CrebB-17A

DEVELOPMENTAL BIOLOGY

Larval and Adult

There is a complex pattern of transcripts apparent in larval stages, and in the heads and bodies of adult flies, with at least 12 different size transcripts apparent. The adult head contains at least six transcripts (Yin 1994).

Adult Fmr1 mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of Fmr1 leads to a lengthened period. Fmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects (Dockendorff, 2002).

One known clock-controlled gene in Drosophila is the cAMP response element binding protein (CREB). To determine if the circadian oscillation of this protein is affected in the Fmr1 mutant flies, Fmr1 mutant flies carrying the CRE-luciferase (CRE-luc) reporter gene were examined in a luminometer continuously in constant daylight (DD) for up to 4 days. Although cycling of the CRE-luc reporter is detected in the Fmr1 mutant background, the amplitude of the oscillations is clearly reduced compared to the oscillations in the control background. This result indicates that dfmr1 affects a known molecular output of the clock. Normal cycling of PDF levels was seen in the termini of the small lateral neurons in the Fmr1 mutant brains. Thus this output of the clock is not affected at the normal site of its release, providing further evidence for normal central clock functioning in the Fmr1 mutant flies (Dockendorff, 2002).

Effects of Mutation

In Drosophila, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, the involvement of a candidate gene, cAMP response-element binding protein (CREB), was investigated. The duration of rest is inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies does not affect the clock, but increases rest rebound. CREB mutants also have a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increases after rest deprivation and remains elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila (Hendricks, 2001).

The daily rest of flies carrying mutations and/or transgenes that alter cAMP signaling was examined at several points in the pathway. dunce flies have a mutation in the phosphodiesterase enzyme and therefore have increased cAMP. The null mutant (dnc^ML) rests significantly less than the background yw strain. Similarly, increasing PKA activity in flies with a heat-shock-inducible transgene of the catalytic subunit of PKA significantly decreases daily rest durations compared to pre-heat-shock rest levels. Decreased adenylyl cyclase enzyme activity and thus decreased cAMP characterize rutabaga (rut) mutants, which rest more than the Canton S background strain. Similarly, S162 flies that carry a mutation that abolishes dCREB2 activity rest more than their comparison group (siblings without the mutation). The mutation is a stop codon just upstream of the basic leucine-zipper motif of the dCREB2 gene (Hendricks, 2001).

Lines of flies with the heat shock-inducible activating (HS-dCREB2a) and blocking (HS-dCREB2b) dCREB2 transgenes were also examined. dCREB2 is a major target of PKA in Drosophila, and these transgenes have effects on long-term memory consolidation in Drosophila. Even without heat shock, baseline rest is increased in flies carrying the HS-dCREB2b transgene, whereas the flies with the inducible activator rest slightly but significantly less, suggesting a leaky heat shock promoter. When the locomotor activity was measured on the same days in all of these lines, three measures of daily activity were not significantly correlated with rest levels, providing evidence that rest is regulated independently of locomotor activity, and that the increase in rest with decreasing cAMP signaling is not due to general debility or sluggishness (Hendricks, 2001).

Because rest is inversely related to the level of cAMP signaling or dCREB2 activity, it seemed that the normal dCREB2 peak might be important for the animal to maintain normal waking. That is, nighttime dCREB2 might have a function for subsequent waking, consistent with the idea that dCREB2 might mediate a restorative function of rest, permitting or fostering sustained waking. Wild-type flies respond to six hours of rest deprivation at night by exhibiting a rest rebound (an increase in rest duration) for the morning six hours of each day of a three-day recovery period. This rebound is related to the duration of rest deprivation, and is not seen when the flies are subjected to the same stimulation during their usual active period. If the nocturnal peak in dCREB2 were necessary for recovery from rest deprivation, blocking the normal dCREB2 activity peak would be expected to impair the ability to recover after rest deprivation, as nighttime dCREB2-dependent gene expression would be abolished. In contrast, overexpressing the activator just before the usual peak might have minimal effects if the normal CREB-mediated transcription is already sufficient for normal function (Hendricks, 2001).

The response to rest deprivation was studied in the wild-type isogenic background strain, flies with the blocker transgene (HS-dCREB2b), and flies with the activator transgene (HS-dCREB2a). Wild-type and transgenic dCREB2 flies were deprived of six hours of rest, with or without heat shock. For each genotype, rest-deprived flies were compared to flies that were allowed to rest undisturbed, and to controls that were subjected to handling but were not rest-deprived. The mixed-model analysis of variance takes into account both between- and within-animal factors. The within-animal factor in this case is the pattern of each individual's rest throughout the study, and between-animal factors are the effects of experimental group, genotype and heat shock. A significant interaction (p < 0.0001) existed in the effect of heat shock and experimental group (resting, handled controls or deprived) genotype. That is, the effect of heat shock on rest duration depended on both the experimental condition and the genotype. By using a series of specific post hoc comparisons using only the data from after heat shock, it was found that inducing the blocker isoform of dCREB2 specifically increases the rest of these flies during recovery from deprivation throughout the entire period after deprivation. In contrast, inducing the activator (dCREB2a) does not significantly alter the rest rebound over the three-day period after deprivation. Heat shock alone does not increase rest in the undisturbed or handled control dCREB2b flies. Thus, rest rebound is enhanced only when dCREB2b induction is combined with rest deprivation. The increased rest during recovery from rest deprivation is detectable in individual dCREB2b flies as well as in the populations. The mean daily rest rebound in HS-dCREB2b flies is increased after deprivation on all three successive days, although the degree of rebound falls over time (from 1.96 hours above baseline on the first day to only 0.67 hour on the third day after deprivation). The ability to move is not differentially changed by heat shock and deprivation in dCREB2b flies compared to wild-type flies, as measured by changes in peak activity during the period after deprivation (Hendricks, 2001).

Because CREB is involved in responses to stress in several systems, blocking CREB activity may somehow alter the flies' response to six hours of stimulation, independent of any rest-related function. The response of dCREB2b flies was studied to the same combination of six hours stimulation and heat shock, applied during the usual daytime active period (heat shock at circadian time [CT] 0, stimulation from CT 6 to 12). The response (change in rest compared to baseline and to handled controls) of dCREB2b and background flies was statistically the same (Hendricks, 2001).

The findings that blocking dCREB2 increases rest rebound, and that rest rebound is associated with an increase in CRE-dependent gene expression, implicate CREB activity in a restorative function of rest. It is hypothesized that CREB activation during rest optimizes waking CNS function. In this context, it is interesting that cAMP signaling, PKA and CREB activity have a conserved role in learning and memory. One putative restorative function of sleep, optimizing neural plasticity, could be evolutionarily ancient. To directly test whether CREB also has a conserved role in states of arousal, CREB mutant mice have been used to study the involvement of CREB in sleep and waking (L. Graves, et al., unpublished observations cited in Hendricks, 2001). Findings support a conserved role of CREB in maintaining normal levels of wakefulness, independent of changes in circadian period. Additional studies to discover whether the rest-related role of CREB is, indeed, related to optimizing plasticity will continue to enhance understanding of the role of this signaling pathway in complex behaviors (Hendricks, 2001).

Because of the array of mutants and transgenics available in Drosophila, it was possible to show that baseline rest duration is inversely related to each component of the classic cAMP-PKA-dCREB2 signaling pathway. However, CREB and the CRE binding site are responsive to signals in addition to the cAMP-PKA pathway, and cAMP signaling has targets in addition to CREB. Multiple signaling pathways may well be involved in the many functions of CREB, and specifically in the rebound response to rest/sleep deprivation. CREB is a complex gene, even in Drosophila, with functions depending on the cellular milieu and developmental stage of the organism. The mammalian CREB/CREM family of transcription factors is critical for development, addiction, neural growth and survival, antidepressant effects, long-term memory consolidation and stress responses. Despite the potential for complex regulation of CREB activity, the data suggests that cAMP and PKA are particularly important in regulating CREB during the rest-activity cycle. An additional link between the circadian system and rest-activity behavior is a gene (the Drosophila NF-1 homolog) previously linked to learning and to cAMP signaling in both Drosophila and mammals, that modulates both the circadian rest-activity cycle and CREB activity. Based on the similarities in the effects of CREB mutations on rest in Drosophila and sleep in mammals, a role for NF-1 may similarly be conserved. Similar to the involvement of fruit fly genetics in identifying the molecular basis of vertebrate circadian rhythms, studies of the molecular mechanisms of Drosophila rest should help to focus studies of mammalian sleep (Hendricks, 2001).

Induction of cAMP response element-binding protein-dependent medium-term memory by appetitive gustatory reinforcement in Drosophila larvae: larval memory depends on both amnesiac and CREB

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).

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References continued: part 2/2

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