The long-term memory trace formed in the Drosophila α/ß mushroom body neurons is abolished in long-term memory mutants.

A prior screen identified dozens of Drosophila melanogaster mutants that possess defective long-term memory (LTM). Using spaced olfactory conditioning, we trained 26 of these mutant lines to associate an odor cue with electric shock and then examined the memory of this conditioning 24 h later. All of the mutants tested revealed a deficit in LTM compared to the robust LTM observed in control flies. We used in vivo functional optical imaging to measure the magnitude of a previously characterized LTM trace, which is manifested as increased calcium influx into the axons of α/ß mushroom body neurons in response to the conditioned odor. This memory trace was defective in all 26 of the LTM mutants. These observations elevate the significance of this LTM trace given that 26 independent mutants all exhibit a defect in the trace, and further suggest that the calcium trace is a fundamental mechanism underlying Drosophila LTM.

A late-phase, long-term memory trace forms in the γ neurons of Drosophila mushroom bodies after olfactory classical conditioning.

Using functional optical imaging in vivo, we demonstrate that the γ mushroom body (MB) neurons of Drosophila melanogaster respond with axonal calcium influx when odors or electric shock stimuli are presented to the fly. Pairing of odor and electric shock stimuli in a single training trial or multiple, massed training trials failed to modify the odor-evoked calcium signal when flies were tested at several different times after training. In contrast, animals that received multiple but spaced odor-shock pairings exhibited a robust increase in calcium influx into the MB axons when tested between 18 and 48 h after training. This time window for the γ neuron memory trace is displaced relative to the modifications that occur between 9 and 24 h after training in the α branch of the α/ß MB neurons. The α/ß and the γ neuron long-term memory traces were both blocked by expressing a repressor of the transcription factor cAMP response element-binding protein or a calcium/calmodulin-dependent kinase II hairpin RNA. These results demonstrate that behavioral long-term olfactory memory is encoded as modifications of calcium influx into distinct MB neurons during overlapping but different windows of time after training.

Drosophila alpha/beta mushroom body neurons form a branch-specific, long-term cellular memory trace after spaced olfactory conditioning.

Functional optical imaging showed that odor or electric shock stimuli presented to the fly causes transient calcium influx into the two major axon branches of alpha/beta mushroom body (MB) neurons. One pairing of odor and electric shock stimuli or multiple, massed pairings did not alter odor-evoked calcium influx. In contrast, animals that received multiple, spaced pairings exhibited a robust increase in calcium influx into the MB axons when tested at 9 or 24 hr after training, but not at 3 hr. This modification occurred only in the alpha branch of the neurons and was blocked by mutation of the amnesiac gene, inhibition of protein synthesis, or the expression of a protein blocker of the transcription factor Creb. Thus, behavioral long-term olfactory memory appears to be encoded as a branch-specific modification of calcium influx into the alpha/beta MB neurons that occurs after spaced training in a protein synthesis-, Creb-, and amnesiac-dependent way.

Roles for Drosophila mushroom body neurons in olfactory learning and memory.

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. We investigated the requirements for the different MB neurons, specifically the alpha/beta versus the gamma neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. We expressed the rutabaga (rut)-encoded adenylyl cyclase in either the gamma or alpha/beta neurons and examined the effects on restoring olfactory associative learning and memory of rut mutant flies. We also expressed a temperature-sensitive shibire (shi) transgene in these neuron sets and examined the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning. Our results indicate that although we did not detect odor-pair-specific learning using GAL4 drivers that primarily express in gamma neurons, expression of the transgenes in a subset of alpha/beta neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shi(ts1).

Aplysia mollusk-derived growth factor is a mitogen with adenosine deaminase activity and is expressed in the developing central nervous system.

Mollusk-derived growth factor (MDGF), the first growth factor to be characterized in Aplysia, was purified and characterized and has both adenosine deaminase activity and stimulates cell proliferation in vitro. MDGF is structurally related to a new subfamily of adenosine deaminase-related growth factors that require enzymatic activity to stimulate cell proliferation, a unique property of known growth factors. We examined the expression of MDGF protein in the CNS since MDGF mRNA increased in the developing CNS, and recent data suggest that inosine is involved in neuronal reorganization and restoration of essential circuitry after CNS injury. MDGF levels transiently increased during embryonic and post-metamorphic development and in the developing CNS, but was undetectable in adult CNS. No effects on morphology or neurite extension of adult Aplysia neurons were observed.