BIOLOGICAL OVERVIEW

The genetic dissection of learning and memory in Drosophila is two decades old. Recently, a great deal of progress has been made towards isolating new mutants as well as a better understanding of those originally isolated. Nighorn's paper reviews the recent developments in the understanding of the structure and function of the gene identified by the first and best-characterized of these mutants, the Drosophila dunce mutant (Nighorn, 1994). R. L. Davis (1996) provides an even more recent review.

Learning in flies is studied using an operant conditioning paradigm involving electric shock and olfactory cues. First an odor (the conditioned stimulus) is paired with electric shock (the unconditioned stimulus). The aversive effects of the shock teach the flies to avoid the odor, or as researchers tend to express it, avoidance of the conditioned stimuli is enhanced. Behavior attributable to learning is measured by testing with a second and different odor. Flies that have learned continue to avoid the conditioned stimulus but not the control odor, both in the absence of the unconditioned stimulus. Proper control includes a second experimental phase: employing the second odor as the conditioned stimulus. When paired with the shock, flies should then avoid the second (control) odor as they did the original conditioned stimulus (Quinn, 1972).

Cyclic AMP has been implicated as the mediator of learning in classical experiments using the marine snail Aplasia (Kandel, 1992). In this system, conditioned stimuli increased Ca++ concentration within neurons. The Ca++ binds to Calmodulin, which in turn binds to adenylate cylase, enhancing the ability of adenylate cyclase to synthesize c-AMP. The Drosophila adenyl cyclase homolog is rutabaga. Mutations in rut result in defective learning. A second gene, dunce, encodes the cAMP phosphodiesterase, the enzyme that degrades cAMP. Mutations in dunce also result in a learning deficit but when combined with the rut mutations in the same fly, the learning defect is reversed. At least in this case it appears that two wrongs make a right. It has been suggested that lower cAMP levels still result in learning despite the double mutation because the cAMP is degraded more slowly.

Protein kinase A, the target of cAMP signaling is a third protein involved in learning . PKA, activated by cAMP, initiates a phosphorylation cascade that in turn activates genes involved in the creation of long term memory. dCREB2, the cyclic AMP response element binding protein is one such gene, a transcription factor associated with long term memory. The Dfos and DJUN genes in Drosophila both have binding sites for CREB, while in mammals, only the fos promoter binds CREB (Davis, 1995, Fagnon, 1995 and Nighorn, 1994). For a discussion of the cellular basis of learning see the rutabaga site.

A novel bioassay system is described that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball is manipulated into contact with cultured Drosophila 'giant' neurons, spontaneous synaptic current-like events are registered. These events are observed within seconds after contact and are blocked by curare and alpha-bungarotoxin, but not by TTX and Cd2+, suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurs not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displays a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses (a synapse between an axon collateral of a neuron and one of the same neuron's dendrites) formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI that disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. These mutations affect the ACh current kinetics, but not the quantal ACh packet, and the release frequency is greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis (Yao, 2000).

It is now well established that Drosophila neurons share many molecular components of the transmitter release machinery with vertebrate neurons. The release of neurotransmitter is a multistep process that involves actions of proteins associated with the synaptic vesicle and the plasma membrane, as well as cytoplasmic proteins. Some of these proteins, e.g., synapsin alphaSNAP, and Ca²⁺ channels are known to be the downstream targets of PKA. Phosphorylation of these proteins may be important for the regulation of vesicle mobilization, docking, and fusion (Yao, 2000).

In Drosophila dnc mutants, increased cAMP levels caused by the disruption of a phosphodiesterase lead to abnormalities in channel function and nerve excitability, synaptic transmission and plasticity, growth cone motility, and nerve arborization. Using the present heterologous detection system, the altered transmitter release process could be examined in developing growth cones of dnc central neurons in isolation from the influence of postsynaptic targets. Examination of PKA-RI neurons suggests that the dnc defects in ACh secretion might be mediated by PKA. These results establish a role for the cAMP cascade in the regulation of the secretion process in developing neurons before synaptogenesis. In light of the profound alterations in synaptic efficacy and activity-dependent modulation observed in mature synapses of dnc mutants, the cAMP pathway may be involved throughout the maturation process of the synapse (Yao, 2000).

The effects of decreased cAMP levels on synaptic transmission have also been extensively studied in Drosophila. Intracellular recordings at the peripheral larval neuromuscular junction have revealed that chronically lowering cAMP causes reduced neurotransmitter release, likely because of reduction of innervation rather than impairment of transmitter release. These results do not contradict results obtained from developing central neurons. It will be important to determine how reduction in cAMP concentration affects neurotransmitter releases in the Drosophila central neurons in future studies (Yao, 2000).

The prolonged ACh currents of dnc and PKA-RI neurons may be attributable to increased ACh diffusion distance and altered presynaptic release mechanisms, as discussed above. A reduced efficiency in the formation of the exocytotic fusion pore and/or a disrupted fusion machinery may account for the prolonged release events for synaptic vesicles containing similar amounts of ACh. Exocytotic efficiency may be regulated by PKA-dependent phosphorylation of vesicular, cytoplasmic, and plasma membrane proteins involved in exocytosis. Additional mutational analysis will be required to identify the specific proteins that are targeted by PKA in this process (Yao, 2000).

Although the spontaneous release in neurons of all genotypes examined does not require Ca²⁺ influx, the activity-dependent increase in release frequency in dnc neurons after repetitive nerve stimulation appears to depend on the external Ca²⁺. It has been proposed that nerve activity regulates cAMP levels, possibly mediated by intracellular accumulation of Ca²⁺ through repetitive nerve spikes, which can trigger the Ca²⁺/CaM activation of adenylyl cyclase. The activity-dependent modification of transmission at mature synapses is altered in dnc mutants. These results suggest that the cAMP pathway may mediate such activity-dependent regulation in developing neurons before synaptogenesis as well, lending support to the notion that the cAMP pathway is important in a wide variety of neuronal processes throughout development (Yao, 2000).

GENE STRUCTURE

The complexity of the dunce locus and its transcription products points to the biological importance of Dunce. The dunce gene encodes multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases). Six different classes have been identified. Exons are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites. Some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons, internal differences arising from the optional inclusion of a 39 base-pair exon, and the alternative use of two 3' splice sites separated by six base-pairs. An unusual feature of the dunce gene is the presence of at least 7 other genes nested between the dunce exons. The significance of this phenomenon is unknown (Qui, 1991 and Nighorn, 1994).

PROTEIN STRUCTURE

Amino Acids - 702, 714 and 777 for three isoforms (Qui, 1991).

Structural Domains

The deduced amino acid sequence is strikingly homologous to the amino acid sequence of a Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase isolated from bovine brain and more weakly related to the predicted amino acid sequence of a yeast cAMP phosphodiesterase. These homologies, together with prior genetic and biochemical studies, provide unambiguous evidence that dunce+ codes for a phosphodiesterase. In addition, the dunce+ gene product shares a seven-amino acid sequence with a regulatory subunit of cAMP-dependent protein kinase that is predicted to be part of the cAMP binding site. There is also a weak homology between a region of the dunce+ gene product and the egg-laying hormone precursor of Aplysia californica (Chen, 1989).

date revised: 29 June 2000 

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