Gene name - rutabaga
Cytological map position - 12F5-13A1
Function - adenylate cyclase
Symbol - rut
Genetic map position - 1-
Classification - Type 1 - Ca++ CaM sensitive
Cellular location - cytoplasmic
rutabaga encodes a calmodulin dependent adenylate cyclase that converts ATP to cyclic AMP. cAMP is a major signal transducer of the cell, and its creation and destruction is involved in just about every response of the cell to environmental changes. Calmodulin is a protein that binds the Ca++ ion, sensing its cellular concentration and interacting with the rutabaga encoded adenyl cyclase to activate adenyl cyclase mediated enzymatic conversion of ATP into cAMP.
The cyclic AMP system plays a critical role in olfactory learning in Drosophila, as does rutabaga, given this gene's crucial involvement in the same system. Calcium ions enter vertebrate cells through a type of glutamate receptor known as the NMDA (N-methyl-D-aspartate) receptor. Neural stimulation results in movement of calcium ions into the cell, leading to activation of adenyl cyclase and a rise in cAMP levels. cAMP, one of the central chemical messengers of the cell, then activates protein kinase A, which in turn initiates a phosphorylation cascade leading to the induction of genes involved in learning (Davis, 1995, Davis, 1996, Fagnon, 1995 and Nighorn, 1995,).
Rutabaga and Dunce are both found expressed at high levels in the mushroom bodies of adult flies. Mushroom bodies are paired groups of about 2500 neurons each, clustered in either hemisphere of the brain. They receive olfactory information from the antennal lobes via dentrites located in the calyx, a region of the brain just ventral to the mushroom bodies. The calyx is a neuropil, a region rich in synapses. It is in the calyx that integrates information from the thoracic ganglion (sensory information) and from the antennal nerve (olfactory information), passing the news on to the mushroom bodies. In classical operant conditioning experiments an electric shock (sensory information) is paired with a particular stimulus (olfactory information) to condition learning (avoidance behavior) upon presentation of the successfully conditioned stimulus, (in these cases, an odor) (Quinn, 1974, Han, 1992 and Nighorn, 1994).
A group of diffuse interneurons in mushroom bodies carry the information to the lateral protocerebrum. Interneurons from the protocerebrum make synaptic contact onto command neurons in a brain structure called the posterior slope, and these response messages are sent out of the brain to direct motor activity. In the case of the odor conditioned to a noxious stimulus like a shock, the message is the equivalent of "get out of here."
Biological systems are built in order to respond to environmental stimulus, whether in nature or in the laboratory. Learning takes this ability to extremes. The cellular basis of operant conditioning is the same, whether one considers Pavlov's dogs, salivating to the sound of a bell, or the conditioned fruit fly, avoiding a particular odor. Coincidence of information from two modalities (smell plus shock sensation) results in heightened neural activity, that is, an influx of Ca++ ions. This in turn stimulates gene activity and the building of new cell connections or the reinforcement of old ones. This is the chemical, genetically driven basis of learning and memory (Davis, 1995).
Memories are thought to be due to lasting synaptic modifications in the brain. The search for memory traces has relied predominantly on determining regions that are necessary for the process. However, a more informative approach is to define the smallest sufficient set of brain structures. In rut mutants, ectopic expression of rutabaga in a spatially restricted fashion, in a defined set of neurons, was used to examine the cellular localization of the memory trait. rutabaga, an enzyme that is ubiquitously expressed in the Drosophila brain and that mediates synaptic plasticity, has been found to be needed exclusively in the Kenyon cells of the mushroom bodies for a component of olfactory short-term memory. This demonstrates that synaptic plasticity in a small brain region can be sufficient for memory formation (Zars, 2000b).
In insects, much attention has been paid to the mushroom bodies as the site for olfactory learning. In Drosophila, they are made up of about 2500 intrinsic neurons (Kenyon cells), receive multimodal sensory input, preferentially from the antennal lobe to the calyx, and send axon projections to the anterior brain where they bifurcate to form the alpha/beta, alpha'/beta', and gamma lobes. Noninvasive intervention techniques can provide mushroom body-less flies. In most respects, these flies show remarkably normal behavior but are deficient in olfactory learning. Genes important for olfactory memory have elevated expression levels in the mushroom bodies (Zars, 2000b and references therein). Additionally, the mushroom bodies are necessary for context generalization in visual learning at the flight simulator and the control of spontaneous walking activity (Martin, 1998; Liu, 1999).
The approach taken is built on the assumption that synaptic plasticity is impaired in general in rut mutants and that it is this cellular defect that causes the various learning deficits. Restoring rut AC in a spatially restricted fashion in a defined set of neurons would furnish synaptic plasticity to only those cells. If in such flies a learning task is rescued, the corresponding memory trace is mapped to the set of neurons expressing the gene, or a subset of these (Zars, 2000b).
Olfactory short-term memory was tested with an apparatus in which flies are sequentially exposed to two odorants, one of which is paired with electric shocks. Shortly after training, ~95% of wild-type flies prefer the odorant not accompanied by punishment. Mutant rut flies show significantly lower memory scores. To test whether the olfactory learning defect of the rut mutant is rescuable, a P-element expressing a wild-type rut cDNA under the control of a GAL4-sensitive enhancer P[UASGAL4-rut+] was combined in the rut mutant with a driver transgene P[elav-GAL4] expressing the yeast GAL4 transcription factor in all neurons. This pan-neuronal expression of rut AC partially restores olfactory learning in the rut mutant. The incomplete rescue could be due to insufficient expression levels of the P[UASGAL4-rut+] transgene, a dominant negative effect of the P[elav-GAL4] element, or a negative effect of ectopically expressing this transgene (Zars, 2000b).
Several GAL4 enhancer trap lines were selected for local rescue because of their expression patterns. Mutant rut flies with the enhancer trap GAL4 elements 247, c772, 30y, 238y, and H24 in combination with the P[UASGAL4-rut+] transgene show memory scores statistically indistinguishable from wild-type flies. The GAL4 line 201y partially rescues the rut learning defect. Finally, rut mutant flies with three other GAL4 enhancer trap elements (c232, 189y, and 17d) and the P[UASGAL4-rut+] effector gene have rut mutant-like short-term memory scores (Zars, 2000b).
Four of the nine enhancer trap lines were previously used to study olfactory learning after locally expressing a constitutively activated G-protein alpha subunit (Galphas*). In the present experiments, the magnitude of rescue was similar to the suppressive effect of the Galphas* protein in the respective lines. In c232, Galphas* has no effect; in 201y, suppression is about 50%, whereas in c772 [the same expression pattern as c747] and 238y, suppression is nearly complete (Zars, 2000b).
Neither the rescuing GAL4 enhancer trap lines without P[UASGAL4-rut+] nor the P[UASGAL4-rut+] line without driver has a dominant rescue effect. Nor do the nonrescuing GAL4 enhancer trap inserts have a negative effect on wild-type flies. Thus, it is the specific interaction of the GAL4 enhancer trap element with the P[UASGAL4-rut+] effector that can rescue the memory defect in rut mutant flies (Zars, 2000b).
Control experiments were conducted with naive wild-type, rut mutant, and potentially rescued rut mutant flies to assure that none of the memory scores were due to changes in shock reactivity or perception of the odorants. All genotypes avoid electric shocks at similar levels. Although 189y and 17d have somewhat reduced shock reactivity, their responses here are similar to that of c772, which shows a wild-type-like learning score. Thus, these shock reactivity scores cannot be responsible for the low memory scores. In addition, all genotypes tested avoid the aversive odorants used in the training protocol (Zars, 2000b).
To determine what brain structures are minimally sufficient for olfactory short-term memory, the expression patterns of the rescue and nonrescue GAL4 enhancer trap lines were examined. Serial sections show that the common structure labeled in all rescuing GAL4 lines is specifically the mushroom bodies. Indeed, comparing expression patterns of rescuing and nonrescuing lines indicates that the gamma lobes may be especially important. In contrast to the GAL4 lines used, the rescuing line 247 lacks expression in the median bundle. The latter, therefore, is not part of the set of minimally sufficient neurons (Zars, 2000b).
On the basis of the current model of how type I ACs function in synaptic plasticity and on the connectivity of the mushroom bodies, the short-term memory trace of odors is localized to a single level in the olfactory pathway: the presynaptic sites in the Kenyon cells contacting extrinsic output neurons and possibly other Kenyon cells in the peduncle and lobes. Modulating neurons carrying the reinforcer must project to the peduncle or lobes and contact presynaptic endings of Kenyon cells there. No rut-dependent synaptic plasticity is required in the antennal lobe or calyx for olfactory learning (Zars, 2000b).
Different brain structures are involved in different learning tasks. In the heat box paradigm, the median bundle, antennal lobes, and ventral ganglion are sufficient for rescue of rut-dependent short-term memory. To find olfactory and heat box memory at different locations was not unexpected, since mushroom body-less flies do well in heat box learning. The task-specific rescue in different GAL4 lines strongly supports the claim that it is the spatial distribution of the rut AC that matters (Zars, 2000b).
Several open questions remain. (1)The conclusions refer only to rut-dependent synaptic plasticity. Although unlikely, the 60% short-term memory remaining in rut mutant flies may reside outside the mushroom bodies. (2) The current understanding of the role of type I ACs in synaptic plasticity and learning is not complete. (3) The P[UASGAL4-TAU] reporter was used to visualize GAL4 expression patterns, and coincidence with P[UASGAL4-rut+] is inferred. (4) Temporal control of transgene expression in these lines is not yet possible, leaving the faint possibility that the behavioral rescue in some cases might be due to developmental expression. (5) Whether all memory traces of odors reside in the mushroom bodies and how memory traces of odors are organized within the mushroom bodies await further investigation (Zars, 2000b).
The technique of restoring synaptic plasticity in minimally sufficient brain regions has, in two cases, revealed simple, locally confined memory traces. This result is probably due to the simplicity of the learning tasks, requiring the animal to store a single sensory modality for a binary orientation response. It will be of considerable interest to map memory traces of more complex learning paradigms (Zars, 2000b).
Exons - 16
Four putative adenylate cyclase genes from Drosophila melanogaster have been identified by virtue of their extensive sequence homology with mammalian cyclases. One is coded for by the learning and memory gene rutabaga and is most similar to the mammalian brain Ca2+/calmodulin (CaM)-responsive cyclase. The hydrophobicity profile of the N-terminal half is similar to that of the mammalian Type I enzyme and suggests a similar arrangement of twelve membrane-spanning segments. There are two potential N-linked glycosylation sites in the predicted extracellular loop between membrane spans 9 and 10. (Levin, 1992).
date revised: 20 FEB 97
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