Olfactory habituation: fresh insights from flies.

H abituation, the reduction in an animal’s response to the repeated occurrence of an unchanging stimulus, is generally regarded as the simplest form of learning (1). Moreover, it is ubiquitous: every animal with a nervous system seems to possess the capacity for habituation (2). Given these facts, one might expect that habituation would be fairly well understood by modern neurobiologists. In reality, however, our understanding of the cellular mechanisms that underlie habituation is meager at best (3). That habituation remains so poorly grasped, neurobiologically, more than 100 years after the initial scientific accounts of this basic behavioral phenomenon (2) is—or should be—a matter of significant embarrassment for the field of learning and memory. However, two articles in PNAS by Ramaswami and colleagues (4, 5) go some way toward easing the embarrassment. They contain important insights into the cellular and molecular mechanisms of olfactory habituation in Drosophila, insights likely to generalize to other forms of habituation in other species, including mammalian species. By way of background, the fly’s olfactory circuit comprises three levels of neurons (6). Odors are initially detected by olfactory sensory neurons (OSNs), whose cell bodies are in the antennae of the head of the fly. There are ≈1,300 OSNs, and each expresses only 1 of 62 receptor proteins. The axons of the OSNs project to the antennal lobe (AL), where they synapse in glomeruli onto odor-specific projection neurons (PNs), as well as onto local excitatory or inhibitory interneurons (LNs); the LNs make both intraglomerular and interglomerular connections with PNs. Finally, the PNs project to neurons (the Kenyon cells) in the mushroom body, a lobed neuropil in the fly’s brain that plays a critical role in associative olfactory learning (7). To induce short-term olfactory habituation in flies, the investigators exposed flies for 30 min to one of two odors [ethyl butyrate (EB) or CO2] that flies normally find aversive. After this training 30–40 flies were placed together in a Y-maze consisting of two glass tubes joined at their base to an entry tube. (Flies are commonly trained and tested en masse in Drosophila learning experiments.) One arm of the maze contained the training odor (EB or CO2), and the other arm contained air, and the flies were allowed to move into either arm from the entry tube. After 1 min the number of flies in each arm was quantified. Other sets of flies that had not received the habituation training (naïve flies) were tested identically. Flies that had been exposed to an odor more readily entered the arm of the Y-maze containing that odor during the test (avoided the odor less) than did naïve flies, and this reduced avoidance, or habituation, lasted ≈30 min. The olfactory habituation was odor-specific: flies exposed to EB, for example, showed habituation to EB during the testing but exhibited normal avoidance to CO2 (4). The basic protocol for inducing long-term habituation (LTH) was similar to that for short-term habituation (STH), except that flies were given 4 d of exposure to an odor during training. The more prolonged training resulted in habituation that persisted for several days (4). A priori, there are two likely candidates for a cellular mechanism of habituation: depression of excitatory connections and potentiation of inhibitory connections. Previous cellular work on habituation has largely provided support for the first mechanism (for example, refs. 3 and 8). Having demonstrated both STH and LTH to olfactory stimuli, Ramaswami and colleagues exploited the well-known power of Drosophila genetics to determine which of the two general mechanisms underlay olfactory habituation. First, the investigators showed that both STH and LTH are defective in mutant flies that lack the gene for rutabaga, which encodes a Casensitive adenylyl cyclase (9). Using rutabaga mutant flies, they systematically expressed a wild-type rutabaga transgene in different classes of neurons in the Drosophila olfactory circuit to identify the type(s) of neurons in which rutabaga must be expressed for normal habituation. Expression of the wild-type transgene in the LN1 class of local interneurons that use the inhibitory transmitter GABA was sufficient to rescue both STH and LTH in the mutant flies; thus, GABA-mediated inhibition within the AL is critical for olfactory habituation (4). To determine whether GABA-mediated inhibition of the responses of PNs to odors underlies the expression of habituation, a specific GABAA receptor, the resistance to dieldrin (RDL) receptor, was selectively reduced in a cell-specific manner through the use of the Drosophila Gal4-UAS binary expression system (10). The targeted knockdown Projection