Scuola Internazionale Superiore di Studi Avanzati / International School for Advanced Studies The interaction between neuronal networks and gene networks Thesis submitted for the degree of Doctor Philosophiae Neurobiology sector October 2008 Candidate Supervisor Silvia Pegoraro Prof. Vincent Torre Ed. Q1 AREA SCIENCE PARK - S.S.14 Km163,5 - 34012 Basovizza (TS) - ITALY II ...a mia mamma... ...a mio papa’... ...ad Andrea... III INDEX 1 Abstract ____________________________________________________________1 2 Introduction _________________________________________________________4 2.1 Experience can alter neural circuitry...............................................................................................5 2.1.1 Dendrites Outgrowth................................................................................................................7 2.1.2 Synapse Maturation/Elimination.............................................................................................7 2.1.3 Synaptic Plasticity....................................................................................................................7 2.2 Models of activity-dependent synaptic plasticity ...........................................................................8 2.2.1 Long-Term Potentiation...........................................................................................................8 2.2.2 Chemically-Induced Long-Term Potentiation......................................................................11 2.2.3 GABAA Receptors Inhibition: A Model of Synaptic Plasticity ..........................................13 2.3 Calcium: a molecule for signalling................................................................................................16 2.4 Routes of calcium entry..................................................................................................................19 2.5 Signalling from synapse to the nucleus.........................................................................................23 2.5.1 Nuclear Factor of Activated T Cells (NFAT).......................................................................24 2.5.2 Downstream Regulatory Element Antagonist Modulator (DREAM).................................24 2.5.3 Calcium Regulated Protein Kinases: CaMKs and ERK ......................................................25 2.6 Activity-regulated genes.................................................................................................................27 2.6.1 Early growth response: Egr family of Transcription Factors ..............................................28 2.6.2 Activity regulated cytoskeletal-associated protein: Arc ......................................................29 2.6.3 Brain-derived neurotrophic factor: Bdnf ..............................................................................30 2.6.4 Homer1a..................................................................................................................................31 2.7 Activity-regulated gene expression profile ...................................................................................32 3 Results ____________________________________________________________37 3.1 Characterization of the time course of the activity pattern and transcriptome ...........................38 in a model of chemical-induced neuronal plasticity triggered by GABAA antagonists 3.2 Calcium control of gene regulation in rat hippocampal neuronal cultures .................................99 4 Conclusions _______________________________________________________ 151 5 References ________________________________________________________ 156 IV Declaration The work described in this thesis has been carried out at the International School for Advanced Studies (SISSA/ISAS, Trieste, Italy) between November 2004 and August 2008. All the work in this thesis arises from my own experiments and data analysis with the exception of the microarrays analysis performed in collaboration with Daniele Bianchini and Claudio Altafini and the MEA analysis performed by Frederic Broccard, in the first work. In the second work the calcium imaging and intracellular recordings were performed by Giulietta Pinato. The results presented in this thesis have been submitted in the following manuscripts: Broccard FD, Pegoraro S, Ruaro ME, Bianchini D, Avossa D, Pastore G, Altafini C, Torre V (2008) Characterization of the time course of the activity pattern and transcriptome in a model of chemical-induced neuronal plasticity triggered by GABAA antagonists. Submit to BMC Neuroscience *Pegoraro S, *Pinato G, Ruaro ME, Torre V (2008) Calcium control of gene regulation in rat hippocampal neuronal cultures. *equally contributed Submit to the Journal of Cellular Physiology V Abbreviation Act D: Actinomycin D BDNF: Brain-derived neurotrophic factor Ca2+: Calcium CaMK: Serine/threonine kinases, Ca2+/calmodulin-dependent kinase cLTP: Chemically-induced long-term potentiation CREB: cyclic adenosine monophosphate (cAMP) response element binding protein D-AP5: d-2-amino-5-phosphonopentanoic acid DG: Dentate gyrus DRB: 5,6-dichloro-1--D-ribofuranosyl benzimidazole E-LTP: Early long-term potentiation ERK: Extracellular signal-regulated kinase E-Sync: Early synchronization GABA:-aminobutyric acid GBZ: Gabazine IEG: Immediate early gene K+: Potassium L-LTP: Late long-term potentiation L-Sync Late: synchronization LTD: Long-term depression LTP: Long-term potentiation MAPK: Mitogen-activated protein kinase MEA: Multielectrode array M-LTP: Medium long-term potentiation mRNA: Messenger RNA NMDA: N-methyl-D-aspartate PKC: Protein kinase C SRE: serum-response element VGCC: Voltage-gated calcium channel VI 1 Abstract 1 Periods of strong electrical activity can initiate neuronal plasticity leading to long-lasting changes of network properties. A key event in the modification of the synaptic connectivity after neuronal activity is the activation of new gene transcription. Moreover, calcium (Ca2+) influx is crucial for transducing synaptic activity into gene expression through the activation of many signalling pathways. In our work we are interested in studying changes in electrical activity and in gene expression profile at the network level. In particular, we want to understand the interplay between neuronal and gene networks to clarify how electrical activity can alter the gene expression profile and how gene expression profile can modify the electrical and functional properties of neuronal networks. Thus, we investigated the neuronal network at three different levels: gene transcription profile, electrical activity and Ca2+ dynamics. Blockage of GABAA receptor by pharmacological inhibitors such as gabazine or bicuculline triggers synchronous bursts of spikes initiating neuronal plasticity. We have used this model of chemically-induced neuronal plasticity to investigate the modifications that occur at different network levels in rat hippocampal cultures. By combining multielectrode extracellular recordings and calcium imaging with DNA microarrays, we were able to study the concomitant changes of the gene expression profile, network electrical activity and Ca2+ concentration. First, we have investigated the time course of the electrical activity and the molecular events triggered by gabazine treatment. The analysis of the electrical activity revealed three main phases during gabazine-induced neuronal plasticity: an early component of synchronization (E-Sync) that appeared immediately after the termination of the treatment persisted for 3 hours and was blocked by inhibitors of the MAPK/ERK pathway; a late component (L-Sync) -from 6 to 24 hours- that was blocked by inhibitors of the transcription. And, an intermediate phase, from 3 to 6 hours after the treatment, in which the evoke response was maximally potentiated. Moreover, gabazine exposure initiated significant changes of gene expression; the genomic analysis identified three clusters of genes that displayed a characteristic 2 temporal profile. An early rise of transcription factors (Cluster 1), which were maximally up-regulated at 1.5 hours. More than 200 genes, many of which known to be involved in LTP were maximally up-regulated in the following 2-3 hours (Cluster 2) and then were down-regulated at 24 hours. Among these genes, we have found several genes coding for K+ channels and the HNC1 channels. Finally, genes involved in cellular homeostasis were up-regulated at longer time (Cluster 3). Therefore, this approach allows relating changes of electrical properties occurring during neuronal plasticity to specific molecular events. Second, we have investigated which sources of Ca2+ entry were involved in mediating the new gene transcription activated in response to bursting activity. Using Ca2+ imaging, a detailed characterization of Ca2+ contributions was performed to allow investigating which sources of Ca2+ entry could be relevant to induce gene transcription. At the same time, changes of gene expression were specifically investigated blocking NMDA receptors and L-, N- and P/Q-type VGCCs. Therefore, the analysis of the Ca2+ contribution and gene expression changes revealed that the NMDA receptors and the VGCCs specifically induced different groups of genes. Thus, the combination of genome-wide analysis, MEA technology and calcium imaging offers an attractive strategy to study the molecular events underlying long- term synaptic modification. 3 2 Introduction 4 2.1 EXPERIENCE CAN ALTER NEURAL CIRCUITRY Sensory experience and the subsequent