| Abstract No.: | B-B2071 |
| Country: | Canada |
| | |
| Title: | MEMORY FORMATION AND SYNAPTIC PLASTICITY IMPAIRED BY REDUCING EXPRESSION OF EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 2 IN DROSOPHILA. |
| | |
| Authors/Affiliations: | 1 Philip Harvey*; 1 David Knight; 1 Kostas Iliadi; 1 Nataly Iliadi; 1 Gabrielle Boulianne;
1 The Hospital for Sick Children, Toronto, ON, Canada
|
| | |
| Content: | Objectives: Drosophila melanogaster is an incredibly useful organism for identifying novel proteins involved in synaptic transmission, plasticity and memory formation. Through a genetic screen a P-element mutation, P124, was found to impair learning as assessed by operant and classical conditioning. This insertion lies between the coding regions for two genes; CG 9596 and equilibrative nucleoside transporter (ent) 2. The objective of the present work was to determine how the insertional mutation P124 impairs memory formation in Drosophila.
Materials and Methods: Temporal expression of the two genes of interest, CG 9596 and ent2, was assessed using quantitative reverse transcriptase PCR (qRT-PCR) on P124 mutants flies and compared to w1118 control lines. Spatial expression of ent2 in the adult brain was analyzed by using an enhancer P-element inserted just upstream of the ent2 coding region to drive GFP expression via the GAL4-UAS enhancer trap system. An allelic series of mutations spanning the promoter and coding regions of the ent2 gene was created by crossing P124 males to females carrying a transposase gene to mobilize the P-element transposon. White-eyed F2 progeny were genotyped to identify precise and imprecise excisions that were then backcrossed to w1118 Canton-S control stocks. Adult homozygous mutants were assessed for learning ability using a classical olfactory conditioning paradigm. Electrophysiology was performed at the neuromuscular junction of wandering third instar larvae. Intracellular recordings were made of spontaneous and evoked potentials in muscle 6 during 0.2 Hz nerve stimulation. Synaptic plasticity was assessed using two paradigms; post-tetantic potentiation and paired-pulse facilitation.
Results: The P124 mutation was found to suppress transcription of the ent2 gene but had no effect on CG9596, as measured by qRT-PCR. Ent2 was further implicated by its localization to the antennal lobes and mushroom bodies of the adult fly brain; regions important for memory formation. Imprecise excision of the P-element yielded deletions removing the promoter region (hypomorphs) or start codon (nulls), with reduced expression confirmed by qRT-PCR. Eliminating ent2 expression (homozygous null) was lethal at late larval third instar stages so behavioural effects could not be assessed in adults. Reduced expression of ent2 produced adult flies with learning deficits identical to the original P124 mutation. Corresponding defects in synaptic transmission were identified at the larval neuromuscular junction, including increased spontaneous activity and evoked potential amplitude. Hypomorphs also failed to exhibit paired-pulse facilitation, a form of synaptic plasticity, although post-tetanic potentiation was normal. Abnormal cyclic AMP (cAMP) levels are known to influence learning in Drosophila. Preliminary measures of cAMP levels show that reduced expression of ent2 increases cAMP concentrations, while ectopic overexpression reduces cAMP.
Conclusions: Ent2 is normally expressed in brain regions know to influence learning in Drosophila, and ent2 hypomorphic mutations have a deleterious effect on learning. Basal synaptic transmission was enhanced in these mutants to the point that short-term facilitation was occluded. Ent2 may be influencing synaptic transmission by buffering the action of adenosine on GPCRs, thus modulating cAMP concentrations. |
| | |
| Back |
|
