rutabaga: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - rutabaga

Synonyms -

Cytological map position - 12F5-13A1

Function - adenylate cyclase

Keywords - neural, cAMP signaling, learning pathway, calcium dependent enzymes

Symbol - rut

FlyBase ID:FBgn0003301

Genetic map position - 1-[46]

Classification - Type 1 - Ca++ CaM sensitive

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Dissel, S., Angadi, V., Kirszenblat, L., Suzuki, Y., Donlea, J., Klose, M., Koch, Z., English, D., Winsky-Sommerer, R., van Swinderen, B. and Shaw, P.J. (2015). Sleep restores behavioral plasticity to Drosophila mutants. Curr Biol [Epub ahead of print]. PubMed ID: 25913403
Given the role that sleep plays in modulating plasticity, this study hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reversed memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

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).

Place memory retention in Drosophila

Some memories last longer than others, with some lasting a lifetime. Using several approaches memory phases have been identified. How are these different phases encoded, and do these different phases have similar temporal properties across learning situations? Place memory in Drosophila using the heat-box provides an excellent opportunity to examine the commonalities of genetically-defined memory phases across learning contexts. This study determines optimal conditions to test place memories that last up to three hours. An aversive temperature of 41°C was identified as critical for establishing a long-lasting place memory. Interestingly, adding an intermittent-training protocol only slightly increased place memory when intermediate aversive temperatures were used, and slightly extended the stability of a memory. Genetic analysis of this memory identified four genes as critical for place memory within minutes of training. The role of the rutabaga type I adenylyl cyclase was confirmed, and the latheo Orc3 origin of recognition complex component, the novel gene encoded by pastrel, and the small GTPase rac were all identified as essential for normal place memory. Examination of the dopamine and ecdysone receptor (DopEcR) did not reveal a function for this gene in place memory. When compared to the role of these genes in other memory types, these results suggest that there are genes that have both common and specific roles in memory formation across learning contexts. Importantly, contrasting the timing for the function of these four genes, plus a previously described role of the radish gene, in place memory with the temporal requirement of these genes in classical olfactory conditioning reveals variability in the timing of genetically-defined memory phases depending on the type of learning (Ostrowski, 2014).

Temperature as an aversive reinforcer interacts with training conditions to induce place memories of different stabilities. Previous work showed that intermittent training for Drosophila in space and place memory increases memory performance up to two hours after training. Shown in this study is that temperatures at or above 41°C are needed for induction of this longer lasting memory. That is, 37°C and below can act as an aversive reinforcer and condition flies to avoid a part of the training chamber, but continued avoidance decays within minutes of training. It is only with a temperature of 41°C that an hours-long memory is induced with massed and intermittent training. This abrupt difference in the length of the memory after training with the higher temperature may reflect a threshold of some sort, the steepness of which is currently unknown. This could arise from a differential input to the reinforcing circuit from separate sensory systems, like the Trp family of receptors, or from altered output from one of these sensory systems. Future studies on different temperature responsive proteins may differentiate between these possibilities (Ostrowski, 2014).

Genetic analysis challenges the use of time as a critical factor in determining a memory phase. Memory phases in the fly were initially examined after classical olfactory conditioning where an odorant is typically paired with an aversive electric shock or a rewarding sugar. Four different memory phases have been classified based roughly on time after training and genetic/pharmacological manipulations. Short-term memory after olfactory learning is measured within minutes of training; long-term memory and anesthesia resistant memory start to be active within hours and are increasingly important for memories at the 24 h range and longer. An intermediate memory is thought to be important in the interval between short-term and long-term memories. That time alone is a critical factor in determining these phases loses support when comparing flies with different mutations in aversive and rewarded olfactory memory. For example, the long-known mutant radish was originally shown to be important in the hours-long range after aversive olfactory training and genetically classified the anesthesia-resistant memory. Interestingly, this gene is important within minutes of training in rewarded olfactory memory (Ostrowski, 2014).

Several genes that are important for early to late phases of classical olfactory conditioning are critical on a finer time scale in place memory. Mutation of both the rut and lat genes leads to reduced aversive olfactory memory tested immediately after training, as well as longer time points. Although it is currently unclear when during the life-cycle these genes are important for place memory, mutation of rut and lat reduces memory directly after training. Furthermore, both the rut and lat products have been implicated in synaptic plasticity at the neuromuscular junction (NMJ), which suggests a role for these genes in early stages of learning and memory. It is pretty straight-forward that the rut-encoded type I adenylyl cyclase is also acting early on in associative processes in place learning. The lat gene encoding a subunit of the origin of replication (orc3) is also localized to the pre-synaptic specializations at the NMJs). The lat-orc3 also acts early-on in associative processes for place learning. How the lat-orc3 product is related to regulation of cAMP levels is, however, not as clear. The rut and lat results add to our understanding of an apparently common set of short-term changes in memory between olfactory and place memory, which include a common function of the S6 kinase II, an atypical tribbles kinase, and the arouser EPS8L3. And, the recently identified role of the foxp transcription factor specifically in operant learning, as tested in a flight simulator, suggests another set of genes that could be important for operant place memory in the minutes range (Ostrowski, 2014).

Late memory phases in classical olfactory conditioning depend on a set of genes that are important for place memory within minutes. The first challenge to a common timing of a memory phase came from the radish gene. In contrast to a role in the hours range after olfactory learning, radish mutant flies have a deficit in operant place memory within minutes of training. Furthermore, the pst gene (CG8588), encoding a novel product, has been previously shown to have a specific defect in aversive olfactory memory 24 h after spaced training. That is, the pst mutant flies have a normal short-term olfactory memory but a defective memory 1 day later. Interestingly, in the heat-box pst mutant flies already show a significant decrement in place memory immediately after training. This place memory defect seems to get worse within the first hour after training, reduced to ~50% of normal after 60 min. Thus, this 'long-term memory gene' is also involved in a memory within minutes of training in a second learning situation (Ostrowski, 2014).

Using the classical aversive olfactory learning paradigm the rac small GTPase has been identified as a key regulator in memory retention. Inhibition of Rac activity slows early olfactory memory decay, leading to elevated memory levels one hour after training, but becoming increasingly important 2 h after training. There does not appear to be an effect of Rac inhibition in olfactory memory in the minutes range after training. Transgenic flies with inhibited Rac function also have an increase in memory retention after place memory training. However, the first evidence of an increase in memory performance is within 10 min. Impressively, significant place memory was still evident up to 5 h after training, far beyond the range that can be typically measured in wild-type flies. Thus, while rac has a more general role in stabilizing memories, the timing of this function depends again on the type of memory trace that is formed (Ostrowski, 2014).

Not all memory genes first identified in other contexts, however, play a significant role in place memory. The DopEcR gene has been implicated in several behaviors, including a 30 min memory after courtship conditioning. This G-protein linked receptor is responsive to both dopamine and the steroid hormone ecdysone. Remarkably, DopEcR has been shown to interact with the cAMP cascade through double mutant and pharmacological tests. Using conditions that induce a robust and lasting place memory, the DopEcR mutant flies do not show a defect in memory directly after training or at 1 h post-training. This is despite the fact that the rut and cAMP-phosphodiesterase genes (dunce) are critical for place memory. It may be that DopEcR is not required for this type of learning and would be consistent with the independence of place memory from dopamine signaling. Alternatively, other redundant pathways may compensate for the reduction in DopEcR function caused by the DopEcRPB1 allele. One might further speculate that other types of behavioral plasticity, such as reversal learning or memory enhancement after unpredicted high temperature exposures in the heat-box might be more sensitive to DopEcR changes. Future experiments will determine if this is the case (Ostrowski, 2014).

Memory stability across learning contexts in Drosophila has some common genetic mechanisms, but the timing for gene action depends on the type of learning. That this study has added several genes here, including lat, pst, and rac as regulators of memory stability in operant place memory suggests that there are at least some common molecular processes in memory stability across different learning types. However, the timing of these genetically-defined phases depends on what is learnt. It is speculated that an ideal system to regulate memory stability would be one that activates its own decline. That is, a given memory type should activate the process of decreasing memory expression. This might work with the recruitment of a reinforcing pathway, like the dopaminergic signal that is important for both the acquisition of an associative olfactory memory and the active process of forgetting that association. In this case an odor associated with shock gives rise to a memory trace in mushroom body neurons that depends on a set of dopamine neurons that is important for both memory acquisition and decline. Whether this type of aminergic-based system applies to other forms of memory is not yet known. However, if an aminergic-based signal is common in memory decline, as appears to be the case with the Rac-based mechanism, differences in the types of aminergic neurons or innervation targets could give rise to the altered stabilities of behaviorally expressed memories (Ostrowski, 2014).


cDNA clone length - 7545

Exons - 16


Amino Acids - 2249

Structural Domains

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).

Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 FEB 97 

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