.3.4 Intravenous drug self-administration in mice ............................................. 35 4.4 Immunohistochemical studies ....................................................................... 36 4.4.1 c-Fos immunohistochemistry ...................................................................... 36 4.5 Statistical tests ................................................................................................ 36 5 RESULTS AND DISCUSSION ............................................................................. 39 5.1 THIP and GAN induced persistent LTP of LTP of glutamatergic synapses on VTA DA neurons....................................................................................... 39 5.1.1 THIP and GAN increased AMPA/NMDA receptor-mediated current ratio in Th-EGFP mice ........................................................................................ 39 5.1.2 THIP and GAN were unable to induce glutamate neuroplasticity in δ-KO mice ............................................................................................................. 41 5.1.3 Mechanism of THIP-induced glutamate neuroplasticity. Electrophysiological study .......................................................................... 42 5.1.4 Tonic inhibition in VTA ............................................................................. 43 5.1.5 Mechanism of GAN-induced glutamate neuroplasticity. Electrophysiological studies ........................................................................ 44 5.2 Behavioral significance of the synaptic plasticity induced in the VTA ...... 45 5.2.1 THIP exerted no reinforcement, but instead induced conditioned aversion in wild-type mice and baboons .................................................................... 46 5.2.2 GAN induced conditioned aversion in wild-type mice, but not in δ-KO mice ............................................................................................................. 47 5.3 Mechanism of THIP-induced glutamate plasticity. The c-Fos study ................ 48 5.4 Aversive THIP effects in abusers. Its limitations to treat primary insomnia .... 50 6 CONCLUSIONS .................................................................................................... 53 7 ACKNOWLEDGEMENTS .................................................................................. 54 8 REFERENCES ....................................................................................................... 56 ABSTRACT Dopamine (DA) neurons of the ventral tegmental area (VTA) are critical for decision- making and motivation and have also been implicated in the development of addictive behaviors. The activity of these neurons and the subsequent changes in DA concentrations in the target regions of the VTA are strictly regulated by both excitatory and inhibitory inputs. Among those inhibitory inputs, GABAergic transmission is mediated by phasic and tonic currents generated through different GABAA receptor subtypes. Although the phasic currents arising through the activation of synaptic GABAA receptors have been well described, much less is known about extrasynaptic GABAA receptors mediating tonic currents and modulating neuronal activity in the VTA. Here, pharmacologically selective receptor modulators, transgenic mouse models and brain slice electrophysiology were all exploited to probe the role of extrasynaptic GABAA receptors in mediating neuroplasticity in VTA DA neurons. Even though they possess distinct molecular sites of action, gaboxadol (THIP) and ganaxalone (GAN) enhanced tonic inhibition by selective targeting of the extrasynaptic δ subunit-containing GABAA receptors located on VTA GABA neurons. The tonic inhibition induced in these neurons appeared to be sufficient to disinhibit DA neurons and induce persistent neuroplasticity in the glutamate synapses on VTA DA neurons, which resulted from insertion of new GluA2 subunit-lacking AMPA receptors into the synapses. Screening of reward-related behaviors associated with VTA DA activity revealed that THIP failed to induce any reinforcement during self-administration either in mice or baboons. Moreover, both THIP and GAN produced conditioned place aversion in mice. The study performed in δ subunit-knockout mice further supported the proposal that tonic inhibition of the VTA GABA neurons contributes to conditioned aversive behavior and THIP- and GAN-induced neuroplasticity. The c-Fos mapping of brain regions, which could take part in THIP-induced aversive behavioral effects and/or neuroplasticity on VTA DA neurons, revealed the bed nucleus of stria terminalis (BNST), a part of the so-called extended amygdala circuitry, as a possible participant in mediating the aforementioned THIP-induced aversive effects. In summary, these studies demonstrate that tonic inhibition mediated by δ subunit-containing GABAA receptors appears to be a significant component of the inhibition in the VTA, and thus important for the control of motivated behavior. ORIGINAL PUBLICATIONS This thesis is based