Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 964 Modulation of Neuronal Functions the Role of SLC10A4 KALICHARAN PATRA ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-8838-3 UPPSALA urn:nbn:se:uu:diva-214162 2014 Dissertation presented at Uppsala University to be publicly examined in B21, Biomedical Centre, Uppsala, Friday, 21 February 2014 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Per Svenningsson (Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden). Abstract Patra, K. 2014. Modulation of Neuronal Functions. the Role of SLC10A4. (SLC10A4- Mediated Modulation of Neuronal Functions). Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 964. 50 pp. Uppsala: Uppsala universitet. ISBN 978-91-554-8838-3. Mental health of a person depends on the correct functioning of the brain. The brain and the spinal cord contain many types of cells, of which one important type are called the neurons. Neurons are special in the way they connect to each other to form large networks. The chemicals called transmitters are packed at the nerve endings into tiny packets called vesicles and when a signal arrives these vesicles fuse immediately to the attached cell surface and release their contents. The role of the synaptic vesicular transporter proteins is to ensure proper packing of transmitter molecules that can be released upon stimulation. Vesicular packing is an important process. The carrier proteins involved in packing work in coordination to determine the amount and type of transmitters to be packed. Missing a carrier protein from the vesicles might lead to improper packing and inaccurate signaliing. These signaling molecules are known for their implications in many psychiatric and neurological disorders like Alzheimer’s disease, Parkinson’s disease, Schizophrenia, and attention deficit to name just a few. How a vesicular transporter can affect the modulatory functions of aminergic neurons is the subject of this thesis. This thesis reports on the effects of the loss of a vesicular orphan transporter. Study I demonstrates the localization of this protein to monoaminergic and cholinergic terminals. It reports the effect of the loss of Slc10A4 on vesicular dopamine uptake, synaptic clearance of dopamine and hypersensitivity of animals to dopamine related psychostimulants. Study I also provides evidence for ATP as a possible ligand for SLC10A4 protein. Study II provides data on the clinical relevance of Slc10A4 in playing a protective role against vulnerability to epilepsy. It reports that loss of Slc10A4 renders the animals hypersensitive to cholinergic drugs. Study III provides a closer look at individual cholinergic synapses at neuromuscular junctions in mice lacking Slc10A4. The structural and electrophysiological properties of the NMJ are found compromised because of the loss of this vesicular protein. Taken together, this thesis presents a SV protein’s perspective of viewing at modulation of synaptic transmission. Keywords: dopamine, acetylcholine, central nervous system, neuromuscular junctions, electrophysiology, monoamine, synaptic transmission, neuromodulation Kalicharan Patra, Department of Neuroscience, Developmental Genetics, Box 593, Uppsala University, SE-75124 Uppsala, Sweden. © Kalicharan Patra 2014 ISSN 1651-6206 ISBN 978-91-554-8838-3 urn:nbn:se:uu:diva-214162 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-214162) The time spans of scientific truths are an inverse function of the intensity of scientific efforts ROBERT M PIRSIG Zen and The Art of Motorcycle Maintenance To The mice that died while still living & The layman List of papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Larhammar, M*., Blunder, M*., Patra, K*., Emilsson, L., Peuckert, C., Arvidsson, E., Pettersson, H., Rönnlund, D., Prebraschenski, J., Birgner, C., Limbach, C., Widengren, J., Blom, H., Hallgren-Martinsson, J., Jahn, R., Wallen-Mackenzie, Å., Kullander, K. (2014) SLC10A4 is a vesicular aminergic-associated transporter modulating monoamine homeostasis. Resubmitted II Zelano, J., Mikulovic, S., Patra, K., Kühnemund, M., Larhammar, M., Emilsson, L., Leão, R., Kullander, K. (2013) The synaptic protein en- coded by the gene Slc10A4 suppresses epileptiform activity and regu- lates sensitivity to cholinergic chemoconvulsants. Experimental Neurol- ogy, 239 (2013): 73-81 III Patra, K*., Lyons, D. J*., Bauer, P., Hilscher, M., Sharma, S., Leão, R. N., Kullander, K. (2013) Role of SLC10A4 in structural remodeling and transmitter release at mouse neuromuscular junctions. Submitted * Equal contribution Reprint was made with permission from Elsevier for paper II. Additional publications I Wootz, H., Fitzsimons-Kantamneni, E., Larhammar, M., Rotterman, T.M., Enjin, A., Patra, K., Andre, E., Van Zundert, B., Kullander, K., Alvarez, F.J. (2013) Alterations in the motor-renshaw cell circuit in the Sod1 (G93A) mouse model. J Comp Neurol. 521(7): 1449-69 II Leão, R.N., Mikulovic, S., Leão, K.E., Munguba, H., Enjin, A., Patra, K., Eriksson, A., Loew, L.M., Tort, A.B., Kullander, K. (2012) OLM in- terneurons differentially modulate CA3 and entorhinal inputs to hippo- campal CA1 neurons. Nat Neurosci. Nov;15(11): 1524-30 III Hånell, A., Clausen, F., Djupsjö, A., Vallstedt, A., Patra, K., Israelsson, C., Larhammar, M., Björk, M., Paixao, S., Kullander, K., Marklund, N. Functional and histological outcome after focal traumatic brain injury is not improved in conditional EphA4 knockout mice. (2012) J Neuro- trauma 29(17): 2660-71 IV Andersson, L.S*., Larhammar, M*., Memic, F*., Wootz, H*., Schwochow, D., Rubin, C.J., Patra, K., Arnason, T., Wellbring, L., Hjälm, G., Imsland, F., Petersen, J.L., McCue, M.E., Mickelson, J.R., Cothan, G., Ahituv, N., Roepstorff, L., Mikko, S., Vallstedt, A., Lind- gren, G., Andersson, L., Kullander, K. (2012) Mutations in DMRT3 af- fect locomotion in horses and spinal circuit function in mice. Nature 488(7413): 642-6 * Equal contribution Content List of papers ................................................................................................... 5 Additional publications ................................................................................... 6 Introduction ..................................................................................................... 9 Background ................................................................................................... 11 The aminergic subsystems ................................................................... 12 Neurotransmission .................................................................................... 13 Pre- and post- synaptic components .................................................... 13 The neuromuscular junction (NMJ) ..................................................... 15 Transmitter release at NMJ .................................................................. 16 Neuromodulation ...................................................................................... 17 Dopaminergic modulation ................................................................... 18 Cholinergic modulation ....................................................................... 18 ATP as a neuromodulator .................................................................... 18 The Solute Carriers ................................................................................... 20 Vesicular solute carriers ....................................................................... 20 SLC10 .................................................................................................. 20 SLC10A4 ............................................................................................. 21 Aims of the studies ........................................................................................ 22 Methodological considerations ..................................................................... 23 Results and discussion ................................................................................... 27 Study I .................................................................................................. 27 Study II ................................................................................................. 28 Study III ............................................................................................... 29 Discussion ................................................................................................. 29 Concluding remarks ...................................................................................... 34 Summary in English ...................................................................................... 35 Sammanfattning ............................................................................................ 37 Acknowledgement ......................................................................................... 39 References ..................................................................................................... 43 Abbreviations ACh Acetylcholine AChR Acetylcholine receptor ATP Adenosine triphosphate ADHD Attention deficit hyperactivity disorder Ado Adenosine AZ Active zone ChAT Choline acetyl transferase ChT Choline transporter CNS Central nervous system DA Dopamine DAT Dopamine transporter EPP Endplate potential KO Knockout