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The role of the voltage-gated potassium channels and their modulators in mechanisms of plasticity underlying learning and memory in Drosophila

Periodic Report Summary 2 - KV CHANNELS & MEMORY (The role of the voltage-gated potassium channels and their modulators in mechanisms of plasticity underlying learning and memory in Drosophila)

The aim of this research proposal was to determine the role of voltage-gated potassium (Kv) channels in mechanisms of plasticity underlying learning and memory using Drosophila. We have set up a highly multidisciplinary approach involving molecular genetics, biochemistry, cell imaging, patch clamp electrophysiology, in vivo calcium imaging, memory and alcohol behaviour assays in order to tackle this question.

During the duration of this proposal we generated or acquired fly stocks mutant for each of the individual Kv channels. Currently only extensive characterisation of Shaker has been performed, these reagents will allow characterisation of the whole Kv channel family revealing how they encode information as a channel family. We have focused and published so far on the function of Kv7 (KCNQ) channels.

We have tested the effect of Kv channel mutations using real time quantitative and semi-quantitative PCR (qRT-PCR) and westerns. The expression pattern of a Kv channel was determined by a combination of immunohistochemistry and in-situ hybridisation. We paid particular interest in expression in the mushroom body and antennal lobe neurons, both structures known to mediate fly memory. We also documented expression in larval motorneuron soma and Neuromuscular junction (NMJ) where we performed our in vivo calcium imaging experiments.

We have used a recombinant expression system that has allowed detailed electrophysiological and pharmacological comparison of Drosophila and homologous mammalian Kv channels using voltage clamp. We also performed initial recordings from adult central clock (large lateral neuron ventral labelled by PDF-Gal4) and Dorsal paired medial (DPM) neurons, which together with a number of other surrounding neurons express Amnesiac-Gal4. DPM are known to be important for memory and alcohol behaviour and their position allowed us to perform somatic recordings.

We have determined the behavioural consequence of modulating the level of Kv channels using olfactory-shock classical conditioning and also looked at the effect of ethanol and memory and effect of Kv channels on ethanol behaviour. We started to look at the effect of PKA and PKC on Kv channel behavioural phenotypes and also looked at the effect of aging on Kv channel function.

We have acquired and generated mutants in all members of the Kv channel family using a range of mutant reagent including chromosomal mutants, deficiencies, full length and RNAi (both conventional and site directed) transgenes.

We have shown the effect of mutation or transgene expression on the level of a given Kv channel using a combination of Western and quantitative Real time polymerase chain reaction (qRT-PCR). We have described the detailed localisation of Kv channels in the brain expression including in the mushroom body and determined the effect of Kv channel specific RNAi knock-down (Hodge and Stanewsky, 2008; Parisky et al., 2008). We have also use qRT-PCR to measure the abundance of specific Kv channels including individual isoforms of specific Kv channels in whole body and brain at different development stages as well as the aged adult brain.

We have electrophysiologically and pharmacologically characterised Kv channels expressed in in Human Embryonic Kidney (HEK) cells using voltage-clamp. We showed that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating potassium (K+) current open at sub-threshold potentials and it has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1 / KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991 and linopirdine) and the opener (zinc pyrithione). We also investigated the molecular basis of the differential selectivity of KCNQ channels to the opener and anti-epileptic retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. Therefore we have shown that dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels (Cavaliere and Hodge, 2011). In addition, we have performed some current and voltage clamp recordings from l-LNv and DPM neurons in the whole adult Drosophila brain.

We have characterised the role of Kv channels in Drosophila memory with a particular focus on KCNQ function in memory. We also have determined the effect of alcohol and aging on Drosophila memory determining the role of KCNQ channels in these novel behavioural assays.

We have determined the effect of changing the level of Kv channels on calcium signalling using in vivo GCaMP3 imaging from larval motorneurons and characterised an effect on neuropeptide and hormone release.

This research has provided novel mechanistic insight into how memories are encoded in neural circuits. The study will also help identify molecular targets for future modulation of excitability to attempt to reverse deficits in learning that occur during disease or aging. Funding has helped me to return to UK and set up my multidisciplinary research program allowing me to successfully import to Europe the extensive knowledge and skill set acquired during my 6 year postdoc in the United States (US). Our work will allow future study of physiological and pathological roles of KCNQ in Drosophila and will allow whole organism screening for new modulators of KCNQ channelopathies that include cardiac arrhythmias, epilepsy, deafness and diabetes.

Our and recent work, suggest KCNQ openers may be a promising new approach to treatment of alcohol and other drug misuse disorders. Therefore, this work has important socioeconomic and health impacts, by isolating a conserved ethanol target that modifies behaviour at a physiologically relevant dose. We also showed Drosophila show age-related memory impairments mediated by KCNQ, this role seems to be conserved in monkeys therefore is likely to be true for humans too. Therefore, KCNQ channels can be considered a new therapeutic target for treating the memory loss associated with aging and dementia, a massive health burden in the European Union.

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