Drosophila has been successfully used as a model animal for the study of the genetic and molecular mechanisms of learning and memory. Although most of the Drosophila learning studies have used the adult fly, the relative complexity of its neural network hinders cellular and molecular studies at high resolution. In contrast, the Drosophila larva has a simple brain with uniquely identifiable neural networks, providing an opportunity of an attractive alternative system for elucidation of underlying mechanisms involved in learning and memory. This paper describes a novel paradigm of larval associative learning with a single odor and a positive gustatory reinforcer, sucrose. Mutant analyses have suggested importance of cAMP signaling and potassium channel activities in larval learning as has been demonstrated with the adult fly. Intriguingly, larval memory produced by the appetitive conditioning lasts medium term and depends on both amnesiac and cAMP response element-binding protein (CREB). A significant part of memory was disrupted at very early phase by CREB blockade without affecting immediate learning performance. Moreover, synaptic output of larval mushroom body neurons is required for retrieval but not for acquisition and retention of the larval memory, including the CREB-dependent component (Honjo, 2005).

The larval olfactory system is significantly simpler than the adult system with only 21 odorant receptor neurons. To find chemicals that are suitable for larval learning assays, 30 odorants were examined for naive larval chemotactic behavior and they were classified into four groups based on their attractiveness. 19 moderate attractants were examined for their effectiveness on larval appetitive olfactory conditioning. Larvae were exposed to an odor for 30 min in association with 1 M sucrose spread on agar. After conditioning, larvae were gently rinsed with distilled water to remove sucrose and tested for olfactory response on the test plate. For 10 of the 19 odorants, animals that received the odor with 1 M sucrose showed enhanced migration to the conditioned odor with significantly higher RI than control larvae, which had been exposed to the same odor but in conjunction with distilled water. Among the odorants examined, linalool (LIN), Pentyl acetate (PA), and gamma-valerolactone (GVA), which gave the largest RI increments in LIN/SUC conditioning, were chosen (Honjo, 2005).

To examine whether the increase of response index after conditioning is attributable to associative learning, a set of control experiments were performed. Significant response index increase was observed only when larvae were trained with LIN in association with SUC (LIN/SUC); response index did not change significantly from naive larvae when larvae are trained with LIN in association with distilled water (LIN) or sucrose alone (SUC). Notably, neither LIN nor sucrose alone resulted in habituation of larval olfactory responses compared with naive animals. Similar results were obtained with PA, except that conditioning with PA in association with distilled water led to slight desensitization. In contrast, conditioning with GVA in association with distilled water led to strong desensitization. However, the associative conditioning with GVA/SUC overcame the suppression (Honjo, 2005).

It was then asked whether the enhancement of larval response requires simultaneous exposure to both the odor and the reinforcer. As a temporal dissociation control, larvae were successively exposed first to sucrose and then to LIN or vise versa. Whereas simultaneous exposure to both LIN and sucrose (conditioning 1) resulted in enhanced olfactory response, the dissociation control, in which larvae were first exposed to sucrose and then to LIN, led to no enhancement compared with the odor alone control (conditioning 2). The requirement of temporal association between odor exposure and sucrose reinforcement was further confirmed in another set of dissociation controls. Exposure to LIN (conditioning 5) led to slightly higher larval response than conditioning 2, which seems a nonassociative effect caused by the delay attributable to the 30 min mock treatment (for delayed nonassociative effects). Nonetheless, simultaneous exposure to LIN and 1 M sucrose (conditioning 4) led to additional response index increment reproducing associative odor learning. In contrast, separate exposures to LIN and then 1 M SUC (conditioning 6) failed to do so (Honjo, 2005).

It was next asked whether the increased larval olfactory response was specific to the exposed odor. To address this question, larval olfactory responses were tested using odorants other than the one used for conditioning. When larvae were trained with LIN/SUC, PA/SUC, or GVA/SUC, only those trained with LIN/SUC showed significant response index increment in the olfactory test with LIN. Similarly, only larvae trained with PA/SUC showed significant response index increment in the olfactory test with PA. These results thus demonstrate that the enhanced larval response with sucrose is specific to the conditioned odor and suggest that Drosophila larvae discriminate the three odors despite their limited olfactory system (Honjo, 2005).

Whereas the above data emphasizes the importance of sucrose as a positive reinforcer, it is not clear whether response index stimulation is attributable to gustatory stimuli or attributable to higher osmotic pressure of 1 M sucrose than that of distilled water. To clarify this point, larvae were trained with LIN in association with 1 MD-sorbitol, a sugar that is tasteless to the flies. Conditioning with LIN in association with D-sorbitol failed to stimulate larval response index compared with the control, in which larvae were exposed to LIN in association with distilled water (Honjo, 2005).

Most studies on Drosophila associative learning have used reciprocal and symmetrical experimental paradigms with two odors. In contrast, the paradigm here uses only a single odor for conditioning and test. Consequently, this asymmetric nature calls for parallel controls to rule out nonassociative learning such as habituation and sensitization. Nonetheless, the paradigm resulted in significant learning only by the associative conditioning, in which both an odor and sucrose were simultaneously presented to larvae; enhanced larval olfactory response was specific to the odor paired with sucrose, excluding nonassociative sensitization to a broad range of odors. Conversely, it should be noted that strong desensitization was observed for certain odors such as GVA. Even with LIN, which showed no desensitization in immediate learning, delayed nonassociative effects on larval olfactory response were detected, emphasizing the importance of odor choice and careful data interpretation (Honjo, 2005).

Because different sets of larvae are used for control experiments for the stimuli involved, reproducibility of larval responses is critical to the paradigm. At this point, select odorants were used for screened for larval olfactory learning. Thus, of 30 chemicals, several odorants were chosen that produced significant response index increment with sucrose. Many odorants, such as 1-octanol and 4-methylcyclohexanol, which have been used in adult studies, failed to produce significant response index increment. The fact that larvae and adult flies exhibit different olfactory responses also highlights the importance of odorant choice for larval experiments (Honjo, 2005).

Despite its asymmetric design, several points are notable with regard to this paradigm: (1) the simple experimental design minimizes stress on larvae, which could affect learning performance; (2) the paradigm generates medium term memory (MTM) that lasts up to 3 h; (3) the paradigm is free from odor discriminative task. Because the larval olfactory system is considerably simpler than the adult system, simultaneous discrimination of different odors could complicate animal responses, although other studies have used two-odor paradigms; (4) because only a single odor is applied to larvae during training, the simple design of this paradigm may be of use in imaging of neural representation of the conditioned odor in the brain during learning and memory (Honjo, 2005).

Adult flies with amn mutations show a reduction in immediate memory as well as a more profound reduction in MTM. In this paradigm, amn larvae show reduced but significant immediate learning/memory. In the adult brain, the AMN peptide is expressed in dorsal paired medial (DPM) neurons that are situated medially to MBs and ramify throughout the MB lobes. Little is known about the network of the DPM neurons and the AMN expression pattern in the larval brain (Honjo, 2005).

Studies with Aplysia, mice, and adult Drosophila flies show that CREB-dependent transcription is required for cellular events underlying LTM. These studies have shown that CREB functions as a conserved molecular switch for LTM, which is thought to be induced several hours after training. Moreover, intervals between trainings or stimulations are generally required to produce CREB-dependent long-term effects (Honjo, 2005).

The larval CREB-dependent memory is stable for only medium term. Moreover, this paradigm continuously exposes larvae to an odor and sucrose during training, a condition similar to massed training of adult flies. Intriguingly, CREB is recruited shortly after learning in larvae; a significant portion of 30 min memory was disrupted by the CREB blocker, whereas immediate learning was not. If the larval MTM is induced after STM as in the adult fly, this very early CREB requirement might imply fast transition of memory phases. Alternatively, the CREB-dependent memory might also be generated independently. Intriguingly, it has been proposed that CREB can be activated independent of STM in long-term synaptic facilitation in Aplysia. In addition, although memory performance becomes undetectable in 3 h, the requirement of CREB activity suggests neural mechanisms that are in part shared with LTM in the adult fly. In fact, memory decay after CREB blockade is somewhat slower than in amn mutants. Furthermore, whereas CREB blocker has been shown to suppress 1 and 7 d memories, whether the blockade has more immediate effects is not known, leaving the possibility that CREB could be recruited early in the adult fly as well. Notably, memory performance in the adult fly tends to be higher with spaced training than with massed training already at several hours (Honjo, 2005).

Biochemically, CREB is activated by phosphorylation in response to diverged extra cellular stimuli. Among them, the protein kinase A (PKA) plays a central role in phosphorylation of CREB1-a, the catalytic subunit. Because the larval memory is completely disrupted in dnc and rut mutants, the cAMP-PKA pathway might be involved in the early activation of CREB in larvae. Alternatively, intracellular pathways other than PKA could also be recruited to mediate CREB activation. Intriguingly, increase of intracellular cAMP is known to activate mitogen-activated protein kinase in Aplysia, which in turn phosphorylates CREB2-b, the regulatory subunit, allowing transcriptional activation by the catalytic CREB isoform in the nuclei (Honjo, 2005).

The adult MBs are highly complex structures with three sets of lobes, each of which might participates in different memory traces. In contrast, the larval MBs exhibit a remarkably simple projection pattern with only a single set of lobes. In addition, recent studies have revealed straightforward organization of the larval olfactory system with only 21 olfactory receptor neurons targeting the 21 antennal lobe glomeruli, from which projection neurons target the larval MB calyx that consists of ~28 glomeruli (Honjo, 2005).

The finding that larval MB output is essential for memory retrieval discloses functional importance of the larval MBs and directly demonstrates anatomical commonality of memory networks between the larval and adult brains. Furthermore, the results that larval MB output is not required for memory acquisition and retention suggest that larval olfactory memory is localized upstream of larval MB synapses, in either MB neurons themselves or upstream circuits such as antennal lobes. Combined with the recent advances in functional neural imaging, the simple and identifiable neural network of the larval olfactory system will help further elucidation of the cellular basis of learning and memory in the brain (Honjo, 2005).