The Interactive Fly

Zygotically transcribed genes

cAMP cascade and other genes involved in learning and memory

  • What is the cyclic AMP second messenger system?
  • Courtship Conditioning and Behavior
  • Global and local missions of cAMP signaling in neural plasticity, learning, and memory - A review
  • Distinct memory traces for two visual features in the Drosophila brain
  • Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation
  • Slow oscillations in two pairs of dopaminergic neurons gate long-term memory formation in Drosophila
  • Analysis of a spatial orientation memory in Drosophila
  • Serum response factor-mediated gene regulation in a Drosophila visual working memory
  • ERK phosphorylation regulates sleep and plasticity in Drosophila
  • Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila
  • Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory
  • Visual place learning in Drosophila melanogaster
  • Place memory retention in Drosophila
  • A subset of dopamine neurons signals reward for odour memory in Drosophila
  • Time of day influences memory formation and dCREB2 proteins in Drosophila
  • A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes

  • Mushroom Bodies - A site for formation and retrieval of olfactory memories
  • A permissive role of mushroom body α/β core neurons in long-term memory consolidation in Drosophila
  • Sequential use of mushroom body neuron subsets during Drosophila odor memory processing
  • Parallel processing of appetitive short- and long-term memories in Drosophila
  • Imaging of an early memory trace in the Drosophila mushroom body
  • Dopaminergic Modulation of cAMP Drives Nonlinear Plasticity across the Drosophila Mushroom Body Lobes
  • Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants
  • Additive expression of consolidated memory through Drosophila mushroom body subsets
  • Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies
  • Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae
  • Basic reversal-learning capacity in flies suggests rudiments of complex cognition

    Genes of the cyclic AMP second messenger system

    Exciting the cyclic AMP pathway

    Other genes involved in behavior, learning and memory but not part of cAMP pathway

    What is the cyclic AMP second messenger system?

    The cyclic AMP second messenger system is often called the learning pathway. But what is the first messenger system? How does cAMP function, and what are the consequences to the cell of cAMP signaling?

    In synaptic transmission the chemical signal sent from cell to cell is a neurotransmitter, a small molecule constituting the first signal (first messenger) received by target cells. Neurotransmitters can act directly on ion channels to modify the permiability of cells to ions. One type of channel allows extracellular Ca2+ ions to enter the cell. Alternatively, mutation in K+ channels, ether à go-go (eag) and Shaker, each affecting different K+ channels, display greatly enhanced nerve activity as a result of reduction in K+ currents. Changes in Ca++ ion levels can result in activation of the cyclic AMP pathway, or, in the case of K+ channels, can result in increased neuronal activity and increase in synaptic structure and branching. K+ channel mutation is thought to increase motoneuron activity and synaptic transmission by increasing the cAMP second messenger signaling.

    Rutabaga is an adenyl cyclase that creates cAMP in response to neurotransmitter signaling. Rutabaga is activated by the protein Calmodulin, an excellent sensor of the level of intracellular Ca2+. When Ca2+ levels are high, Rutabaga converts ATP into cAMP, which in turn activates PKA, the cyclic AMP dependent protein kinase. PKA in turn transduces the cAMP signals to downstream targets by phosphorylating (modifying with a phosphate residue) target proteins capable of effecting biological activity, such as transcription factors.

    Where does Amnesiac fit in this system? Amnesiac is a neuropeptide, the kind of protein that interacts with non-channel receptors called G-protein coupled receptors. Instead of serving as ion channels, G-protein coupled receptors signal through G-proteins. G-proteins are biochemical marvels. Upon receptor activation, heterotrimeric G-proteins bind GTP and dissociate into constituent parts which in turn initiate further signal transduction. In vertebrates, one target of G-proteins is the enzyme adenyl cyclase which converts ATP, the prevelent store of energy of the cell, into the second messenger, cyclic AMP.

    We are thus brought by a second route to Rutabaga, an adenyl cyclase of the fly, and its potential role in transmitting G-protein coupled receptor signaling to activate cell responses. How does all this lead to learning? This is discussed in the dunce and rutabaga sites and at sites for other genes involved in the cAMP second messenger pathway.

    Just as the cyclic AMP pathway is involved in learning, it is also involved in determining the strength of the neuromuscular junction. In fact, the neuromuscular junction, because of its accessability and the ease with which is observed, is used as a model system for studying the synaptic changes that take place on learning. For more information about the neuromuscular junction, see the Dunce, Fasciclin II, Discs large, MEF2 and Rutabaga sites.

    Global and local missions of cAMP signaling in neural plasticity, learning, and memory - A review

    The fruit fly Drosophila melanogaster has been a popular model to study cAMP signaling and resultant behaviors due to its powerful genetic approaches. All molecular components (AC, PDE, PKA, CREB, etc) essential for cAMP signaling have been identified in the fly. Among them, adenylyl cyclase (AC) gene rutabaga and phosphodiesterase (PDE) gene dunce have been intensively studied to understand the role of cAMP signaling. Interestingly, these two mutant genes were originally identified on the basis of associative learning deficits. Findings on the role of cAMP in Drosophila neuronal excitability, synaptic plasticity and memory are summarized in this review. It mainly focuses on two distinct mechanisms (global versus local) regulating excitatory and inhibitory synaptic plasticity related to cAMP homeostasis. This dual regulatory role of cAMP is to increase the strength of excitatory neural circuits on one hand, but to act locally on postsynaptic GABA receptors to decrease inhibitory synaptic plasticity on the other. Thus the action of cAMP could result in a global increase in the neural circuit excitability and memory. Implications of this cAMP signaling related to drug discovery for neural diseases are also described (Lee, 2015; Full text online).

    Distinct memory traces for two visual features in the Drosophila brain

    The fruit fly can discriminate and remember visual landmarks. It analyses selected parts of its visual environment according to a small number of pattern parameters such as size, colour or contour orientation, and stores particular parameter values. Like humans, flies recognize patterns independently of the retinal position during acquisition of the pattern (translation invariance). The central-most part of the fly brain, the fan-shaped body, contains parts of a network mediating visual pattern recognition. Short-term memory traces have been identified of two pattern parameters -- elevation in the panorama and contour orientation. These can be localized to two groups of neurons extending branches as parallel, horizontal strata in the fan-shaped body. The central location of this memory store is well suited to mediate translational invariance (Liu, 2006).

    A fly tethered to a torque meter, with its head (and hence its eyes) fixed in space, can control its orientation with respect to the artificial scenery in a flight simulator. In this set up, the fly is conditioned to avoid certain flight directions relative to virtual landmarks and recognizes these visual patterns for up to at least 48 h. Visual pattern recognition in Drosophila has been studied in some detail. Flies store values of at least five pattern parameters: size, colour, elevation in the panorama, vertical compactness, and contour orientation. Moreover, they memorize spatial relations between parameter values. The neuronal substrate underlying visual pattern recognition is little understood in any organism (Liu, 2006).

    In Drosophila, memory traces can be localized to groups of neurons in the brain. Using the enhancer GAL4/UAS expression system, short-term memory traces of aversive and appetitive olfactory conditioning have been assigned to output synapses of subsets of intrinsic neurons of the mushroom bodies (MBs). The Rutabaga protein -- a type 1 adenylyl cyclase that is regulated by Ca2+/Calmodulin and G protein, and is considered a putative convergence site of the unconditioned and conditioned stimulus in olfactory associative learning, selectively restores olfactory learning if expressed in these cells in an otherwise rutabaga (rut)-mutant animal. Moreover, expressing a mutated constitutively activating Galphas protein (Galphas*) in the MBs interferes with olfactory learning. Blocking the output from these neurons during memory retrieval has the same effect, while blocking it during acquisition has no effect. Interestingly, memory traces for other learning tasks seem to reside in other parts of the brain: for remembering its location in a dark space, the fly seems to rely on a rut-dependent memory trace (Zars, 2000) in neurons of the median bundle and/or the ventral ganglion (Liu, 2006).

    The present study localizes short-term memory traces for visual pattern recognition to the fan-shaped body (FB), the largest component of the central complex (CX; also called the central body in other species). The CX is a hallmark of the arthropod brain. It has been characterized functionally as a pre-motor centre with prominent, but not exclusive, visual input. In the locust, large-field neurons sensitive to the e-vector orientation of polarized light have been described in the CX. Because of its repetitive structure and the precisely ordered overlay of fiber projections from the two hemispheres in the FB, neighbourhood relations of visual space might still be partially preserved at this level (retinotopy). Using the genetic approach, this study shows that a small group of characteristic stratified neurons in the FB house a memory trace for the pattern parameter 'elevation', and a different set of neurons forming a parallel stratum contain a memory trace for 'contour orientation' (Liu, 2006).

    Of ten mutants with structural abnormalities in the CX, all were impaired in visual pattern recognition. They were able to fly straight and to avoid heat, yet they failed to remember the patterns. Did they really lack the memory or had they lost their ability to discriminate between patterns? Fortunately, individual flies often display spontaneous preferences for one of the patterns. In three lines, these preferences were consistent enough to reveal intact pattern discrimination, suggesting that aberrant circuitry of the central complex can affect visual learning independent of visual pattern discrimination (Liu, 2006).

    Since the developmental and structural defects in these mutants are not well characterized, the GAL4/UAS system was used to acutely interfere with CX function. A GAL4 driver line (c205-GAL4) was used with expression in parts of the CX and, the gene for tetanus toxin light chain (CntE) was used as the effector. CntE blocks neurons by cleaving neuronal Synaptobrevin, a protein controlling transmitter release. For temporal control, the temperature-sensitive GAL4-specific silencer GAL80 was added under the control of a tubulin promoter (tub-GAL80ts). Flies (UAS-CntE/+; tub-GAL80ts/c205-GAL4) were raised at 19 °C, and were transferred for 14 h to the restrictive temperature (30 °C) just before the behavioural experiment to induce GAL4-driven toxin expression. Flies kept at the low temperature showed normal memory scores, while after inactivation of GAL80ts no pattern memory was observed. Again, flight control and heat avoidance were normal, and Fourier analysis confirmed that flies at the high temperature had retained their ability to tell the patterns apart. As with the structural mutants, interrupting the circuitry of the CX by tetanus toxin expression seemed to specifically interfere with visual pattern memory. In addition, the use of tub-GAL80ts excluded the possibility that toxin expression in unknown tissues during development might cause the memory impairment in the adult. These results do not, as yet, address the question of memory localization (Liu, 2006).

    Visual pattern memory in the flight simulator requires an intact rut gene. Mutant rut flies (rut2080) showed normal visual flight control, heat avoidance and pattern discrimination. To confirm that the defect was indeed due to the mutation in the rut gene rather than an unidentified second-site mutation, rut was rescued by the expression of the wild-type rut cDNA (UAS-rut+) using the pan-neuronally expressing driver line elav-GAL4. Indeed, flies of the genotype rut2080/Y;elav-GAL4/UAS-rut+ have normal memory (Liu, 2006).

    Visual pattern memory in the flight simulator has been shown to depend upon at least two kinds of behavioural plasticity: (1) an associative classical (pavlovian) memory trace is formed linking a particular set of values of pattern parameters to heat; (2) the fly's control of the panorama operantly facilitates the formation of this memory trace (Brembs, 2000). Either of the two processes might depend upon the Rut cyclase (Liu, 2006).

    To address this issue, rut mutant flies were tested in a purely classical variant of the learning paradigm. During training, panorama motion was uncoupled from the fly's yaw torque and the panorama was slowly rotated around the fly. Heat was made contingent with the appearance of the 'punished' pattern in the frontal quadrant of the fly's visual field. All other parameters were kept as described. For testing memory, panorama motion was coupled again to yaw torque and the fly's pattern preference was recorded as usual. Even in the absence of operant facilitation, visual pattern memory required the intact rut gene. Therefore, the rut-dependent memory trace investigated in this study represents the association of a property of a visual pattern with the reinforcer (Liu, 2006).

    As a first step in localizing the memory trace, it was asked in which neurons of the rut mutant expression of the wild-type rut gene would be sufficient to restore learning. To this end, a total of 27 driver lines expressing GAL4 in different neuropil regions of the brain was used to drive the UAS-rut+ effector gene in the rut mutant background. The parameter 'elevation' was measured. With seven of the driver lines, pattern memory was restored (104y, 121y, 154y, 210y, c5, c205 and c271) (Liu, 2006).

    Comparison of the expression patterns of the 27 lines allowed the putative site of the memory trace to be narrowed down to a small group of neurons in the brain. The seven rescuing lines all showed transgene expression in a stratum in the upper part of the FB. In three of them staining is rather selective. It comprises, in addition to the FB, only a layer in the medulla, several cell clusters in the subesophageal ganglion and a few other scattered neurons (Liu, 2006).

    Evidently, rut+ expression in the MBs is neither necessary (104y, c5, c205, 154y) nor sufficient for rescue. This result is in line with the earlier observation that elimination of more than 90% of the MBs by hydroxyurea treatment of first-instar larvae has no deleterious effect on visual pattern memory. The MBs were ablated in one group of rescue flies (rut2080/Y;UAS-rut+/ +;c271/+). They showed full visual pattern memory (Liu, 2006).

    Although GAL4 expression in the optic lobes is prominent in all seven rescuing lines, it occurs in distinctly different layers that do not overlap. For instance, in 104y expression is restricted to layer 2, whereas in 210y it is found only in the serpentine layer (layer 7). A similar situation is found for the s ganglion, although there the staining patterns are more difficult to evaluate. Finally, expression in the ellipsoid body is again not necessary (104y, c5, c205, 154y) or sufficient (c232, 78y, 7y, and so on) for rescue. Thus, the expression patterns favour the conclusion that the neurons of the upper stratum of the FB might be the site of the memory trace for the parameter 'elevation' in visual pattern memory (Liu, 2006).

    Neurons in this stratum, labelled in all seven rescuing lines, have a very characteristic shape. Their cell bodies are located just lateral to the calyces. Their neurites run slightly upward in an antero-medial direction, forming an upward-directed tufted arborization just behind the alpha/alpha'-lobe of the MB. From there, the fiber turns sharply down and backward towards the midline just in front of the FB. Finally, it turns horizontally backward, spreading as a sharp stratum through all of the FB across the midline. These neurons have been described in Golgi preparations. They belong to a larger group of tangential FB neurons called F neurons. Besides the stratum in FB, most of them have an arborization in a particular part of the unstructured neuropil. The layer stained in 104y, and the other six rescuing lines, is tentatively classify as layer 5 (from bottom upward), and hence provisionally the neurons are called F5, although, without further markers, it is difficult to reliably number the layers. In summary, expression of Rut cyclase in F5 neurons rescues the rut-dependent memory defect for pattern elevation, whereas no rescue effect is observed in any of 20 strains without expression of Rut cyclase in F5 neurons (though Rut cyclase was expressed in other regions of the brain). Hence, a rut-dependent memory trace for pattern elevation may reside in F5 neurons (Liu, 2006).

    This finding does not exclude the possibility that memory is redundant, and that other rut-dependent memory traces for pattern elevation might be found elsewhere. Therefore, it was asked whether plasticity in the F5 neurons is necessary for visual pattern memory. The Rut cyclase is regulated by G protein signaling, and olfactory learning/memory can be blocked by a constitutively active form of the Galphas protein subunit (Galphas*). The Galphas* mutant protein was expressed in the FB using the driver line c205, and the flies were tested for their memory of 'elevation'. Memory was fully suppressed. Since in olfactory learning, overexpression of the wild-type protein does not interfere with learning, these results support the hypothesis that continuous upregulation of Rut cyclase in the F5-neurons interferes with visual short-term memory, implying that F5 neurons are the only site of a rut-dependent memory trace for pattern elevation (Liu, 2006).

    The patterns used in the experiments so far exclusively addressed the parameter 'elevation' (upright and inverted Ts or horizontal bars at different elevations). It was of interest to discover whether the mutant defect in rut and the Rut rescue in the F5 neurons affects only this parameter, or whether it applies to other pattern parameters as well. Therefore, the study looked at to two further parameters: 'size' and 'contour orientation'. Three driver lines -- c205, NP6510 and NP2320 -- were chosen showing different expression patterns in the FB. In the line NP6510, as in c205, a group of F neurons is marked. They are putatively classified as F1, since their horizontal stratum lies near the lower margin of the FB. Their cell bodies form a cluster in the dorso-frontal cellular cortex above the antennal lobes. Like the F5 neurons, they have large arborizations in the dorsal unstructured neuropil. The line NP2320 expresses the driver in columnar neurons running perpendicular to the strata of F neurons, with their cell bodies scattered singly or in small groups between the calyces. Since they seem to have no arborizations outside the FB, they are tentatively classified as pontine neurons (Liu, 2006).

    Initially, it was shown that pattern memory requires the rut gene for each of the three parameters. Next, the Rut rescue flies were studied (for example, rut2080/Y;c205/UAS-rut+). In the line c205, memory was restored only for 'elevation', not for 'size' or 'contour orientation'. Correspondingly, the memory impairment by expression of dominant-negative Galphas* in this driver line should be specific for 'elevation', as is indeed the case. With the driver line NP6510, memory was not restored for either 'elevation' or for 'size,' but memory was restored for 'contour orientation'. The third driver line, NP2320, labelling columnar neurons of the FB, did not restore the memory for any of the three pattern parameters. Among the 27 GAL4 lines, a second was found with a very similar expression pattern as NP6510 (NP6561). The P-element insertions in the two lines are only 124 nucleotides apart from each other. Like NP6510, NP6561 restores the memory for 'contour orientation' but not for 'size' or 'elevation'. These results strongly suggest that memory traces for distinct visual pattern parameters are located in different parts of the FB, and that, in addition to the memory trace in F5 neurons, a memory trace for the parameter 'contour orientation' is located in F1 neurons (Liu, 2006).

    A pertinent question in rescue experiments is whether the rescue is due to the provision of an acute function in the adult or to the avoidance of a developmental defect. Therefore, the tub-GAL80ts transposon was added to the system. The driver lines c205 and NP6510 were chosen. Groups of adult males (for example, rut2080/Y;+/tub-GAL80ts;NP6510/UAS-rut+), raised at 19°C, were kept as adults for 14 h at 19°C or 30°C. Afterwards, pattern memory for the corresponding pattern parameter was tested. In both cases, flies that had been kept at 30°C showed normal memory, indicating that Rut cyclase induced just a few hours before the experiment had restored an immediate neuronal function rather than preventing a developmental defect. This conclusion was further supported by the finding that Galphas* expression in the adult (using tub-GAL80ts) was sufficient to disrupt memory (Liu, 2006).

    Several conclusions can be drawn from the above results. Memory traces in Drosophila are associated with specific neuronal structures: odor memories with the MBs, visual memories with the CX, and place memory (tentatively) with the median bundle. Memory traces are not stored in a common all-purpose memory centre. Even within the visual domain, memories for distinct pattern parameters are localized within distinct structures: a rut-dependent short-term memory trace for the pattern parameter 'elevation' to F5 neurons, and a corresponding memory trace for 'contour orientation' to F1 neurons. Moreover, if the constitutively activating Galphas* protein indeed interferes with the regulation of Rut cyclase, it follows that the brain contains no other redundant rut-dependent memory traces for these pattern parameters. The Rut-mediated plasticity is necessary and sufficient, at least in F5 neurons. As in the earlier examples, the memory traces are confined to relatively small numbers of neurons. At least in flies, and probably in insects in general, memory traces appear to be part of the circuitry serving the respective behaviour (Liu, 2006).

    This study provides a first glimpse of the circuitry within a neural system for visual pattern recognition. Though the picture is far from complete, it invites (and may guide) speculation. The FB is a fiber matrix of layers, sectors and shells. The F1- and F5-neurons form two sharp parallel horizontal strata in this matrix. If the width of the FB represents the azimuth of visual space as has been proposed, the horizontal strata of the F neurons would be well suited to mediate translation invariance. In any case, it is satisfying to find a translation invariant memory trace in the CX where visual information from both brain hemispheres converges. These first components of the circuitry may encourage modelling efforts for pattern recognition in small visual systems (Liu, 2006).

    Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation

    Memories are formed, stabilized in a time-dependent manner, and stored in neural networks. In Drosophila, retrieval of punitive and rewarded odor memories depends on output from mushroom body (MB) neurons, consistent with the idea that both types of memory are represented there. Dorsal Paired Medial (DPM) neurons innervate the mushroom bodies, and DPM neuron output is required for the stability of punished odor memory. Stable reward-odor memory is also DPM neuron dependent. DPM neuron expression of amnesiac (amn) in amn mutant flies restores wild-type memory. In addition, disrupting DPM neurotransmission between training and testing abolishes reward-odor memory, just as it does with punished memory. DPM-MB connectivity was examined by overexpressing a DScam variant that reduces DPM neuron projections to the MB α, β, and γ lobes. DPM neurons that primarily project to MB α′ and β′ lobes are capable of stabilizing punitive- and reward-odor memory, implying that both forms of memory have similar circuit requirements. Therefore, these results suggest that the fly employs the local DPM-MB circuit to stabilize punitive- and reward-odor memories and that stable aspects of both forms of memory may reside in mushroom body α′ and β′ lobe neurons (Keene, 2006).

    It is widely believed that memory is encoded as changes in synaptic efficacy between neurons in a network. This concept of synaptic plasticity predicts that it will be possible to localize memory to discrete synapses in neural networks in the brain. The relatively small brains of insects are well suited to this endeavor, and genetic manipulation in the fruit fly Drosophila has greatly aided neural circuit mapping of odor memory. Flies can be taught to associate an odor conditioned stimulus (CS) with either a punitive electric shock or a rewarding sugar unconditioned stimulus (US). Strikingly, learning and memory with these opposing unconditioned stimuli requires differential transmitter involvement: sugar-rewarded odor memory is dependent on intact octopamine signaling (see Tyramine β hydroxylase), while shock-punished (punitive) odor memory is dependent on dopamine signaling. However, despite the differential requirement for these monoamine transmitters, blocking MB output during retrieval impairs both punitive- and reward-odor memories, implying that these memories rely on overlapping brain regions. Stability of reward-odor memory is reliant on the same MB extrinsic neurons that are required for stability of punitive-odor memory (Keene, 2006).

    amnesiac mutant flies can associate odors with a punitive or a rewarding US, but they quickly forget this information, which suggests that amn might be generally involved in memory. The amn gene is expressed throughout the brain and strongly in Dorsal Paired Medial neurons—two large modulatory neurons that appear to ramify throughout the approximately 5000 neurons of the MBs. Prolonged DPM neuron output is required for the stability of punitive-odor memories. Since DPM neurons heavily ramify in the MBs, these data support the importance of the MB as a crucial locus for memory and also suggested that the neural network involving MB and DPM neurons could be critical for all MB-dependent memory. Therefore whether the circuitry involving DPM neurons was involved in the stability of rewarded olfactory memory was tested (Keene, 2006).

    It was first confirmed that amn mutant flies have a memory defect when conditioned with odors and sugar reward. A modified protocol was used that more closely resembles the odor-shock conditioning protocol and that produces robust memory that lasts for more than 6 hr. In brief, approximately 100 starved flies were exposed to an odor for 2 min in the absence of sugar, followed by a clean air stream for 30 s and a second odor with sugar reward for 2 min. Olfactory memory was tested 3, 60, 180, and 360 min after training. Flies homozygous for the strong amn alleles—amn1 or amnX8—learn to associate the appropriate odor with sugar reward (they have a small but significant initial performance defect), but they forget this association within 60 min of training. These data are consistent with the earlier report that amn1 flies have defective reward-odor memory (Keene, 2006).

    Since amn mutant flies forget quickly when trained with either a punitive or a rewarding US, it was of interest to see whether similar neural circuitry was involved in both types of memory. Although the amn gene is expressed throughout the brain, expressing the amn gene in DPM neurons restores punitive odor memory performance to amn mutant flies. Therefore whether restoring amn expression in DPM neurons of amn mutant flies would rescue the reward-odor memory defect was tested. The c316 {GAL4} line was used to transgenically express the amn gene in DPM neurons of amn mutant flies. 3 hr memory of amnX8/amn1;c316/uas-amn and amn1;c316/uas-amn flies was similar to wild-type flies and was statistically different from the memory of amnX8 and amn1; uas-amn mutant flies. These data demonstrate that amn expression in DPM neurons is sufficient to restore reward-odor memory to amn mutant flies and suggest that DPM neurons are generally critical for olfactory memories (Keene, 2006).

    Next an acute role of DPM neurons in reward-odor memory was directly tested by temporally blocking their output during the course of the experiment. The dominant temperature-sensitive shibirets1 transgene was tested in DPM neurons by using the c316{GAL4} and Mz717{GAL4} drivers and a sugar reward conditioning experiment was performed at either the permissive (25°C) or the restrictive (31°C) temperature. At the restrictive temperature, shibirets1 blocks vesicle recycling and thereby blocks synaptic vesicle release. At 25°C, reward-odor memory of c316; uas-shits1 and Mz717; uas-shits1 flies was comparable to memory of wild-type and uas-shits1 flies. However, at 31°C, memory of c316; uas-shits1 and Mz717; uas-shits1 flies was statistically different from wild-type and uas-shits1 flies. Therefore, DPM synaptic release is necessary for stable reward-odor memory as it is with punitive-odor memory (Keene, 2006).

    Stable punitive-odor memory requires prolonged DPM output between acquisition and retrieval, and DPM output is dispensable during training and testing. Therefore whether DPM neurons were similarly required for reward-odor memory was tested. Again DPM output was blocked by expressing uas-shits1 with c316{GAL4}, but this time the inactivation was restricted to either the training, testing, or storage period. Blocking DPM neurons during acquisition did not produce memory loss; memory of c316; uas-shits1 flies was comparable to wild-type and uas-shits1 flies. Similarly, DPM neuron output was not required during memory retrieval; memory of c316; uas-shits1 flies was comparable to wild-type and uas-shits1 flies. However, blocking DPM output for 30 min after training significantly reduced reward-odor memory; memory of c316; uas-shits1 flies is statistically different from wild-type and uas-shits1 flies. These data parallel results with punitive-odor memory and suggest that there is a similar requirement for DPM neuron output to stabilize both punitive- and reward-odor memory. DPM block from 30 to 60 min after training decreased punitive-odor memory similar to a 0–30 min block. However, disrupting DPM neuron output from 30 to 60 min had an insignificant effect on reward-odor memory. These data imply that the role of DPM neurons is diminished at 30–60 min for reward-odor memory. The Mz717 driver was used to increase the confidence that the temporal uas-shits1 disruptive effect can be ascribed to blocking DPM neurons. Blocking DPM output for 60 min after training with Mz717 significantly reduced reward-odor memory. Memory of Mz717; uas-shits1 flies is statistically different from wild-type and uas-shits1 flies (Keene, 2006).

    DPM neurons innervate all the lobes of the MBs, and previous imaging studies suggest that the DPM projections there may be both transmissive and receptive. In addition, expression of n-syb::GFP in DPM neurons has been reported to label DPM projections to the MB lobes. In an attempt to gain further insight into DPM neuron organization, a collection of pre- and postsynaptic compartment markers was overexpressed in DPM neurons. However, no clear evidence was seen for asymmetry within DPM neurons or between projections to individual MB lobes. Therefore, understanding DPM polarity and organization will require further work (Keene, 2006).

    During this analysis, it was found that expression of the DScam17-2::GFP fusion protein (which has been described to label the presynaptic compartment when overexpressed in certain neurons, in DPM neurons, with c316{GAL4}, affected DPM neuron development and resulted in DPM neurons that predominantly project to the MB α′ and β′ lobe subsets. Coexpressing uas-DScam17-2::GFP and uas-CD2 or uas-lacZ in DPM neurons reveals that DScam17-2::GFP labels the remaining projections rather than a subset of existing projections. To identify projections to MB α/β neurons versus α′/β′ neurons, brains were costained with anti-FASII, which labels α/β and γ neurons , and anti-TRIO, which labels α′/β′ and γ neurons (Keene, 2006).

    A functional role for the MB α′ and β′ lobes in memory has not been reported. Therefore, uas-DScam17-2::GFP; c316 flies were used to assess the role of DPM neuron projections to the MB α′ and β′ lobe subset in punitive- and reward-odor memory. Heterozygous uas-DScam17-2::GFP flies were included as a control as well as wild-type and amnX8 flies for comparison. The presence of the uas-DScam17-2::GFP transgene did not significantly affect punitive-odor memory. Remarkably, DPM neurons that primarily project to the α′ and β′ lobe subsets retain punitive-memory function. Memory of uas-DScam17-2::GFP; c316 flies was similar to memory of uas-DScam17-2::GFP flies and was significantly greater than that of amnX8 flies. Therefore, DPM neuron projections to the α′ and β′ lobes of the MB are apparently sufficient for punitive-odor memory. Next the function of uas-DScam17-2::GFP; c316 flies in reward-odor memory was tested. Again, memory of uas-DScam17-2::GFP; c316 flies was similar to memory of uas-DScam17-2::GFP flies and was significantly greater than that of amnX8 flies. These data indicate that the DPM neuron projections to the α′ and β′ MB lobe subsets are also apparently sufficient for reward-odor memory and imply that the circuit requirements for the stability of rewarded and punished odor memory are very similar. Although redundancy of DPM projections or retention of a few critical projections to the α, β, and γ lobes cannot currently be ruled out, these data are consistent with the notion that DPM projections to the α′ and β′ MB lobes are sufficient for stabilizing memory. The data also suggest that DScam may play a role in wiring the DPM-MB circuit (Keene, 2006).

    In Drosophila, there is a striking dissociation of monoamine transmitters for reward and punishment. Dopamine is required for aversive-odor memory formation, whereas octopamine is necessary for appetitive-odor memory. Octopaminergic and dopaminergic neurons project throughout the brain including to the MBs. Although it is not known whether the MB arborization of these monoaminergic neurons is required for odor memories, blocking MB output is required to retrieve both aversive- and appetitive-odor memory (Keene, 2006).

    DPM neurons ramify throughout the MB lobes and provide a general stabilizing mechanism for both punitive- and reward-odor memory. This DPM neuron analysis enhances resolution of memory processing and provides further weight to the idea that components of both punitive- and reward-odor memory reside at synapses within MB neurons. Imaging studies suggest that DPM neurons are both receptive and transmissive to MB neurons, and a model is favored where DPM neurons represent recurrent feedback neurons that consolidate conditioned changes in synaptic weight in MB neurons. However, if MB neurons provide drive to DPM neurons, one would expect MB neuron output and DPM neuron output to have similar temporal requirements. Current published data conclude that MB neuron output is not required during memory storage (but is exclusively required for retrieval) when DPM neuron output is required. However, the work described here suggests a role for MB α′ and β′ lobe neurons in memory stability, and it is noteworthy that MB studies did not employ GAL4 drivers with extensive expression in MB α′/β′ neurons. Further detailed analysis of the role of MB α′/β′ neurons in memory should resolve this conundrum (Keene, 2006).

    Slow oscillations in two pairs of dopaminergic neurons gate long-term memory formation in Drosophila

    A fundamental duty of any efficient memory system is to prevent long-lasting storage of poorly relevant information. However, little is known about dedicated mechanisms that appropriately trigger production of long-term memory (LTM). This study examined the role of Drosophila dopaminergic neurons in the control of LTM formation, and they were found to act as a switch between two exclusive consolidation pathways leading to LTM or anesthesia-resistant memory (ARM). Blockade, after aversive olfactory conditioning, of three pairs of dopaminergic neurons projecting on mushroom bodies, the olfactory memory center, enhanced ARM, whereas their overactivation conversely impaired ARM. Notably, blockade of these neurons during the intertrial intervals of a spaced training precluded LTM formation. Two pairs of these dopaminergic neurons displayed sustained calcium oscillations in naive flies. Oscillations were weakened by ARM-inducing massed training and were enhanced during LTM formation. These results indicate that oscillations of two pairs of dopaminergic neurons control ARM levels and gate LTM (Plaçais, 2012).

    In terms of neural circuitry, the inputs of MV1 and MP1 neurons are unknown, raising the question of whether they are isolated self-oscillators or are part of a larger oscillating circuit. The fact that MV1 and MP1 oscillations are in phase, both in the same hemisphere and across hemispheres, favors the second hypothesis (Plaçais, 2012).

    The molecular pathways by which dopaminergic neurons activity could regulate ARM remain elusive. ARM regulation by dopaminergic neurons was found not to rely on the products of the rsh gene. Activation of protein kinase A has been shown to inhibit ARM in the mushroom body. Dopamine release from the MV1 and MP1 neurons could trigger cAMP production and increased protein kinase A activity in the mushroom body, thereby inhibiting ARM (Plaçais, 2012).

    In the framework of memory phases, it has been proposed that ARM and LTM are exclusive consolidated memories, a model that has been debated. The results support the idea that ARM is inhibited after spaced conditioning, when LTM is formed. Why is ARM inhibited when LTM is formed? When the three pairs of ARM-inhibiting dopaminergic neurons were blocked between the multiple cycles of a spaced training, LTM formation was voided. Consistent with these results, in vivo calcium imaging showed that a single cycle and, more strongly, a spaced training fostered MV1 and MP1 oscillatory activity, whereas a massed training inhibited MV1 and MP1 oscillations (Plaçais, 2012).

    These results consistently point to a plausible model of consolidated memory phases in Drosophila, in which oscillations of dopaminergic neurons gate the formation of LTM by tuning the ARM pathway. In this model, two parallel mutually inhibiting pathways can lead to the formation of day-lasting ARM or LTM. After a single cycle or massed conditioning, the ARM pathway is activated and prevents LTM formation. During the rest intervals of the spaced conditioning MV1 and MP1 oscillations are enhanced and the ARM pathway is therefore inhibited and LTM can form in relevant mushroom body neurons (Plaçais, 2012).

    Although this study has identified ARM-regulating neurons, the mechanisms by which ARM, or physiological events leading to it, prevents LTM formation remain to be elucidated. The spacing effect, that is, the fact that stronger memory is formed when multiple trainings are spaced over time compared with the same number of trainings without spacing, is widely established in the animal kingdom. Notably, the gating of LTM formation occurred during the intertrial intervals (ITIs) of spaced training in these experiments. Recently, it has been shown that the duration of the ITI required to form LTM in Drosophila is regulated by the corkscrew gene through waves of Ras/mitogen-activated protein kinase activity. It will be interesting to investigate a putative interaction between mitogen-activated protein kinase waves and MV1 and MP1 oscillatory activity, and to determine whether stimulating oscillations during the ITI facilitates LTM formation, for example, with shorter ITIs or with fewer conditioning cycles (Plaçais, 2012).

    This study identified dopaminergic neurons whose activity inhibits ARM and therefore, as in mammals, positively affects LTM formation. That regulation of memory consolidation seems to involve precisely cadenced oscillations in MV1 and MP1 neurons is of particular conceptual interest. Indeed, it was recently suggested that a subset of hypothalamic dopaminergic neurons in rats, robustly oscillating at 0.05 Hz, may be responsible for lactation inhibition. Thus, inhibition through rhythmic oscillations appears to be a widespread functional feature of dopaminergic networks. In addition, a slow oscillatory firing mode, in the 0.5-1.5-Hz frequency range, has been identified in the dopaminergic neurons in the ventral tegmental area of rats. This oscillatory firing pattern would underlie subsecond synchronization between ventral tegmental area and prefrontal cortex, an area of major importance in learning and memory. Thus, and although such an assumption remains quite speculative, LTM regulation by dopaminergic neurons in mammals might involve mechanisms similar to those described in this study in Drosophila (Plaçais, 2012).

    Analysis of a spatial orientation memory in Drosophila

    Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Fruit flies possess a similar spatial memory during locomotion. Using a new detour setup, flies are shown to be able to remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, it was found that the GABAergic (stainable with antibodies against GABA) ring neurons (Hanesch, 1998) of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. The protein kinase S6KII (ignorant; Putz, 2004) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies (Neuser, 2008).

    Previous studies have shown that walking flies heading for an object maintain their direction even when the target disappears. This persistence of orientation can last for several seconds, indicating that flies store the position of, or the path towards, the hidden object for further targeting. It is therefore proposed that flies form a spatial memory for objects that is similar to the working memory in vertebrates. To investigate this putative memory in Drosophila a detour paradigm for walking flies was established. Single flies were put into a cylindrical virtual-reality arena, in which two dark vertical stripes were presented at opposite sides. Normally, flies patrol between the two visual objects for a considerable length of time. In the new paradigm, the stripes disappeared when the fly crossed the invisible midline of the circular walking platform, and a new target appeared laterally at a 90° angle to the fly. In most cases wild-type flies turned towards this new target if it was presented for more than 500 ms. After the fly had oriented itself towards the new object (deviation of the fly's longitudinal body axis from the ideal course to the stripe below +/-15°), this target also disappeared within 1 s and no objects were visible to the fly. It was then determined whether the fly turned back to continue its approach to its initial, but still invisible, target. The walking traces reveal a direct course towards the former location of the first target. The flies therefore retained positional information on the former object, although it was no longer present in the environment (Neuser, 2008).

    Wild-type (Canton-S) flies recall the old target and integrate it into a guided behaviour with a median frequency of 80% as measured in ten consecutive trials for each fly. Longer presentation of the distracter stripe did not significantly change the percentage of positive choices. These data strongly suggest that flies stored the relative position of the first target in a spatial orientation memory for at least 4 s. To exclude the possibility that flies used chemical traces of former runs for their orientation the absolute positions of the stripes were randomly changed after each trial. As a result of this randomization, flies had to update their memory continuously. Moreover, no training effect could be observed, because the frequency of positive turns did not change during the ten consecutive trials. Similar performances were observed when two opposing distracters were presented to the fly. This orientation memory for vanished objects is considered to be to be idiothetic. Because no visible landmarks were presented to the fly after the distracter disappeared, the fly could not use a stored reference picture of the environment for its guidance. It is therefore suggested that the fly uses online stored information of its own angle towards the former target, a strategy known as path integration. Path integration has been shown to be used by other insects, such as ants and bees, to navigate through a familiar landscape (Neuser, 2008).

    In an attempt to localize this type of memory to discrete parts of the insect brain, several mutant lines with structural central-complex defects of Drosophila were analysed. The central complex is composed of four different neuropils and has been implicated in supervising motor output during locomotion. First tests showed that the persistence of orientation towards a removed target is reduced or lost whenever the ellipsoid body of the central complex was defective. Therefore the ellipsoid body open mutant (eboKS263) was tested in the detour paradigm; these flies did not show a preference for the first target after the detour, suggesting that an intact ellipsoid body is required for establishing a spatial orientation memory. In contrast, the use of hydroxyurea to ablate the mushroom bodies, which are important in olfactory memory, did not disturb the orientation memory (Neuser, 2008).

    One prominent type of neuronal cells of the ellipsoid body is the group of GABAergic ring neurons. The fibres of these neurons run in a prominent tract, the RF tract (ring-neuron and tangential fan-shaped-body neuron tract), and form bushy thin endings in the ipsilateral lateral triangle and bleb-like endings in the ellipsoid body. Four different kinds of ring neuron (R1-R4) can be distinguished by their arborization pattern around the ellipsoid body canal. R1-R3 neurons project outwards from the ellipsoid body canal, whereas the arborization of R1 is restricted to the inner zone, that of R2 to the outer zone, and that of R3 to both zones. R4 neurons project from the periphery inwards and arborize in the outermost zone. It was next proposed that the ring neurons might be necessary for the orientation memory. The GAL4/UAS system was used to silence distinct subsets of ring neurons through the expression of tetanus toxin (TNT) by using the GAL4 driver lines c232, c481 and c105. For temporal control, TNT was induced conditionally by using the temperature-sensitive GAL4 repressor GAL80ts under the control of the ubiquitous Tubulin promoter (Tub-GAL80ts). Experimental and control flies were raised at 18°C, tested within the detour paradigm, and retested after the induction of TNT. Pairwise comparison revealed that the preference for the original target was lost whenever the toxin was expressed in ring neurons of the ellipsoid body. This finding confirms the hypothesis that the ellipsoid-body ring neurons are necessary components of the orientation memory (Neuser, 2008).

    To investigate which molecular pathways are involved in this kind of memory, focus was placed on the cyclic-AMP signalling pathway. Variable levels of cAMP have been shown to have a crucial function in memory formation during associative learning in Drosophila. cAMP levels are modulated by the opposing actions of adenylyl cyclases and cAMP phosphodiesterases. Mutants for the adenylyl cyclase gene rutabaga (rut1 and rut2080) were unable to target visual objects and could not be tested in the paradigm. Therefore mutants of the dunce gene (dnc), which encodes a cAMP phosphodiesterase, were tested in the detour paradigm. The dnc1 mutant is a hypomorph and displays about half of the enzyme activity in the wild type. dnc1 mutant flies show deficits in several paradigms of associative classical learning and operant conditioning. In contrast, dnc1 mutants showed no defects in the detour paradigm, indicating that a tight modulation of cAMP levels might not be critically required for spatial orientation memory (Neuser, 2008).

    Another molecule involved in memory formation in Drosophila is a member of the ribosomal serine kinase family. ignorant (ign) encodes the S6 kinase II (S6KII), which interacts with mitogen-activated protein (MAP) kinase signalling in Drosophila. S6KII does not seem to be involved in cAMP signalling pathways. The null allele ign58/1 has been shown to be defective in classical aversive conditioning and operant learning (Putz, 2004). Therefore ign58/1 flies were tested in the detour paradigm. Although the mutants readily targeted visible objects, they showed no directional preference for the position of the original target after it disappeared, suggesting that they had lost their memory. In contrast, walking speed, walking activity and orientation towards visual objects were similar to those of the wild type. Next whether ign is required in the ring neurons targeted by c232-GAL4 was tested with the use of a UAS-ign RNA-mediated interference (RNAi) effector line. RNAi silencing in these ring neurons decreased the performance by half. This decrease in memory constitutes only a partial phenocopy of the null mutant, because the performance was not significantly different from that of the wild type or ign58/1. Nevertheless, this result is interpreted to suggest that ign is required in the ring neurons for spatial orientation memory (Neuser, 2008).

    To address the question of whether restoring S6KII levels is sufficient for regaining memory, neuron-specific rescue experiments were performed in the ign58/1 mutant background. S6KII was expressed pan-neuronally with Appl-GAL4 and elav-GAL4, and also specifically in the R3 and R4 ring neurons with c232-GAL4. In all three cases a complete rescue was observed. Next, whether ign function in the R3 and R4 ring neurons is acutely required for orientation memory was examined. Therefore, again use was made of the GAL80ts transgene to rescue the ign phenotype only in the adult. Conditional expression of S6KII only in the R3 and R4 ring neurons resulted in a perfect rescue of the ign mutant. This result -- that acute S6KII expression in the R3 and R4 ring neurons accomplished a complete rescue -- reveals that this very narrow subset of cells is sufficient for regaining a functional orientation memory. It has been reported that Drosophila S6KII negatively regulates extracellular signal-regulated kinases (ERKs) by acting as a cytoplasmic anchor of the MAP kinase. Further studies will determine whether the MAP kinase signalling pathway is required for this kind of memory task (Neuser, 2008).

    It is concluded that the relevant ring neurons use the inhibitory neurotransmitter GABA. Their circuitry and interconnections within the ellipsoid body are not yet known. Expression of the dDA1 dopamine receptor in the ellipsoid body has recently been shown. It is therefore possible that the same neurotransmitter systems as those used for visual-spatial memory in the monkey prefrontal cortex are used to establish orientation memory in the central complex of flies (Neuser, 2008).

    Serum response factor-mediated gene regulation in a Drosophila visual working memory

    Navigation through the environment requires a working memory for the chosen target and path integration facilitating an approach when the target becomes temporarily hidden. Previous studies have shown that this visual orientation memory resides in the ellipsoid body, which is part of the central complex in the Drosophila brain (see Neuronal architecture of the central complex in Drosophila melanogaster in Niven's Visuomotor control: Drosophila bridges the gap). Former analysis of foraging and ignorant (ign) mutants have revealed that a hierarchical PKG and RSKII kinase signaling cascade in a subset of the ellipsoid-body ring neurons is required for this type of working memory in flies. This study shows that mutants in the ellipsoid body open (ebo) gene, which encodes the actin-binding protein Exportin 6, exhibit excessive nuclear accumulation of actin during development and in the adult brain. ebo mutants lack the orientation memory independent of the structural defect in the ellipsoid-body neuropil, and EBO activity in any type of adult ring neurons is sufficient for orientation-memory function. Moreover, genetic interaction studies revealed that nuclear actin accumulation in ebo mutants inhibits the Drosophila coactivator myocardin-related transcription factor A (dMRTF) and therefore the transcriptional activator serum response factor (dSRF). dSRF also functions in different ring neurons, suggesting that it regulates abundance of a diffusible factor that enables a working memory in ellipsoid-body ring neurons. To date, SRF has only been implicated in longer forms of memory formation like synaptic long-term potentiation and depression. This study provides the first evidence that SRF-mediated gene regulation is also required for a working memory that lasts only for a few seconds (Thran, 2013).

    The central complex (CX) of the adult fly brain consists of four compartments that interconnect the protocerebral hemispheres: the protocerebral bridge, the fan-shaped body, the ellipsoid body (EB), and the ventrally located paired noduli. The analysis of several Drosophila mutant strains with structural defects in one or more neuropils of the CX has suggested that the CX represents a higher control center for locomotion and orientation behavior. These mutants walk slower than wild-type flies, have a delayed reaction to changing stimuli during flight, and show deficits in the orientation behavior toward landmarks. A major sensory input region for external stimuli, especially visual information, appears to be the ellipsoid body and the fan-shaped body, and in larger insects this includes information on the orientation of polarized light, which is used for navigation. In Drosophila, it has been shown that the protocerebral bridge is required for step-length control in walking flies and visual targeting during climbing events. Visual input is also processed in the fan-shaped body, which mediates visual pattern recognition to memorize different objects during flight control], a function that has also been attributed to the EB. The EB also holds a memory trace for the position of landmarks, as revealed in a Morris water maze-like paradigm for visual place learning in flies. In addition, the analysis of the ellipsoid body open (ebo) mutant has shown that the EB is necessary to establish a visual orientation memory for a vanishing object in walking Drosophila (Thran, 2013).

    In this so-called detour paradigm, a fly approaching an attractive target is lured out of the way by a dark stripe on one side while simultaneously the original object vanishes. After the distracting stripe has also been removed, the fly is left without any visual cue. However, in over 80% of the cases, wild-type flies use idiothetic information on their past movements with respect to the original target to resume their originally intended approach. This type of working memory lasts about 4 s and must be updated during every turn the fly takes (Neuser, 2008). Analysis of two Drosophila memory mutants in ignorant (ign) and foraging (for), which encode the ribosomal-S6 kinase II (RSKII) and cGMP-dependent protein kinase (PKG), respectively, revealed that both kinases share one signaling pathway that is required in a specific type of EB ring neurons to display an orientation memory (Kuntz, 2012; Thran, 2013).

    To further elucidate the structural and biochemical components that enable the EB to hold an orientation memory, the ebo mutants were genetically and molecularly analyzed. This study reports that the ebo gene encodes the actin-binding protein Exportin 6, which is required for the export of globular actin (G-actin) from the nucleus, thus preventing actin-filament formation there. This analysis of the ebo mutant confirms these findings because elevated levels of an Actin-GFP fusion protein can be found in nuclei of the mutant. Interestingly, excessive G-actin has been shown to inhibit the myocardin- related transcription factor A in vertebrates (MRTF-A), a coactivator of the transcriptional regulator serum response factor (SRF). This genetic interaction analysis of ebo with the Drosophila ortholog of MRTF, as well as rescue experiments of the dSRF mutant blistered (bs), revealed that elevated levels of nuclear actin in EB neurons and the subsequent malfunction of the dMRTF/dSRF transcription regulator complex prevent the visual orientation memory in flies (Thran, 2013).

    In vertebrates, more than 200 putative target genes of SRF have been postulated, most of them involved in cytoskeleton dynamics and cell motility, but immediate early genes like c-fos have also been identified. Unfortunately, downstream targets of Drosophila dSRF in neuronal cells are yet unknown, complicating the identification of further gene products that are involved in development of the EB and a functional visual orientation memory in adult flies. Based on the current results, it is hypothesized that the effect of EBO on the transcriptional activity of the dMRTF/dSRF complex is responsible for both the developmental and behavioral phenotype (Thran, 2013).

    Although transheterozygous mutants for hypomorphic bs alleles displayed no structural EB defect and the morphological defects of homozygous MrtfD7 mutants are less prominent than those of ebo mutants, it nevertheless is possible that dMRTF/dSRF-mediated gene regulation is also required in EB development. For instance, in vertebrates, transcription of profilin, an F-actin promoting factor, is activated by SRF, and the structural ebo phenotype could indicate that dSFR is also promoting transcription of the profilin encoding gene chickadee in Drosophila. Alternatively, nuclear actin in the ebo mutant could sequester profilin in the nucleus, thus reducing levels at the growth cone required in axonal outgrowth (Thran, 2013).

    However, it is obvious that a memory that lasts only a few seconds cannot depend on changes in transcriptional regulation. The rescue experiments described in this study have established that EBO and dSRF can restore memory function of the respective mutant independent of the specific ring-neuron subtype. This is surprising, considering the biochemical activity of EBO and dSRF, which are definitively cell autonomous. Therefore, it is proposed that dSRF promotes gene expression that ultimately results in the production of a diffusible factor that has to be delivered to the ring neurons. Presumably, this factor feeds into pathways that enable rapid changes in synaptic transmission of the ring neurons necessary to encode an orientation memory. For instance, ring neurons might need a higher density of synaptic release sites, a highly efficient synaptic vesicle reserve pool, or elevated expression of ion channels for prolonged excitation. Similarly, a high density of dendritic neurotransmitter receptors and elevated levels of second messenger molecules at the postsynaptic site of the ring neurons could be necessary to exert their specific function in working memory formation and/or retrieval. Finding the dSRF target genes that mediate the orientation memory in flies might lead to new insights how working memories are orchestrated in general (Thran, 2013).

    Sequential use of mushroom body neuron subsets during Drosophila odor memory processing

    Drosophila mushroom bodies (MB) are bilaterally symmetric multilobed brain structures required for olfactory memory. Previous studies suggested that neurotransmission from MB neurons is required only for memory retrieval. An unexpected observation that Dorsal Paired Medial (DPM) neurons, which project only to MB neurons, are required during memory storage but not during acquisition or retrieval, led a revisiting og the role of MB neurons in memory processing. This study shows that neurotransmission from the α'β' subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory, but is dispensable during memory retrieval. In contrast, neurotransmission from MB αβ neurons is required only for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in an MB α'β' neuron-DPM neuron loop is required to stabilize memories formed in the MB αβ neurons (Krashes, 2007).

    It is often said that form follows function. While this postulate would argue the striking multi-lobed arrangement of the insect mushroom bodies implies functional differences between the different types of MB neurons: αβ, α'β' and γ, very limited data exists describing the individual function of these anatomical subdivisions. Although several complex behaviors in insects appear to require the MBs and a differential role for distinct MB neuron groups has been suggested, most conceptual models of memory treat the MBs as a single unit (Krashes, 2007).

    One of the most detailed examinations of MB function has been in the context of Drosophila aversive olfactory memory, where flies are trained to associate specific odors with the negative reinforcement of electric shock. Genetic studies over the last thirty years have suggested that the MBs play an essential role in fly olfactory memory, but the role of the MBs in memory acquisition, storage, and retrieval has been examined only recently. Taking advantage of a dominant, temperature-sensitive dynamin transgene, uas-shits1, a number of laboratories concluded that MB output was required only for recall, but not acquisition or storage. These and other findings have led to a simple model whereinDrosophila olfactory memory is formed and stored at MB output synapses (Krashes, 2007).

    Functional studies of DPM neurons, MB extrinsic neurons that ramify throughout the MB lobes, demonstrated they were specifically required during consolidation, but not acquisition or storage. Furthermore, genetically-modified DPM neurons that primarily innervate the MB α'β' lobes retain function implying that MB α'β' neurons might also have a similar function in memory consolidation (Keene, 2006; Krashes, 2007 and references therein).

    Examination of the GAL4 enhancer trap lines used to express the uas-shits1 transgene in the earlier MB studies revealed that c309, c747, and MB247 only express in a few MB α'β' neurons compared to αβ and γ neurons, while c739 expresses exclusively in αβ neurons. Thus it seems likely that prior studies utilizing these drivers did not observe requirements for MB activity during olfactory memory acquisition and storage because of insufficient expression in α'β' neurons (Krashes, 2007).

    Subsequently two GAL4 enhancer traps that strongly express in MB α'β' neurons were identified in order to test this hypothesis. The expression of c305a appears to be entirely restricted to α'β' neurons within the MBs whereas c320 expresses in α'β', αβ, and a few γ neurons. Both of these lines also express in additional non-MB neurons so a MB{GAL80} tool was used to more rigorously test the requirement for MB activity, when the neurons labeled with either {GAL4} line were manipulated. With these new reagents the role of MB α'β' neurons in memory was investigated and it was found that MB α'β' neuron output during and after training is critical to form, and consolidate, both appetitive and aversive odor memory from a labile to a more stable state. For comparison the requirements for MB αβ neurons were also examined using c739, confirming the results of McGuire (2001). Thus, output from the MB α'β' neuron subset is required for memory acquisition and stabilization whereas, as previously described (McGuire, 2001), output from αβ neurons is apparently dispensable during training and consolidation but is required for memory retrieval (Krashes, 2007).

    Based on c305 and c739 data, it was recognized that c320 flies, which express in both α'β' and αβ neurons, might be expected to exhibit memory loss if MB neuron output was blocked during both the consolidation and recall time windows. However, it is possible that a retrieval effect was not observed with c320 because it expresses GAL4 in fewer αβ neurons, or is in a different subset of αβ neurons, relative to the c739 driver (Krashes, 2007).

    Despite this caveat, it is believed that the data suggest that different lobes of the MB have different roles in memory and therefore provide a significant shift in understanding of the role of the MB in memory. Older models implied that MB αβ, α'β' and γ neurons are largely interchangeable and that each of the MB neurons that respond to a particular odor receive coincident CS and US input and modify their presynaptic terminals to encode the memory. The present data suggest that MB αβ and α'β' neurons are functionally distinct (Krashes, 2007).

    This study did not investigate the role of the unbranched γ lobe neurons. Previous work with c309, c747 and MB247 suggests that neurotransmission from γ neurons is likely dispensable for acquisition and consolidation. In addition, another study indicated that γ neurons are minimally involved in MTM and ARM. However, it is possible that experiments to date have not employed odors that require γ neuron activation. The response of γ neurons may be tailored to ethologically relevant odors such as pheromones. It is notable that fruitless, a transcription factor required for male courtship behavior, is expressed in MB γ neurons and blocking expression of the male-specific fruM transcript in the MB γ neurons impairs courtship conditioning. If the relevant odors can be identified, it will be interesting to determine if MB α'β' neurons and DPM neurons are required to stabilize these odor memories in the γ neurons. Recent work by Akalal (2006) is supportive of the idea that odor identity is an important factor in determining the requirement for the function of distinct subsets of MB neurons in olfactory learning (Krashes, 2007).

    Stable aversive and appetitive odor memory requires prolonged DPM neuron output during the first hour after training and DPM neuron output is dispensable during training and retrieval. DPM neurons ramify throughout the MB lobes but DPM neurons that have been engineered to project mostly to the MB α'β' lobes retain wild-type capacity to consolidate both aversive and appetitive odor memory (Keene, 2006). This study has demonstrated that similar to wild-type DPM neurons, blocking output from these modified DPM neurons for one hour after training abolishes memory. Thus finding a specific role for both DPM neuron output to MB α'β' lobes and MB α'β' neuron output during the first hour after training is consistent with the notion that a direct DPM-MB α'β' neuron synaptic connection is important for memory stability. It should be reiterated that the focus of this paper has been on protein-synthesis-independent memory and whether or not a similar processing circuit is utilized for protein synthesis-dependent LTM remains an open question (Krashes, 2007).

    Beyond simply attributing an additional function to the MBs, when taken in conjunction with work on the role of DPM neurons in memory, the data presented in this study suggest a new model for how olfactory memories are processed within the MBs. It is proposed that olfactory information received from the second-order projection neurons is first processed in parallel by the MB αβ and α'β' neurons during acquisition. Activity in α'β' neurons establishes a recurrent α'β' neuron-DPM neuron loop that is necessary for consolidation of memory in αβ neurons and subsequently memories are 'stored' in αβ neurons, whose activity is required during recall. It is plausible that MB α'β' neurons are directly connected to MB αβ neurons and/or that DPM neurons provide the conduit between MB neurons. However, the finding that DPM neurons that project primarily to MB α'β' neurons are functional implies that only a few connections from DPM neurons to MB αβ neurons are necessary (Krashes, 2007).

    The requirement for α'β' neuron output during training also potentially provides a source for the activity that drives DPM neurons. DPM neuron activity is not required during training and the current data are consistent with the idea that olfactory conditioning triggers activity in MB α'β' neurons that in turn elicits DPM neuron-dependent activity. It is proposed that after training recurrent MB α'β' neuron-DPM neuron activity is self-sustaining for 60-90 minutes (Yu, 2005). This recurrent network mechanism is similar to models for working memory in mammals (Durstewitz, 2000). It is also conceivable that MB α'β' neurons receive prolonged input after training from the antennal lobes (AL) via the projection neurons (PN). Olfactory conditioning has been reported to alter the odor response of Drosophila PNs in the AL but the observed effects are short-lived. Nevertheless, AL plasticity for a few minutes after training could contribute to the required MB α'β' neuron activity. If continued activity from the AL is required for consolidation, blocking PN transmission with shibirets1 for one hour after training should abolish memory. The bee AL and MB are clearly involved in olfactory memory and may function somewhat independently in learning and memory consolidation respectively. However, biochemical manipulation of the bee AL can also induce LTM and therefore it is possible that either plasticity in the AL alone can support LTM, or that the AL and MB interact during acquisition and consolidation. A differential role for the AL and MBs has also been suggested from neuronal ablation studies of courtship conditioning in Drosophila. Short-term courtship memory can be supported by the AL but memory lasting longer than 30 min requires the MBs (Krashes, 2007 and references therein).

    This work also has significant implications for the organization of aversive and appetitive odor memories in the fly brain. Stability of both appetitive and aversive memory is dependent on DPM neurons (Keene, 2006) and MB α'β' neurons. It therefore appears that processing of aversive and appetitive odor memories may bottleneck in the MBs. Schwaerzel (2003) demonstrated that aversive memory formation requires dopaminergic neurons whereas appetitive memory relies on octopamine providing a possible mechanism to distinguish valence. However, Schwaerzel also found that MB output is required to retrieve aversive and appetitive odor memory suggesting that both forms of memory involve MB neurons and that both US pathways may converge on MB neurons. It will be important to understand how the common circuitry is organized to independently process the different types of memory and to establish if, and how, such memories co-exist (Krashes, 2007).

    These data imply that stable memory may reside in MB αβ neurons because blocking output from MB αβ neurons impairs retrieval of MTM and ARM (both components of 3-hour memory). It has been previously proposed that AMN peptide(s) released from DPM neurons cause prolonged cAMP synthesis in MB neurons that is required to stabilize memory. The finding that genetically-engineered DPM neurons mostly projecting to the MB α'β' lobes, are functional (Keene, 2006) taken with the idea that stable memory resides in MB αβ neurons is somewhat inconsistent with the notion that crucial AMN-dependent memory processes occur in MB αβ neurons. However, it is plausible that AMN, or another DPM product that is released in a shibire-dependent manner, could diffuse locally from the aberrant DPM neurons to MB αβ neurons (Krashes, 2007).

    This work demonstrates that MB αβ neurons and α'β' neurons have different roles in memory. Beyond gross structural and gene expression differences, it will be essential to establish the precise connectivity, relative excitability and odor responses of the different MB neurons. Future study may also reveal further functional subdivision within the MB lobes and it should be possible to refine current MB α'β' neuron GAL4 lines with appropriate GAL80 transgenes and FLP-out technology (Krashes, 2007).

    In the mammalian brain, memories that initially depend on the function of the hippocampus lose this dependence when they are consolidated. This transient involvement of the hippocampus has led to the idea that consolidation of memory results in the transfer of memory from the hippocampal circuits to the cortex. An alternate view is that aspects of the memory are always in the cortex but they are dependent on the hippocampus because recurrent activity from cortex to hippocampus to cortex is required for consolidation. Hence, disrupting hippocampal activity during consolidation leads to memory loss (Krashes, 2007).

    The current data suggest the simpler fruit fly brain similarly employs parallel and sequential use of different regions to process memory. MB α'β' neuron activity is required to form memory, MB α'β' neurons and DPM neurons are transiently required to consolidate memory and output from αβ neurons is exclusively required to retrieve memory. It is therefore proposed that aversive and appetitive odor memories are formed in MB αβ neurons and are stabilized there by recurrent activity involving MB α'β', DPM neurons and the MB αβ neurons themselves (Krashes, 2007).

    It is becoming increasingly apparent that neural circuit analysis will play an important role in understanding how the brain encodes memory. The ease and sophistication with which one can manipulate circuit function in Drosophila, combined with the relative simplicity of insect brain anatomy should ensure that the fruit fly will make significant contributions to this emerging discipline (Krashes, 2007).

    Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila

    This study distinguishes the memory response of flies to appetitive vs. aversive long-term memory. In Drosophila, formation of aversive olfactory long-term memory (LTM) requires multiple training sessions pairing odor and electric shock punishment with rest intervals. In contrast, this study shows that a single 2 min training session pairing odor with a more ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. Appetitive LTM has some mechanistic similarity to aversive LTM in that it can be disrupted by cycloheximide, the dCreb2-b transcriptional repressor, and the crammer and tequila LTM-specific mutations. However, appetitive LTM is completely disrupted by the radish mutation that apparently represents a distinct mechanistic phase of consolidated aversive memory. Furthermore, appetitive LTM requires activity in the dorsal paired medial neuron and mushroom body α'β' neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval, suggesting that appetitive middle-term memory and LTM are mechanistically linked. Finally, this study describes experiments in which feeding and/or starving flies after training reveal a critical motivational drive that enables appetitive LTM retrieval (Krashes, 2008).

    A single 2 min training session pairing odor with sucrose forms appetitive memory that lasts for days. The term 'session' rather than 'trial' is used cautiously because, although the conditioned odor stimulus is continuously presented for 2 min, it is not known how often the flies sample the sugar unconditioned stimulus. One session of the established aversive training paradigm presents 12 shocks at 5 s intervals overlapping with 1-min-long odor exposure, and therefore neither protocol is strictly 'single-trial' learning. Nevertheless, the results present a profound difference between the training protocol requirements to form aversive and appetitive LTM in flies. Formation of aversive LTM requires 5-10 training sessions with rest intervals, whereas a single 2 min session is sufficient to form robust protein synthesis-dependent appetitive LTM. Appetitive LTM is disrupted by cycloheximide (CXM) feeding, inhibition of CREB-dependent transcription, and the crammer (Comas, 2004) and tequila (Didelot, 2006) genes, which suggests that it is bona fide LTM. Furthermore, these data indicate some mechanistic parallel between aversive and appetitive LTM. Appetitive conditioning forms more distributed memory traces in the brain and more efficiently forms LTM than aversive conditioning. It is speculated that these properties of appetitive memory result from the ethological relevance of feeding and the salience of sucrose reinforcement. Furthermore, the salience is likely to be enhanced in hungry flies because they are motivated to seek food. There are a few other reports of single-trial training forming LTM. With the notable exception of fear conditioning in rodents, most involve feeding behavior and the gustatory pathway. In conditioned taste aversion experiments, rodents develop a long-lasting avoidance of a novel tastant after a single exposure of the tastant and delayed drug-induced malaise. Similarly, pond snails develop long-lasting conditioned taste aversion if carrot juice is paired with salt exposure, and 1-d-old chicks develop LTM to avoid pecking a colored bead if that bead was tainted with a bitter tasting compound when first presented. There are also examples in which single-trial conditioning forms appetitive LTM. Rats deficient in thiamine can be trained to prefer non-nutritious saccharin-flavored water by pairing it with delayed an intramuscular thiamine injection. Pond snails form appetitive LTM for the odorant/tastant amylacetate after a single trial of appetitive conditioning pairing it with sucrose. Last, a single trial of appetitive conditioning in honeybees forms robust day-long memory that, surprisingly, does not require new protein synthesis after training. Therefore, it is possible that the innate importance of food-seeking behavior and memory makes it particularly prone to fast consolidation to LTM (Krashes, 2008).

    The single training session appetitive LTM assay provides a unique advantage for the study of memory consolidation because one can manipulate the brain immediately after training during the initial period of memory formation. In contrast, 10 cycles of aversive spaced training takes 150 min to complete, and therefore one cannot perturb neural processing during this period without also interfering with acquisition. Using cold-shock anesthesia, it was found that appetitive memory is quickly, and perhaps entirely, consolidated to anesthesia-resistant forms within 2 h after training (Krashes, 2008).

    Previous work in flies suggests that cold shock-resistant memory can be broken into two independent components, ARM that depends on the radish (rsh) gene and is resistant to CXM and LTM that is unaffected by rsh and is sensitive to CXM. Feeding flies CXM disrupted appetitive LTM and produced a statistically significant defect 6 h after training, suggesting that protein synthesis-dependent LTM guides behavior at that time. Although the effect of CXM feeding is estimated to inhibit only 50% of global protein synthesis and has to be partial, these data are consistent with the notion that consolidated memory before 6 h might be ARM. However, whereas aversive LTM requires protein synthesis and is not affected by rsh, appetitive LTM requires new protein synthesis and rsh, suggesting appetitive LTM and rsh-dependent appetitive memory do not represent separable memory phases. This result highlights a potentially major mechanistic difference between aversive and appetitive LTM, and that the relationship between ARM and LTM is worth revisiting. Unfortunately, the cloning of rsh does not provide any mechanistic insight because its primary sequence does not contain any known functional domains (Krashes, 2008).

    These data reveal a slight discrepancy in the notion that rsh, dCreb-dependent transcription and new protein synthesis are all necessary components of appetitive LTM. Cold-shock anesthesia indicates that appetitive memory consolidation is nearly complete 2 h after training and rsh mutant flies display defective performance 3 h after training, but neither dCreb2-b repressor transgene nor CXM feeding produced a significant difference in memory performance 3 h after training. It is speculated that expression of early forms of appetitive LTM (E-LTM) depend on rsh and that because Radish protein immunolocalizes to neuropil, Radish might function in a synaptic tagging process that marks the relevant synapses for capture of dCreb2-dependent transcripts. This idea provides a plausible reason why radish is required both for E-LTM and for later appetitive LTM (L-LTM), whereas dCreb2-b (Cyclic-AMP response element binding protein B) only interferes with L-LTM. Similarly, it is posited that CXM feeding blocks the translation of mRNAs that are direct and indirect targets of CREB and that are necessary for L-LTM. Similar models have been proposed based on work in rodents and Aplysia (Krashes, 2008).

    Previous work has determined that stable olfactory memory (MTM) observed 3 h after aversive and appetitive training requires the sequential involvement of different MB neuron subsets. MB α' β' neurons are required during and after training to acquire and stabilize memory (Krashes, 2007), whereas MB αβ neuron output is required only to retrieve the memory. Stable aversive and appetitive MTM also requires the action of MB-innervating dorsal paired medial (DPM) neurons during the first hour after training. Similarly timed manipulation of these distinct neural circuit elements strongly impairs appetitive LTM, suggesting a tight mechanistic link between appetitive MTM and LTM (Krashes, 2008).

    Finding that consolidation of appetitive memory to a protein synthesis-dependent form requires the DPM-MB neural circuitry and that retrieval requires MB αβ neuron output is consistent with the idea that consolidated memory is represented in MB αβ neurons themselves. Several studies have now reported that MB neuron output is required to retrieve olfactory memory, and a few have indicated that MB αβ neurons are particularly important to retrieve aversive and appetitive MTM. A recent live-imaging study provided additional evidence that consolidated aversive LTM is represented in MB αβ neurons (Yu, 2006). Flies that had been space trained with odor and shock exhibited enhanced odor-evoked Ca2+ signals in the vertical α branch of MB αβ neurons 9-24 h after conditioning. The development of this memory 'trace' was disrupted by CXM administration, by mutations in the amnesiac gene, and by expressing a transgenic dCreb2-b in MB αβ neurons. Furthermore, expression of the dCreb2-b transgene in MB αβ neurons also impaired aversive LTM behavior. These data are highly consistent with the current findings for appetitive LTM after a single training session and therefore indicate that there are common mechanistic components to aversive and appetitive LTM. It is also worth noting that radish is strongly expressed in MB αβ neurons. Therefore, this collection of findings provides strong evidence that consolidated aversive and appetitive LTM involves MB αβ neurons (Krashes, 2008).

    These results do not support the recently proposed idea that LTM consolidation involves transfer from MB to EB (Wu, 2007). Although an appetitive memory assay was used, it was found that Feb170;uas-shits1 flies have a pronounced locomotor defect and therefore these flies are not suitable for memory analysis. Furthermore, Ruslan GAL4 (Wu, 2007) and c305a (Krashes, 2007) express in EB ring neurons, but blocking these neurons does not affect appetitive LTM retrieval. These data are instead consistent with the notion that the transfer of the MB lobe requirement within the first few hours after training may be the fly equivalent of systems consolidation (Krashes, 2008).

    These data clearly demonstrate that flies have to be hungry to effectively retrieve appetitive memory. Feeding them ad libitum after training suppressed memory performance, but restarving them restored memory performance. It is proposed that this apparent context dependence of appetitive memory retrieval reflects a motivational state to seek food and therefore it is predicted to be regulated by neuromodulatory systems that signal hunger (Krashes, 2008).

    Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory

    A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, it was postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. This study found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory (Majundar, 2012).

    Learning changes the efficacy and number of synaptic connections. Memory is the maintenance of that altered state over time. Synaptic modification is likely to include both quantitative and qualitative changes in local protein composition. However, this model of memory raises a fundamental question that remains unanswered: how does the altered protein composition of a synapse persist for years when the molecules that initiated the process should disappear within days (Majundar, 2012)?

    The protein composition of a synapse can be altered in several ways including synthesis of new proteins. Local synthesis of new proteins at the synapse has been shown to be essential for stabilizing the functional changes and physical growth of the activated synapse. Previously, a family of RNA-binding proteins, known as cytoplasmic polyadenylation element-binding proteins (CPEBs), were identified as regulators of activity-dependent protein synthesis at the synapse. In the sea snail Aplysia, a neuron-specific variant of CPEB, ApCPEB, is required not for the initial changes in synaptic efficacy or growth following serotonin stimulation but for the maintenance of these changes beyond 24 hr). In Drosophila, reduction in Orb2, a member of the CPEB protein family, does not affect short-term memory (< 3 hr) but does prevent the memories from persisting beyond 12 hr. In mice, deletion of the CPEB-1 gene reduces long-term potentiation evoked by theta-burst stimulation and growth-hormone applicatio. Together, these observations suggest that CPEBs play a role in stabilizing activity-dependent changes in synaptic efficacy. However, how CPEB-dependent changes in molecular composition of the synapse persist over time is unknown (Majundar, 2012 and references therein).

    Previously, based on the self-sustaining amyloidogenic (prion-like) properties of Aplysia CPEB, it was hypothesized that the activity-dependent conversion of CPEB to the amyloidogenic state provides a self-sustaining mechanism for the persistent change in molecular composition of the synapse and thereby persistence of memory. Consistent with this idea, in Aplysia sensory neurons ApCPEB forms amyloidogenic aggregates when overexpressed, and the number of aggregates increases following stimulation with serotonin. Moreover, an antibody that recognizes oligomeric ApCPEB selectively blocks the persistence of long-term facilitation of the sensory-motor neuron synapse beyond 24 hr. However, the central question of whether such conversion of neuronal CPEB to the amyloid-like state is necessary for the persistence of memory remains unanswered. To address the behavioral significance of the amyloid-like state of CPEB, Drosophila Orb2 was studied. Drosophila Orb2 protein carry a prion-like domain and target synaptic growthrelated proteins, suggesting that Orb2 is not only structurally but also functionally similar to ApCPEB (Majundar, 2012 and references therein).

    This paper has asked two specific questions. First, does the Orb2 protein form amyloid-like oligomers in the adult Drosophila brain in an activity-dependent manner? Second, is this oligomerization necessary for long-term memory? It was found that Drosophila Orb2 forms stable SDS-resistant, amyloid-like oligomers upon neuronal stimulation, and Orb2 mutant defective in activity-dependent oligomerization is specifically impaired in forming stable long-term memories. These observations support the hypothesis that self-sustaining amyloid-like conversion of neuronal CPEB is involved in the persistence of memory (Majundar, 2012).

    Previously, based on studies primarily with Aplysia CPEB, it was postulated that self-sustaining amyloidogenic oligomers, similar to yeast prion-like proteins, might be the basis of the persistence of activity-dependent increase in synaptic efficacy and the persistence of memory. However some of the earlier analysis was performed under overexpression or in heterologous conditions. This study found that like other amyloids, in physiological concentration, in the adult Drosophila brain, Orb2 forms tetramers or hexamers that are resistant to heat, SDS, and chaotropic reagents. Stimulation of behaviorally relevant neurons increases the level of oligomeric Orb2, which is enriched in the synaptic membrane fraction. These observations suggest that the unusual amyloidogenic oligomerization of Orb2/CPEB is conserved across species, and the oligomer may indeed act to stabilize activity-dependent increase in synaptic efficacy (Majundar, 2012).

    A mutation in the rare Orb2A isoform that results in reduced oligomerization, without lowering the amount of Orb2B protein, produces a selective deficit in the stabilization of memory beyond 24 hr. This is different than loss of both isoforms, which leads to earlier memory deficit. One interpretation of these data is that Orb2B activity is required for the formation of long-term memory, whereas Orb2A activity is required for the persistence of memory beyond 24 hr. However, both Orb2A and Orb2B form amyloidlike oligomers and when overexpressed in Dorb2/+ background can rescue the male courtship suppression memory as well as olfactory-reward memory at 24 hr, suggesting functional similarity. How can these observations be reconciled (Majundar, 2012)?

    When expressed in S2 cells, both Orb2A and Orb2B act as translation repressor (Mastushita-Sakai, 2010). In the adult brain, the Orb2B protein is constitutively expressed in a large number of neurons, but the Orb2A protein is 100-fold less abundant, expressed in fewer neurons, and deletion of Orb2A reduces overall Orb2 oligomerization in vivo. Together, these results suggest the following model. Orb2B-mediated translation repression is critical for the formation and consolidation of memory up to 24 hr, and when ectopically expressed, Orb2A rescues this repressive function of Orb2B. However, regulated conversion of Orb2 proteins to the oligomeric state is necessary for long-term stabilization of memory beyond 24 hr, and the Orb2A protein regulates this conversion. This model implies that Orb2A and Orb2B have nonredundant functions in long-term memory, and neurons in which Orb2 oligomerizes are the site for long-term memory storage in Drosophila (Majundar, 2012).

    The recent modENCODE project has reported four new protein isoforms in the Orb2 locus that would be affected in the Orb2 deletion mutants. However, the Orb2 oligomers or the behavioral phenotypes observed in this study are not dependent on these isoforms. The Orb2 antibodies used in this study do not recognize the common region between Orb2B and the new isoforms. Moreover, Orb2A and Orb2B cDNA as well as a genomic construct that does not code for any of these new proteins isoforms can rescue behavioral deficit. The function of these new isoforms has yet to be determined (Majundar, 2012).

    Although the amyloidogenic forms of PrP and other proteins are pathogenic, it is now evident that amyloids can underlie epigenetic heritable phenotypes in yeast and can serve normal physiological functions in other organisms. However, in most cases it is unclear how amyloid formation is regulated, if at all. The low level of Orb2 oligomers in the adult brain and their virtual absence from the body raise the possibility that although Orb2B protein is widely expressed, Orb2 oligomerization per se is limited, perhaps only in neurons in which Orb2A is expressed. The Orb2A protein, due to its propensity to oligomerize, may form the seed that recruits Orb2B protein, resulting in the temporally and spatially restricted conversion of Orb2A/Orb2B into self-sustaining oligomers. In this regard, Orb2 oligomerization may resemble seeded formation of curli amyloid on the surface of bacteria, in which oligomerization of the major curli subunit CsgA is seeded by the membrane-bound minor subunit CsgB (Majundar, 2012 and references therein).

    Curiously, this study found that Orb2A mRNA in the adult brain retains an intronic sequence with stop codons. Among age-matched pCasper-Orb2AEGFP flies, heterogeneity in the Orb2A protein level was observed, particularly in the synaptic-neuropil region. The low abundance, presence of unprocessed mRNA, and immense propensity to oligomerize imply that in the adult head, expression of Orb2A is regulated and may constitute the rate-limiting step in Orb2 oligomerization and thereby long-lasting memory formation. Moreover, although Orb2A mRNA is present in the body, the Orb2A protein was undetectable, suggesting that it is present either in very low levels or only in certain cell types. What function Orb2A serves outside the nervous system is not known (Majundar, 2012).

    It is now evident that a number of proteins with very different primary amino acid sequences can form self-templating amyloids. What sequence and structural features distinguish a regulated functional amyloid from unregulated inactive or pathogenic amyloids? Although these studies were initiated with the observation that Aplysia CPEB contains a Q-rich unstructured domain, this study found that the Q-rich region of Orb2 is important for the stability but not formation of oligomer. Similarly, a coiled-coil domain outside the amyloid-forming domain of Aplysia CPEB regulates its oligomerization. These observations suggest that identification of functional amyloid based on primary amino acid sequence is challenging. Highlighting this point, it was found that a single nonpolar to polar amino acid change in the N-terminal 8 amino acids of Orb2A affects not only the efficiency of oligomerization but also the nature of the amyloid oligomer. Structural analysis of wild-type and mutant Orb2 proteins may help us to understand what features distinguish functional amyloid from nonfunctional amyloid (Majundar, 2012).

    Imaging of an early memory trace in the Drosophila mushroom body

    Extensive molecular, genetic, and anatomical analyses have suggested that olfactory memory is stored in the mushroom body (MB), a higher-order olfactory center in the insect brain. The MB comprises three subtypes of neurons with axons that extend into different lobes. A recent functional imaging study has revealed a long-term memory trace manifested as an increase in the Ca2+ activity in an axonal branch of a subtype of MB neurons. However, early memory traces in the MB remain elusive. This paper reports learning-induced changes in Ca2+ activities during early memory formation in a different subtype of MB neurons. Three independent in vivo and in vitro preparations were used, and all of them showed that Ca2+ activities in the axonal branches of α'/β' neurons in response to a conditioned olfactory stimulus became larger compared with one that was not conditioned. The changes were dependent on proper G-protein signaling in the MB. The importance of these changes in the Ca2+ activity of α'/β' neurons during early memory formation was further tested behaviorally by disrupting G-protein signaling in these neurons or blocking their synaptic outputs during the learning and memory process. The results suggest that increased Ca2+ activity in response to a conditioned olfactory stimulus may be a neural correlate of early memory in the MB (Wang, 2008).

    Functional Ca2+ imaging was used in three different preparations to search for memory traces in the MB after a single-cycle training that produces short-term memory (STM), middle-term memory (MTM), anesthesia-resistant memory (ARM). Flies were trained in groups in the standard T-maze or individually while held immobilized. These two different settings in training both led to enhanced Ca2+ activity in response to trained odors in the axonal branches of the α'/β' neurons. The enhancement was specific to the odor that was paired with electric shock and was detected at ~1 hour after conditioning. The semi-in vivo preparation that mimicked the single-cycle olfactory training with paired stimulation of the antennal nerves (ANs) and the ventral nerve cord (VNC) led to a similar observation. In addition, an immediate enhancement in Ca2+ response in α'/β' lobes after the paired event was observed in this preparation. Targeted disruption of the G-protein signaling in the MB blocked the enhancement of Ca2+ response after paired stimulation of AN and VNC. These results indicate that the enhanced Ca2+ response in α'/β' lobes after conditioning may represent a memory trace associated with early forms of memory. This was further confirmed in a behavioral experiment in which the 3 min and 1 h memory was impaired by disrupting the G-protein signaling in the α'/β' neurons or blocking synaptic output from these neurons (Wang, 2008).

    This finding does not preclude the possible existence of memory traces in other output branches of the MB neurons. There are a number of possibilities why the enhanced Ca2+ response to trained odors was only observed in axonal branches of the α'/β' neurons. Memory traces in the α/β and γ neurons may appear in forms other than alteration of Ca2+ response. It is known that a Ca2+-independent PKC signaling can potentiate synaptic transmission presynaptically. Another possibility is that for α/β and γ neurons, memory traces may occur postsynaptically in downstream neurons. Postsynaptic mechanisms play a key role in long-term potentiation and long-term depression. A third possibility is that the low G-CaMP responses in α/β and γ lobes are making it difficult to detect changes in these lobes. Activities in MB neurons are sparse, and the γ neurons tend to show a lower probability of response. Changes in a small amount of activity may be hard to detect with the imaging approach that was used. Finally, learning may lead to changes in the fine temporal pattern of neuronal activities in the MB that cannot be detected by Ca2+ imaging (Wang, 2008).

    Olfactory learning and memory in Drosophila seem to involve multiple brain structures in parallel and sequential pathways. Blocking the output from the MB impairs retrieval of memory at multiple stages that encompass STM, MTM, ARM, and LTM. This requirement of the MB output has been further dissected and assigned to different subtypes of MB neurons for different stages of memory processing. The initial study with the Shits has indicated that retrieval of 3 min and 3 h memory may be exclusively through α/β neurons. The most recent study on 3 h memory confirmed the role for α/β neurons in retrieval and further revealed that output of the α'/β' neurons is required for acquisition and consolidation. Several places outside the MB are also involved in olfactory learning and memory. Output from a pair of neurons called dorsal paired medial (DPM) neurons that innervate the entire MB lobes are required for 3 h memory in a time window of 30-150 min after conditioning. One of the NMDA receptor subunits, NR1, which is preferentially expressed in a small number of neurons that innervate the MB dendritic region, affects learning and LTM consolidation. Several genes expressed in a small number of neurons outside the MB were identified in a large-scale mutagenesis to affect 1 d memory. Dopaminergic and octopaminergic neurons, which both innervate the MB lobes, are believed to carry the aversive and appetitive unconditioned stimulus reinforcement, respectively (Wang, 2008).

    Correspondingly, learning and memory-associated cellular changes, collectively called memory traces, have been found in some of these structures at different stages of memory processing. In dorsal paired medial (DPM) neurons, odor-evoked Ca2+ response in the branch that innervates the MB vertical lobes (α and α') is enhanced 30 min after conditioning. In the MB, enhanced odor-evoked Ca2+ response is observed in the α lobe 24 h after spaced training (multiple training sessions with a rest between sessions), which produces LTM. In addition, a memory trace appearing as recruitment of new projection neurons (PNs) in the AL occurs immediately after conditioning but lasts only a few minutes, disappearing at 7 min after conditioning (Wang, 2008).

    How these memory traces interact with each other is not yet clear. The memory trace in the AL PNs and that was observed in the MB α'/β' neurons may appear in parallel after conditioning so STM may be formed in two independent places. The following observations support this possibility. (1) The memory trace in PNs is observed in the presynaptic specializations of PNs localized in the AL glomeruli. (2) No change was observed in the amount of odor-evoked Ca2+ response after olfactory training in the calyx, where synaptic connections are made between MB neurons and PNs. The spatial distribution of activity in the calyx was also not changed after olfactory training. Therefore, the recruited new synaptic activities in the AL may not be transmitted to the MB to drive activity changes there at time points studied (Wang, 2008).

    The involvement of DPM neurons in memory consolidation relies on their projections to the axonal branches of the MB α'/β' neurons. This suggests that memory consolidation may be performed by local interactions between DPM neurons and the MB α'/β' neurons, and the memory may reside in the α'/β' neurons during this process. This idea is also supported by the finding that the α'/β' neurons are required for memory acquisition and consolidation. The observation of a memory trace in the axonal branches of the MB α'/β' neurons also provides a strong support to the idea that the memory initially resides in the α'/β' lobes. From this perspective, it is interesting that the memory trace in the DPM neurons is only observed in branches that innervate the MB vertical lobes. It is not known whether this memory trace is restricted further to branches that innervate the α' lobe or is expressed in all branches that innervate vertical lobes. The memory trace observed in the current study in α'/β' neurons seems to appear before the memory trace in the DPM neurons, which was detected ~30 min after conditioning. Currently, it is not known whether there is any causal link between the two memory traces or whether they may exist independently. It should be interesting to see how the two memory traces are affected by manipulating activities of the DPM neurons and α'/β' neurons (Wang, 2008).

    The LTM trace in the α lobe of the α/β neurons start to appear at 9 h after spaced training. It will be interesting to investigate whether the early memory trace observed in the α'/β' neurons also exists after spaced training and how the STM trace is converted into the LTM trace. The LTM trace is blocked by mutation of the amnesiac gene, which is expressed in the DPM neurons. Given the facts that the amnesiac mutation also blocks the intermediate memory trace in DPM neurons and that DPM projections to the axonal branches of the α'/β' neurons are sufficient for its role in memory consolidation, there is a very good possibility that the amnesiac mutation will disrupt the memory trace in the α'/β' neurons and this disruption results in the elimination of the LTM trace in the α/β neurons. Future studies addressing these issues should help derive a better understand how memory is formed and maintained in the brain (Wang, 2008).

    A permissive role of mushroom body α/β core neurons in long-term memory consolidation in Drosophila

    Memories are not created equally strong or persistent for different experiences. In Drosophila, induction of long-term memory (LTM) for aversive olfactory conditioning requires ten spaced repetitive training trials, whereas a single trial is sufficient for LTM generation in appetitive olfactory conditioning. Although, with the ease of genetic manipulation, many genes and brain structures have been related to LTM formation, it is still an important task to identify new components and reveal the mechanisms underlying LTM regulation. This study shows that single-trial induction of LTM can also be achieved for aversive olfactory conditioning through inhibition of highwire (hiw)-encoded E3 ubiquitin ligase activity or activation of its targeted proteins in a cluster of neurons, localized within the α/β core region of the mushroom body. Moreover, the synaptic output of these neurons is critical within a limited posttraining interval for permitting consolidation of both aversive and appetitive LTM. It is proposed that these α/β core neurons serve as a 'gate' to keep LTM from being formed, whereas any experience capable of 'opening' the gate is given permit to be consolidated into LTM (Huang, 2012).

    The current study began with the finding that 24 hr memory resulting from single session was enhanced in two hiw mutant alleles. This enhanced memory component was identified as facilitated LTM, given that it was sensitive to protein synthesis inhibition. The behavioral effect of hiwδRING and the presence of a hiw-GAL4 line allowed mapping the neural circuitry to a cluster of MB α/β core neurons, within which Hiw and its downstream targets regulate LTM. Furthermore, it was shown that the MB α/β core neurons are involved in the consolidation of both aversive and appetitive LTM. In conclusion, the observations that the MB α/β core neurons are capable of both facilitating and limiting LTM suggests a working model in which the ability to form LTM is gated through these neurons. The significance of these results is further elaborated below (Huang, 2012).

    Not only synthesis but also degradation of proteins plays a critical role in the remodeling of synapses, learning, and memory. Altered memory formation in ubiquitin ligase mutants has been reported in mice and Drosophila. This study reports that Hiw, an evolutionarily conserved E3 ubiquitin ligase, negatively regulated LTM formation through restraining its downstream target Wallenda (Wnd). The results indicated Hiw function as an inhibitory constraint, as the memory suppressor gene, on LTM formation. Removal of this suppressor or direct activation of its downstream signals could lead to the facilitated LTM induction without the repetitive training that is normally required. So far, the physiological consequence of Hiw or Wnd in core neurons still remains an open question. Because of the extensively shared components with hiw’s function in synaptic growth and transmission, the attenuated Hiw activity may elevate Wnd level and then lead to the excessive synaptogenesis or abnormal synaptic activity in core neurons. It will also be interesting to test whether the hiw-mediated LTM facilitation shares some common molecular components with the corkscrew-regulated spacing effect in LTM induction, in which the MB α/β lobes also play an important role (Huang, 2012).

    In a recent report, Hiw was shown to regulate the axon guidance in MB (Shin, 2011). The morphological defect was also observed in MB α/β lobes in a portion of hiw mutant flies. It is striking that hiw mutants with MB defect can form LTM even more efficiently, given the observation of LTM impairment in another MB structural mutant, ala. However, comparing to the total loss of vertical lobes (including α and α') in ala mutant, most hiw mutants had the abnormal thickness of α/β lobes caused by the unequal distribution of the MB axonal projections between the α and β lobes, and about 39% of hiwDN mutant had the shortened α lobe. One of the possible explanations is that the remaining function of α/β lobe in hiw mutants is sufficient to support the LTM. Moreover, expression of Hiw dominant-negative protein or acutely increasing Wnd protein level in MB was sufficient to promote LTM but did not give rise to any observable gross morphological change in MB. Thus, it is suggested that Hiw mediates memory phenotype through a different mechanism from the one that led to the structure change in the MB (Huang, 2012).

    The involvement of MB in the hiw-mediated LTM facilitation led an examination of the function of this structure in LTM regulation. It has been well documented that the MB, a bilateral brain structure that consists of approximately 2,500 neurons in each hemisphere, plays the central role in olfactory memories, both aversive and appetitive. Intrinsic MB neurons are organized into physically distinct α, β, α', β' and γ lobes. All three lobes exhibit different functions in memory processing, such that the output of α/β lobes is required for retrieval of memory, α' β' lobes are transiently required to stabilize memory or to retrieve immediate memory, and γ lobe mediate a rutabaga-dependent mechanism and dopaminergic signal to support short-term memory (STM) and LTM formation. Moreover, memory traces mapped to different lobes exhibit different temporal features (Huang, 2012).

    Through gene expression patterns and enhancer trap lines, each lobe of MB can be classified into more specific subgroups such as the posterior, surface, and core regions in the α/β lobes. The current work shows that α/β core neurons play a distinct role in LTM induction. The synaptic outputs of these neurons are critical during consolidation of LTM for both aversive and appetitive conditioning, but these neurons are not involved in LTM cellular consolidation per se because a landmark of LTM cellular consolidation, CREB-mediated protein synthesis, occurs in non-MB neurons. Thus, one of the roles for this cluster of neurons can be viewed as simply providing connections to channel learning information to the downstream neurons in which LTM is formed. However, the remarkable feature of enabling single-trial induction of aversive LTM through targeted genetic manipulation of this cluster of neurons suggests that they play a unique permissive role in determining whether an experience should be consolidated (Huang, 2012).

    This newly identified function for permitting an experience to be consolidated leads to proposal of a gating theory. This theory proposes that the α/β core neurons serve as a 'gate,' and activation of this gating mechanism functions as a checkpoint that keeps LTM from being formed for general experiences, whereas only specific experiences, capable of 'opening' this gate, can and are bound to trigger LTM consolidation and to form LTM ultimately. There is little survival advantage in committing never-to-be-repeated episodes to memory, particularly because the very act of LTM formation may be deleterious to the fly. In contrast, repetitively occurring experience, such as spaced repetitive aversive conditioning, and events critical for survival, such as finding food or single-trial appetitive conditioning, would be able to 'open' the gate, and therefore, LTM is formed for such experiences (Huang, 2012).

    A neural circuit mechanism integrating motivational state with memory expression in Drosophila

    Behavioral expression of food-associated memory in fruit flies is constrained by satiety and promoted by hunger, suggesting an influence of motivational state. This study identified a neural mechanism that integrates the internal state of hunger and appetitive memory. Stimulation of neurons that express neuropeptide F (dNPF), an ortholog of mammalian NPY, mimics food deprivation and promotes memory performance in satiated flies. Robust appetitive memory performance requires the dNPF receptor in six dopaminergic neurons that innervate a distinct region of the mushroom bodies. Blocking these dopaminergic neurons releases memory performance in satiated flies, whereas stimulation suppresses memory performance in hungry flies. Therefore, dNPF and dopamine provide a motivational switch in the mushroom body that controls the output of appetitive memory (Krashes, 2009).

    Drosophila can be efficiently trained to associate odorants with sucrose reward. Importantly, fruit flies have to be hungry to effectively express appetitive memory performance (Krashes, 2008). This apparent state dependence implies that signals for hunger and satiety may interact with memory circuitry to regulate the behavioral expression of learned food-seeking behavior. The mushroom body (MB) in the fly brain is a critical site for appetitive memory. Synaptic output from the MB α′β′ neurons is required to consolidate appetitive memory whereas output from the αβ subset is specifically required for memory retrieval (Krashes, 2007; Krashes, 2008). This anatomy provides a foundation for understanding neural circuit integration between systems representing a motivational state and those for memory (Krashes, 2009).

    Neuropeptide Y (NPY) is a highly conserved 36 amino acid neuromodulator that stimulates food-seeking behavior in mammals. NPY messenger RNA (mRNA) levels are elevated in neurons in the arcuate nucleus of the hypothalamus of food-deprived mice. Most impressively, ablation of NPY-expressing neurons from adult mice leads to starvation. NPY exerts its effects through a family of NPY receptors and appears to have inhibitory function. NPY therefore must repress the action of inhibitory pathways in order to promote feeding behavior. Drosophila neuropeptide F (dNPF) is an ortholog of NPY, which has a C-terminal amidated phenylalanine instead of the amidated tyrosine in vertebrates. Evidence suggests that dNPF plays a similar role in appetitive behavior in flies. dNPF overexpression prolongs feeding in larvae and delays the developmental transition from foraging to pupariation. Furthermore, overexpression of a dNPF receptor gene, npfr1, causes well-fed larvae to eat bitter-tasting food that wild-type larvae will only consume if they are hungry (Krashes, 2009 and references therein).

    This study exploited dNPF to identify a neural circuit that participates in motivational control of appetitive memory behavior in adult fruit flies. Stimulation of dNPF neurons promotes appetitive memory performance in fed flies, mimicking the hungry state. npfr1 is required in dopaminergic (DA) neurons that innervate the MB for satiety to suppress appetitive memory performance. Directly blocking the DA neurons during memory testing reveals performance in fed flies, whereas stimulating them suppresses performance in hungry flies. These data suggest that six DA neurons are a key module of dNPF-regulated circuitry, through which the internal motivational states of hunger and satiety are represented in the MB (Krashes, 2009).

    It is critical to an animal's survival that behaviors are expressed at the appropriate time. Motivational systems provide some of this behavioral control. Apart from the observation that motivational states are often regulated by hormones or neuromodulatory factors, little is known about how motivational states modulate specific neural circuitry. Hungry fruit flies form appetitive long-term memory, after a 2 min pairing of odorant and sucrose, and memory performance is only robust if the flies remain hungry (Krashes, 2008). Therefore, this paradigm includes key features of models for motivational systems: the conditioned odor provides the incentive cue predictive of food, there is a learned representation of the goal object (odorant/sucrose), and the expression of learned behavior depends on the internal physiological state (hunger and not satiety). This study identified a neural circuit mechanism that integrates hunger/satiety and appetitive memory (Krashes, 2009).

    The signals that ordinarily control dNPF-releasing neurons is unknown. In mammals, NPY-expressing neurons are a critical part of a complex hypothalamic network that regulates food intake and metabolism. In times of adequate nutrition, NPY-expressing neurons are inhibited by high levels of leptin and insulin that are transported into the brain after release from adipose tissue and the pancreas (Figlewicz, 2009). In hungry mice, leptin and insulin levels fall, leading to loss of inhibition of NPY neurons. Flies do not have leptin, but they have several insulin-like peptides, that may regulate dNPF neurons. Some NPY-expressing neurons are directly inhibited by glucose (Levin, 2006). Fly neurons could sense glucose with the Bride of Sevenless receptor (Kohyama-Koganeya, 2008). In blowflies, satiety involves mechanical tension of the gut and abdomen. Lastly, it will be interesting to test the role of other extracellular signals implicated in fruit fly feeding behavior, including the Hugin and Take-out neuropeptides (Krashes, 2009).

    NPY inhibits synaptic function in mammals, and the data from this study suggest that dNPF promotes appetitive memory performance by suppressing inhibitory MB-MP neurons [named according to the regions of the MB that they innervate: medial lobe and pedunculus (MP)]. A model is proposed in which MB-MP neurons gate MB output. Appetitive memory performance is low in fed flies because the MB αβ and γ neurons are inhibited by tonic dopamine release from MB-MP neurons. Hence, when the fly encounters the conditioned odorant during memory testing, the MB neurons encoding that olfactory memory respond, but the signal is not propagated beyond the MB because of the inhibitory influence of MB-MP neurons. However, when the flies are food deprived, dNPF levels rise, and dNPF disinhibits MB-MP neurons, and other circuits, through the action of NPFR1. dNPF disinhibition of the MB-MP neurons opens the gate on the MB. Therefore, when hungry flies encounter the conditioned odorant during memory testing, the relevant MB neurons are activated and the signal propagates to downstream neurons, leading to expression of the conditioned behavior (Krashes, 2009).

    Satiety and hunger are not absolute states. Sometimes above-chance performance scores are observed in fed flies, and shorter periods of feeding after training suggest that inhibition of performance is graded. This could be accounted for by a competitive push-pull inhibitory mechanism between dNPF and MB-MP neurons (Krashes, 2009).

    By gating the MB through the MB-MP neurons, hunger and satiety are likely affecting the relative salience of learned odor cues in the fly brain. However, MB-MP neurons are unlikely to change the sensory representation of odor in the MB because flies trained with stimulated MB-MP neurons perform normally when tested for memory without stimulation. Therefore, odors are likely perceived the same irrespective of MB-MP neuron activity. Furthermore, the MB-MP neurons did not affect naive responses to the specific odorants used. It will be interesting to test whether MB-MP neurons change responses to other odorants and/or modulate arousal, visual stimulus salience, and attention-like phenomena (Krashes, 2009).

    There are eight different morphological classes of DA neurons that innervate the MB (Mao, 2009), and the current data imply functional subdivision. Previous studies concluded that DA neurons convey aversive reinforcement (Krashes, 2009).

    This study specifically manipulated the MB-MP DA neurons. MB-MP neurons are not required for acquisition of aversive olfactory memory, consistent with a distinct function in controlling the expression of appetitive memory. Since several studies have implicated the MB α lobe in memory, other DA neurons in protocerebral posterior lateral 1 (PPL1) that innervate the α lobe (like those labeled in MBGAL80;krasavietz) may provide reinforcement. The MB-MP neurons may also be functionally divisible and independently regulated to gate MB function. The idea that a specific DA circuit restricts stimulus-evoked behavior is reminiscent of literature tying dopamine to impulse control in mammals. Previous studies of DA neurons in Drosophila have simultaneously manipulated all, or large numbers of DA neurons. The current data suggest that the DA neurons should be considered as individuals or small groups (Krashes, 2009).

    Flies have to be hungry to efficiently acquire appetitive memory, but whether this reflects a state-dependent neural mechanism or results from the failure to ingest enough sugar is unclear. Stimulation of MB-MP neurons in hungry flies did not impair appetitive memory formation, and therefore MB-MP neurons are unlikely to constrain learning in fed flies. Other dNPF-regulated neurons may provide this control since NPY has been implicated in learning (Krashes, 2009).

    The dNPF-expressing neurons innervate broad regions of the brain and may simultaneously modulate distinct neural circuits to promote food seeking. MB-MP neurons represent a circuit through which the salience of learned food-relevant odorant cues is regulated by relative nutritional state. Given the apparent role of the MB as a locomotor regulator, MB-MP neurons may also generally promote exploratory behavior. There are likely to be independent circuits for other elements of food-seeking behavior including those that potentiate gustatory pathway sensitivity and promote ingestion (Krashes, 2009).

    NPY stimulates feeding but inhibits sexual behavior in rats. Modulators exerting differential effects could provide a neural mechanism to establish a hierarchy of motivated states and coordinate behavioral control. dNPF may potentiate activity in food seeking-related circuits while suppressing circuits required for other potentially competing behaviors, e.g., sexual pursuit (Krashes, 2009).

    This study has provided the first multilevel neural circuit perspective for a learned motivated behavior in fruit flies. The work demonstrates a clear state-dependence for the expression of appetitive memory. Odorants that evoke conditioned appetitive behavior in hungry flies are ineffective at evoking appetitive behavior in satiated flies. Therefore, the fly brain is not simply a collection of input-output reflex units and includes neural circuits through which the internal physiological state of the animal establishes the appropriate context for behavioral expression (Krashes, 2009).

    It has been proposed that a satiated fly receives maximum inhibitory feedback so that sensory input is behaviorally ineffective. As deprivation increases inhibition wanes and sensory input becomes increasingly effective in initiating feeding. The current data provide experimental evidence that this prediction is also likely to be accurate for expression of appetitive memory in the fruit fly where the mechanism involves neuromodulation in the central brain. The DA MB-MP neurons inhibit the expression of appetitive memory performance in satiated flies, whereas dNPF disinhibits the MB-MP neurons in food-deprived flies. The likelihood that appetitive behavior is triggered by the conditioned odorant is therefore determined by the competition between inhibitory systems in the brain. The concept that continuously active inhibitory forces in the insect brain control behavioral expression has also be proposed many years ago. This study provides evidence that these neurons exist and that their hierarchical arrangement is a key determinant of behavioral control (Krashes, 2009).

    Visual place learning in Drosophila melanogaster

    The ability of insects to learn and navigate to specific locations in the environment has fascinated naturalists for decades. The impressive navigational abilities of ants, bees, wasps and other insects demonstrate that insects are capable of visual place learning but little is known about the underlying neural circuits that mediate these behaviours. Drosophila is a powerful model organism for dissecting the neural circuitry underlying complex behaviours, from sensory perception to learning and memory. Drosophila can identify and remember visual features such as size, colour and contour orientation. However, the extent to which they use vision to recall specific locations remains unclear. This study describes a visual place learning platform and demonstrate that Drosophila are capable of forming and retaining visual place memories to guide selective navigation. By targeted genetic silencing of small subsets of cells in the Drosophila brain, it was shown that neurons in the ellipsoid body, but not in the mushroom bodies, are necessary for visual place learning. Together, these studies reveal distinct neuroanatomical substrates for spatial versus non-spatial learning, and establish Drosophila as a powerful model for the study of spatial memories (Ofstad, 2011).

    Vision provides the richest source of information about the external world, and most seeing organisms devote enormous neural resources to visual processing. In addition to visual reflexes, many animals use visual features to recall specific routes and locations, such as the placement of a nest or food source. When leaving the nest, bees perform structured 'orientation flights' to learn visual landmarks. If subsequently displaced from their outbound flight, bees take direct paths back to their nests using these learned visual cues. However, it is not clear how insects, which have relatively compact nervous systems, perform these navigational feats. In mammals, the identification of place, grid and head direction cells suggests the existence of a 'cognitive map'. Unfortunately, little is known about the cellular basis of invertebrate visual place learning. To identify the neurons and dissect the circuits that underlie navigation, place learning was studied in Drosophila (Ofstad, 2011).

    To test explicitly for visual place learning in Drosophila, a thermal-visual arena inspired by the Morris water maze and a heat maze, used with cockroaches and crickets, was developed. In the Drosophila place learning assay, flies must find a hidden 'safe' target (that is, a cool tile) in an otherwise unappealing warm environment. Notably, there are no local cues that identify the cool tile. Rather, the only available spatial cues are provided by the surrounding electronic panorama that displays a pattern of evenly spaced bars in three orientations. To assay spatial navigation and visual place memory, fifteen adult flies are introduced in the arena and confined to the array surface by placing a glass disk on top of a 3-mm-high aluminium ring. During the first 5-min trial, nearly all flies (94%) eventually succeed in locating the cool target. In subsequent trials, the cool tile and the corresponding visual panorama are rapidly shifted to a new location (rotated by either 90° clockwise or 90° anticlockwise, chosen at random). Importantly, the target and visual panorama are coupled so that although the absolute position of the cool tile changes, its location relative to the visual panorama remains constant. The results demonstrate that over the course of ten training trials flies improve dramatically in the time they require to locate the cool tile. This improvement is accomplished by taking a shorter, more direct route to the target, without noteworthy changes in the mean walking speed. To ensure that social interactions between flies were not influencing place learning (for example flies following each other to the safe spot), single flies were also trained, and it was found that flies tested individually show equivalent place learning. As would be predicted for bona fide visual place learning, the improvement in place memory is critically dependent on the visual panorama. Flies tested in the dark show no improvement in the time, path length or directness of their routes to the target (Ofstad, 2011).

    To verify that flies are using the spatially distinct features of the visual panorama to direct navigation, flies were also tested using an uncoupled condition whereby the cool tile was still randomly relocated for each trial but the display remained stationary throughout. With this training regime, the visual panorama provides no consistent location cues, but idiothetic and weaker spatial cues such as the distance and local orientation of the arena wall are still available to the flies. The results demonstrate that flies trained with the uncoupled visual panorama show little improvement in the time taken to find the cool tile and no improvement in the directness of their approaches. Thus, spatially relevant visual cues are required for flies to learn the location of the target (Ofstad, 2011).

    As a further test of visual place memory, flies were challenged immediately after training with a probe trial in which the visual landscape is relocated as usual but no cool tile is provided (to determine whether the flies will go to the non-existent safe spot). It was proposed that if the flies learned to locate the cool tile by using the peripheral visual landmarks, then they should bias their searches to the area of the arena where the visual landscape indicates the cool tile should be, even when the target is absent. Indeed, flies preferentially search in the arena quadrant where they have been trained to locate the now 'imaginary' cool tile. In contrast, if flies were trained in the dark or with an uncoupled visual landscape, conditions that contain no specific information about the location of the cool tile, the flies instead searched the arena uniformly during the probe trial. Together, these results demonstrate that fruit flies can learn spatial locations on the basis of distal visual cues and use this memory to guide navigation. By varying the time between the end of a single round of training (ten trials) and testing during a probe trial, it was also shown that flies retain these visual place memories for at least 2h (Ofstad, 2011).

    Next it was considered where spatial memories are processed (or stored) in the Drosophila brain. It was reasoned that specific regions of the fly brain would function as the neuroanatomical substrate for visual place learning, and therefore animals were tested in which different brain areas were selectively inactivated using the GAL4/UAS expression system. In essence, small subsets of neurons were conditionally silenced in adult flies by targeting expression of the inward-rectifying potassium channel Kir2.1 to limit potential side-effects of Kir2.1 expression during development, a temperature-sensitive GAL80ts was used that blocks Kir2.1 expression when flies are reared at 18°C but allows expression when the temperature is raised to 30°C before testing. GAL4 driver lines were selected for expression in two areas: the mushroom bodies and the central complex. The mushroom bodies have been the subject of extensive studies of learning and memory in Drosophila , and have been shown to be essential for associative olfactory conditioning but not for some other forms of learning, such as tactile, motor and non-visually guided place learning. The central complex is thought to be a site of orientation behaviour, multisensory integration and other 'high-order' processes. In some social insects, the mushroom bodies have been implicated in visual place learning, and in the cockroach bilateral surgical lesions to these structures abolish spatial learning. However, no evidence was seen for involvement of the mushroom bodies in the assay. In fact, silencing mushroom body intrinsic neurons using the GAL4 drivers R9A11, R10B08, R67B04, had no significant effect on the performance of flies in visual place learning. The differing requirement for the mushroom bodies between Drosophila and other species may be explained by the observations that mushroom body inputs in Drosophila are predominantly olfactory. In sharp contrast, silencing subsets of neurons with projections to the central complex ellipsoid body did have a significant effect. Notably, silencing a different subset of ring neurons with line R38H02 leaves visual place learning intact. Thus, specific circuits within the ellipsoid body (but not the entire structure) are necessary for visual place learning (Ofstad, 2011).

    To confirm that silencing the ellipsoid body neurons in lines R15B07 and R28D01 produces a specific impairment in visual place memory, these flies were tested in a series of behavioural paradigms and shown to display normal locomotor, optomotor, thermosensory and visual pattern discrimination behaviours. In addition, it was reasoned that if these flies have a general defect in memory (or in processing thermally driven learned behaviours), then they should show impairment in multiple types of learning (or in using thermal signals to drive learning and memory). Thus, a novel olfactory conditioning paradigm was developed using temperature (rather than electric as the unconditioned stimulus. As expected, silencing the mushroom bodies leads to a total loss of odour learning. In contrast, silencing subsets of neurons in the ellipsoid body has no effect on olfactory learning yet ablates visual place learning. Taken together, these results demonstrate that subsets of cells in the ellipsoid body are specifically required for visual place learning and substantiate the presence of distinct neuroanatomical substrates for visually guided spatial (place) versus non-spatial (olfactory) learning in Drosophila (Ofstad, 2011).

    Mammals probably use place, grid and head direction cells to solve and perform navigational tasks. The tight correlation between place cell activity and an animal's position in space has established the hippocampus as the substrate for a cognitive map. This map is probably informed by head direction cells (indicating an animal's orientation) and grid cells that tile the surrounding environment and could support path integration. Although it is not known whether there are direct correlates to these cells in flies, invertebrates are capable of solving similarly challenging navigational feats and do so using significantly smaller brains. Indeed, flies are able to use idiothetic cues, and path integration, to aid navigation. The current studies demonstrate that Drosophila can learn and recall spatial locations in a complex visual arena and do so with remarkable efficacy (Ofstad, 2011).

    It was also shown that subsets of neurons in the fly brain (ring neurons of the ellipsoid body) are critical for visual place learning, probably by implementing, storing, or reading spatial information. Strikingly, flies in which ellipsoid body neurons were silenced have a basic 'circling' search routine that is reminiscent of the behaviour displayed by rats with hippocampal lesions. Imaging of neuronal activity in the fly brain while the animal is executing a navigation task should help further define the role of the central complex, and ellipsoid body neurons in particular, in spatial memory (for example in a head-fixed preparation with a virtual-reality arena. Ultimately, elucidating the cellular basis for place learning in Drosophila will help uncover fundamental principles in the organization and implementation of spatial memories in general (Ofstad, 2011).

    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).

    A subset of dopamine neurons signals reward for odour memory in Drosophila

    Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. This study shows that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. It is therefore proposed that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. These results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs (Liu, 2012).

    Reward is positive reinforcement and drives the formation of appetitive associative memory. In insects, octopamine was shown to be involved in reward, whereas specific sets of dopamine neurons were identified to mediate aversive reinforcement. Recent studies in Drosophila suggest that dopamine in the MBs is involved in appetitive odour memory, but the specific role of dopamine and the underlying circuit are unclear (Liu, 2012).

    To examine whether the activation of dopamine neurons can substitute for a rewarding stimulus in the formation of an appetitive odour memory, the expression of a thermosensitive cation channel dTRPA1 was targeted to different, but overlapping sets of, dopamine neurons by using two GAL4 drivers, TH-GAL4 and DDC-GAL4. Activation of dTRPA1 in DDC-GAL4 flies during the presentation of an odour resulted in a weak appetitive memory, but robust aversive memory in TH-GAL4 flies. The same activation on starvation induced a much greater appetitive memory in DDC-GAL4/UAS-dTrpA1 flies. Activation of dTRPA1 that was not paired with an odour did not induce appetitive memory. Thermo-activation with the driver HL9-GAL4, a variant of DDC-GAL4, induced similar appetitive memory. Furthermore, TH-GAL80 did not significantly suppress induced memory in DDC-GAL4/UAS-dTrpA1 flies, suggesting that the neurons labelled in DDC-GAL4 but not in TH-GAL4 flies are responsible for signalling reward. As in appetitive memory with sugar, a single thermo-activation using DDC-GAL4 induced persistent appetitive memory, which lasted for up to 24h (Liu, 2012).

    To address when starvation is required for the dTRPA1-induced memory performance, examined the effect of changing motivational states was examined before either training or test by a brief feeding. Appetitive memory was induced on thermo-activation despite feeding before training. If applied before the test, feeding fully suppressed the behavioural expression of 12-h memories. These results suggest that starvation is required for the retrieval, but not the acquisition, of appetitive memory induced by thermo-activation (Liu, 2012).

    To explore the role of DDC-GAL4-labelled neurons in mediating the sugar reward, the output of these neurons was blocked using Shits1, which inhibits neuronal output at high temperature. Unlike another known type of dopamine neurons that restricts appetitive memory retrieval, blocking the DDC-GAL4-labelled neurons did not release memory expression in fed flies. Instead, the blockade impaired the acquisition, but not the expression, of the sugar-induced memory. Neither memory performance at the permissive temperature nor sugar preference at the restrictive temperature was impaired (Liu, 2012).

    Attempts were made to identify the cells responsible for reward processing. DDC-GAL4 heavily labels the PAM cluster neurons, whereas this cluster is sparsely labelled by TH-GAL4. For selective manipulation of the PAM cluster neurons, a collection of GAL4 driver lines was screened, and R58E02-GAL4 was identified. This driver strongly labels the PAM cluster neurons and glial cells in the optic lobes with little expression elsewhere. Arbours of the PAM neurons in the MBs are largely localized to the medial lobes. The enhancer of R58E02-GAL4 is derived from the first intron of the Drosophila dopamine transporter gene. Consistently, the PAM neurons labelled in R58E02-GAL4 as well as in DDC-GAL4 flies are dopamine immunoreactive with no detectable serotonin labelling. Thermo-activation of the PAM neurons with the use of R58E02-GAL4 induced robust appetitive odour memory in starved flies, whereas the activation itself did not cause any obvious reflexive appetitive behaviour (Liu, 2012).

    DDC-GAL4 labels many neurons outside the PAM cluster, including those projecting to the s ganglion, where sweet taste neurons terminate. To address the contribution of the non-PAM cells in DDC-GAL4 flies, R58E02-GAL80, a GAL80 line using the same enhancer integrated at the same genomic location as in R58E02-GAL4, was generated. Combination of R58E02-GAL80 with DDC-GAL4 suppressed transgene expression in most PAM neurons in DDC-GAL4 flies. Thermo-activation with DDC-GAL4/R58E02-GAL80 did not induce appetitive memory, demonstrating the importance of PAM neurons in reward signalling (Liu, 2012).

    A transient Shits1 block of the PAM neurons by R58E02-GAL4 impaired the acquisition, but not the expression, of sugar-induced memory. Furthermore, blocking the PAM neurons did not impair the reflexive choice of sugar. Consistently, R58E02-GAL80 rescued the memory impairment of DDC-GAL4/UAS-shits1 flies. Thus, the PAM neurons are necessary and sufficient for signalling the sugar reward (Liu, 2012).

    Expression of a presynaptic marker using R58E02-GAL4 demonstrated that input and output sites of the PAM neurons are highly segregated, with presynaptic terminals localized predominantly in the MBs. To address whether the signal from the PAM neurons is mediated by dopamine receptors, these neurons were activated in the background of dumb2, a mutant for the dDA1 gene (also known as DopR), which encodes a D1-type dopamine receptor. The previously reported role of dDA1 in the Kenyon cells of the MBs for sugar-induced appetitive memory was confirmed. Because it was hoped to use a GAL4 driver to express dDA1 in Kenyon cells simultaneously with dTRPA1 in the PAM neurons, a LexA driver R58E02-LexA::p65 was generated. It recapitulated the expression pattern in R58E02-GAL4 and was able to induce appetitive memory using LexAop2-dTrpA1. Activation of the PAM neurons failed to induce marked appetitive memory in flies lacking dDA1. Driving wild-type dDA1 expression in α/β and γ Kenyon cells by using the driver MB247-GAL4 restored appetitive memory in R58E02-LexA/LexAop2-dTrpA1 flies. These results indicate the importance of dopamine signalling in the MBs for reward processing, but do not exclude a role for other possible co-transmitters released by the PAM neurons (Liu, 2012).

    MB-M3 neurons in the PAM cluster have been identified as important for aversive memory formatio. Both MB-M3 and the reward-signalling PAM neurons were labelled in the same brain, and no overlap was found. This highlights the functional heterogeneity of individual cell types in the PAM cluster (Liu, 2012).

    Similarly, different populations of dopamine neurons were made that signal appetitive and aversive reinforcement visible by using R58E02-LexA and TH-GAL4, respectively, and the distribution of their projections in the MBs was examined. The terminals of the PAM and protocerebral posterior lateral (PPL)1 clusters are largely non-overlapping in the MBs and together cover the entire lobes despite the simultaneous expression of R58E02-LexA and TH-GAL4 in a few PAM cluster neurons. Thus, axonal compartments of Kenyon cells are targeted by functionally different dopamine neurons (Liu, 2012).

    Given the importance of octopamine signalling in reward processing, the PAM cluster neurons were activated in TβH mutants, which lack octopamine. No marked effect of TβH on appetitive memory induced by activation of the PAM neurons was found, indicating that the PAM neurons act in parallel with or downstream of, but not upstream of, octopamine signalling. Consistently, double labelling of the octopamine and PAM cluster dopamine neurons revealed potential direct contacts of these arbours in the spur of the γ lobe and protocerebral regions, where the putative input and output sites of the PAM and octopamine neurons, respectively, are located. This suggests that octopamine may regulate reward processing by directly modulating the activity of the PAM cluster neurons (Liu, 2012).

    To test whether the PAM neurons respond to the sugar reward, in vivo calcium imaging was performed in starved flies expressing the fluorescent calcium reporter GCaMP3. A gustatory stimulation protocol was devised with the unrestrained proboscis that enabled confocal imaging of the PAM terminals in the MBs. Sugar ingestion caused stronger calcium responses than water or a bitter caffeine solution. It was found that the calcium response of the PAM neurons on stimulation with sugar was greatly reduced when flies were fed. Flies can sense sweet taste with their tarsi, but stimulating tarsi with sugar barely activated the PAM neurons, suggesting that sweet substances need to be ingested to trigger the reward signal (Liu, 2012).

    These data suggest the existence of a reward circuit in which the PAM neurons integrate gustatory reward and other relevant regulatory inputs, and then convey the summed positive value signal to specific subdomains of the MBs. The MB lobes can be anatomically divided into 35 subdomains that are defined by specific combinations of intrinsic and extrinsic neurons. Distinct sets of dopamine neurons may provide functionally independent local circuits within the MBs, potentially allowing appetitive and aversive modulation of the same odour. The PAM neurons may drive positive associative modulation of concomitant olfactory signals of the Kenyon cells. The dual processing of appetitive and aversive stimuli may be a conserved function of dopamine, highlighting the physiological pleiotropy of a neurotransmitter (Liu, 2012).

    dCREB2-mediated enhancement of memory formation

    CREB-responsive transcription has an important role in adaptive responses in all cells and tissue. In the nervous system, it has an essential and well established role in long-term memory formation throughout a diverse set of organisms. Activation of this transcription factor correlates with long-term memory formation and disruption of its activity interferes with this process. Most convincingly, augmenting CREB activity in a number of different systems enhances memory formation. In Drosophila, a sequence rearrangement in the original transgene used to enhance memory formation has been a source of confusion. This rearrangement prematurely terminates translation of the full-length protein, leaving the identity of the 'enhancing molecule' unclear. This report shows that a naturally occurring, downstream, in-frame initiation codon is used to make a dCREB2 protein off of both transgenic and chromosomal substrates. This protein is a transcriptional activator and is responsible for memory enhancement. A number of parameters can affect enhancement, including the short-lived activity of the activator protein, and the time-of-day when induction and behavioral training occur. The results reaffirm that overexpression of a dCREB2 activator can enhance memory formation and illustrate the complexity of this behavioral enhancement (Tubon, 2013).

    This report has shown that a 28 kDa protein initiates from the internal ATG2 codon, that it functions as a CRE-dependent transcriptional activator both in vitro and in vivo, and is responsible for the original report of memory enhancement. Although ATG2 is infrequently used, and the resulting protein is expressed at low levels, its existence has been shown using multiple antibodies and different two-step enrichments (EMSA supershifts and Western identification of proteins on EMSA complexes) (Tubon, 2013).

    he ATG2 codon is also used on endogenous dCREB2-encoded mRNAs, since all of the sequenced dCREB2 cDNAs contain ATG1 and ATG2 on the same molecule. Interestingly, internal translation initiation is also used on both of the mammalian CREM and CREB genes. 'Intronic' or internal ATGs can become positioned to be the first start codons through alternative promoter usage and alternative splicing. The mammalian CREB β isoform is a minority species that becomes upregulated upon deletion of the α and Δ isoforms. This study has not caracterized the transcriptional regulation of the dCREB2 gene, so it is possible that ATG2 is the first initiation codon on a minor, currently uncharacterized, dCREB2 transcript (Tubon, 2013).

    A number of related issues have complicated molecular analysis of dCREB2-encoded protein isoforms, and are likely to be relevant in the characterization of these proteins in all species. First, the number and variety of posttranslational modifications that occur on dCREB2-encoded proteins is large. The KID region contains up to 7 phosphorylation sites, and other modifications, including O-GlcNac glycosylation, SUMOylation, ubiquitylation, and cysteine oxidation and/or nitrosylation, occur elsewhere on CREB proteins. These posttranslational modifications can dramatically affect the apparent mobility of protein species, and make it difficult to determine whether Western blots that contain many bands are due to a nonspecific or specific recognition of dCREB2-encoded proteins. A related observation is that these modifications can alter the binding affinity of many of the antibodie, suggesting that any given antibody reports a specialized subpool of protein. Finally, the blocker (40 kDa doublet) and activator (22-35 kDa cluster) species seem to be differentially modified, further complicating detailed analysis. It is likely that combinations of modifications are used to regulate the complex subcellular localization and activity of dCREB2 protein isoforms (Tubon, 2013).

    Various parameters contribute to the inconsistency of memory enhancement. The expression of the 28 kDa protein off of the original 572 transgene is low but detectable. However, this modest level of expression is not responsible for inconsistent enhancement of olfactory avoidance memory, since the 807 transgenic fly (which has consistently higher levels of expression) also sporadically enhances memory formation. Instead, the limited duration of dCREB2-mediated transcriptional activation can place serious timing constraints on the requisite interval between transgene induction and behavioral training (the temporal window). A second temporal parameter is the time-of-day when induction and behavioral training occur. There is a growing awareness that the time-of-day-of training can affect memory formation, and this literature highlights the importance of circadian/sleep-related physiological processes and their relationship with the neuroanatomy and molecular machinery of memory formation. Careful control of these different timing issues greatly increases the reproducibility of behavioral enhancement using the olfactory avoidance assay. The consistent enhancement of the courtship behavior reinforces the original observation that the 28 kDa protein can enhance memory formation (Tubon, 2013).

    Why does 807 enhance memory of courtship suppression reliably, but affects memory of olfactory avoidance less consistently? Comparing two diverse behavioral paradigms is difficult, since there are many parameters that differ. However, this type of approach is necessary, and will be useful. Another behavioral paradigm was developed using conditioned place preference. In the place preference behavioral assay, the 807 transgene enhances memory formation consistently, reinforcing the conclusion that the 28 kDa protein can have important effects on memory formation. Current experiments are directed at determining what behavioral factor(s) differ between courtship suppression and place preference (where consistent enhancement is seen) and olfactory avoidance (where enhancement is less consistent). One possibility is that enhancement in flies specifically requires a 'behavioral state' that is difficult to control experimentally, and which can be epistatic to the other parameters such as expression levels, activity windows, and the time-of-day of training. This behavioral state appears to be an 'all-or-nothing' group effect, with all of the flies in a given experiment affected similarly (Tubon, 2013).

    Recent work using acute interventions in mice and other systems has shown that increasing CREB activity increases the intrinsic excitability of neurons, while interfering with CREB activity has the opposite effect (see for example Liu, 2011 and Suzuki, 2011). The CREB-dependent increase in excitability is correlated with memory enhancement, and vice versa. If dCREB2 enhances memory formation partially through affecting the excitability of relevant neurons, then the 'excitable state' of those neurons at the time of training might determine whether additional dCREB2 protein has enhancing potential or not (Benito, 2010). Since excitability is saturable, there are two simple outcomes, depending upon the state of the neurons at the time of training. If neurons are more quiescent, dCREB2 induction can increase excitability, and enhancement will occur in response to training (relative to equally quiescent neurons that just receive training). However, if the neurons are already excitable at the time of training, then extra dCREB2 will not have any effect, since excitability is saturable. The pretraining handling and housing of flies differs between various behaviors, and is somewhat variable even with the same behavior. These parameters could affect the baseline excitability of the flies, and indirectly affect enhancement. The behavioral data are consistent with this view, since enhancement usually becomes significant when the control fly population has lower memory scores, rather than the experimental population having higher memory scores. The effect of the time-of-day on enhancement also is consistent with this general hypothesis, since excitability is known to vary across the circadian cycle, at least for certain neurons. This possibility and its relevance has important implications for the role that dCREB2 plays in memory formation are currently being tested in a non-transgenic fly (Tubon, 2013).

    Time of day influences memory formation and dCREB2 proteins in Drosophila

    Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. This study shows that the time-of-day (TOD) of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation. The results demonstrate notable correlations between the TOD behavioral effects and the circadian profile of dCREB2 proteins. At ZT = 20, there is a significant depression in memory formation, an event which coincides with apparent increases in blocker-related species clearly visible on the Western blots. At ZT = 16, a significant increase was measured in performance. This time point correlates with the end of a window (ZT = 13-15) when nuclear levels of the activator are elevated. Based on these relationships, it is hypothesized that the dynamics of dCREB2 protein levels contribute to the TOD effects on memory formation (Fropf, 2014).

    A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes

    The ability to integrate experiential information and recall it in the form of memory is observed in a wide range of taxa, and is a hallmark of highly derived nervous systems. Storage of past experiences is critical for adaptive behaviors that anticipate both adverse and positive environmental factors. The process of memory formation and consolidation involve many synchronized biological events including gene transcription, protein modification, and intracellular trafficking: However, many of these molecular mechanisms remain illusive. With Drosophila as a model system this study used a nonassociative memory paradigm and a systems level approach to uncover novel transcriptional patterns. RNA sequencing of Drosophila heads during and after memory formation identified a number of novel memory genes. Tracking the dynamic expression of these genes over time revealed complex gene networks involved in long term memory. In particular, this study focuses on two functional gene clusters of signal peptides and proteases. Bioinformatics network analysis and prediction in combination with high-throughput RNA sequencing identified previously unknown memory genes, which when genetically knocked down resulted in behaviorally validated memory defects (Bozler, 2017).

    Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

    The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. Using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, this study demonstrates that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). A dual regulatory role of FSK on GABAergic transmission was observed, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, it was shown that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short-term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, this study suggests that alterations in cAMP-mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning (Ganguly, 2013).

    Ca2+/CaM dependent adenylate cyclase (AC) produces cAMP and is also known to function as a co-incidence detector during learning in both Drosophila and Aplysia. In addition, AC-dependent cAMP activation changes the strength of Drosophila excitatory synapses which may be the cellular mechanism underlying learning and memory. Although inhibitory synaptic transmission is equally important for proper neuronal communication, the effects of cAMP at the inhibitory GABAergic synapses have remained unexplored. This study shows that forskolin (FSK), an activator of cAMP, suppresses the frequency of inhibitory GABAergic IPSCs in Drosophila primary neuronal cultures. A concentration dependent effect of FSK on GABAergic IPSCs was observed in the same physiological range as described in recent imaging studies in intact fly brains. Further cAMP was shown to decrease GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism (Ganguly, 2013).

    Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive., 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).

    GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).

    There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).

    Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).

    Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).

    The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).

    Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).

    Additive expression of consolidated memory through Drosophila mushroom body subsets

    Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α'β', and γ neurons. It has been previously demonstrated that two parallel pathways mediate ARM consolidation: the serotonergic dorsal paired medial (DPM)-αβ neurons and the octopaminergic anterior paired lateral (APL)-α'β' neurons. This study shows that blocking the output of αβ neurons and that of α'β' neurons each impairs ARM retrieval, and blocking both simultaneously has an additive effect. Knockdown of radish and octβ2R in αβ and α'β' neurons, respectively, impairs ARM. A combinatorial assay of radish mutant background rsh1 and neurotransmission blockade confirms that ARM retrieved from α'β' neuron output is independent of radish. The MB output neurons MBON-β2β'2a and MBON-β'2mp were identified as the MB output neurons downstream of αβ and α'β' neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, α'β' neurons can be functionally subdivided into α'β'm neurons required for ARM retrieval, and α'β'ap neurons required for ARM consolidation. These data demonstrate that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval (Yang, 2016).

    The key finding in this study is the identification of two parallel neural pathways that additively express 3-h aversive ARM through Drosophila MB αβ and α'β' neurons. After training, Radish in MB αβ neurons and octopamine signaling in α'β' neurons independently consolidate ARM, which is additively retrieved by αβ-MBON-β2β'2a and α'β'm-MBON-β'2mp circuits for memory expression. Five lines of evidence support this scenario. First, the output from αβ or α'β' neurons is required for ARM retrieval, and the effect of blocking αβ output and that of blocking α'β' output during retrieval are additive. Second, knockdown of radish in αβ neurons, but not in α'β' neurons, impaired ARM, while knockdown of octβ2R in α'β' neurons further impaired the residual ARM in rsh1 mutant flies. Third, blocking output from α'β' neurons, but not from αβ neurons, during retrieval further impaired the residual ARM in rsh1 mutant flies. Forth, glutamatergic output from neurons downstream of the αβ or α'β' neurons, i.e., MBON-β2β'2a or MBON-β'2mp neurons, is required for ARM retrieval, and the effects of knockdown of VGlut are additive. Finally, output from α'β'm neurons, but not α'β'ap neurons, is required for ARM retrieval, consistent with the dendritic distribution of MBON-β'2mp neurons (Yang, 2016).

    The parallel pathways for 3-h ARM expression were spatially defined by the requirements of neurotransmission from two sets of circuits during retrieval, the αβ-MBON-β2β'2a neurons and the α'β'm-MBON-β'2mp neurons. In addition, blocking neurotransmission from αβ or α'β' neurons during retrieval reduced ARM expression by about 50% whereas simultaneous blockade produced an additive effect that completely abolished ARM expression. Similar additive effects were repeatedly observed in experiments that utilize manipulations in both pathways: an rsh1 mutant background plus octβ2R RNAi knockdown or plus retrieval blockade in α'β' neurons and knockdown of VGlut in MBON-β2β'2a plus MBON-β'2mp neurons. Thus, total four lines of evidence support the additive expression of 3-h ARM (Yang, 2016).

    The parallel pathways for 3-h ARM expression shown in this study differ from the degenerate parallel pathways for the stomatogastric ganglion of the crab or CO2 avoidance in the fly, as the latter enable mechanisms by which the network output can be switched between states. In the current study, the two parallel neural pathways additively contribute to the expression of 3-h ARM. The nature of the ARM parallel pathways may be similar to that for cold avoidance behavior in the fly, where parallel pathways in the β' and β circuits additively contribute but only the β circuit allows age-dependent alterations for potential benefits against aging (Shih, 2015). Considering the robustness of ARM through the course of senescence, it's unlikely to be age-dependent alterations in ARM system (Yang, 2016).

    In studies of Drosophila neurobiology, C305a-GAL4 is a common GAL4 line for α'β' neurons. In this study, by examining three different zoom-in sections of the MB lobes and counting the cells, the following GAL4 lines expressing in α'β' neurons were extensively characterized: VT30604-GAL4 and VT57244-GAL4, which cover most α'β'ap and α'β'm neurons; VT37861-GAL4 and VT50658-GAL4, which cover α'β'ap neurons; and R42D07-GAL4 and R26E01-GAL4, which cover most α'β'm neurons. In contrast, C305a-GAL4 sporadically expresses in about half as many MB neurons as VT30604-GAL4 or VT57244-GAL4 does. Although covering both subsets of α'β' neurons, the expression pattern of C305a-GAL4 in α'β'm neurons is too few and/or weak to lead to a perturbation of synaptic transmission. This is shown by the data that retrieval of 3-h ARM was disrupted by shibire manipulation using all-α'β' neurons driver or α'β'm-specific driver, but neither α'β'ap-specific driver nor C305a-GAL4 for 3-h memory. Note that the GFP signals were acquired from flies carrying two copies of 5XUAS-mCD8::GFP reporter and without any immunostaining-mediated amplification. With the assistance of immunostaining and/or advanced reporter such as increasing copy number of UAS or incorporating a small intron to boost expression, some studies have shown appreciable GFP signal in most α'β' neurons. Given that shibire-mediated neurotransmission blockade and RNAi-mediated knockdown require high enough expression level, the imaging method adopted in this study can faithfully reflect the regions that were effectively manipulated in these behavioral assays. Regarding the pervasive use of C305a-GAL4 for shibire or RNAi manipulation, some functional studies of α'β' neurons might need to be carefully revisited. This study showed, by close examination and cell counting, that VT30604-GAL4, VT37861-GAL4, and R42D07-GAL4 are useful GAL4 lines to study α'β', α'β'ap, and α'β'm neurons, respectively, especially when split-GAL4 lines that span the second and third chromosomes are not genetically feasible (Shih, 2015).

    ARM was thought to be diminished in radish mutant flies, in which a truncated RADISH is expressed. It's noteworthy that radish mutants still show a residual 3-h ARM with a PI of roughly 10, which is equal to the 3-h ARM score in wild-type flies fed with an inhibitor of serotonin synthesis to hinder the serotonergic DPM neurotransmission. Interestingly, feeding radish mutant flies with the drug didn't make the 3-h memory score worse, which has already implied that RADISH mediates the consolidation of ARM in the serotonergic DPM-αβ neurons circuit. Indeed, in this study advantage was taken of RNAi-mediated knockdown to identify αβ neurons with RADISH-mediated ARM consolidation. However, only the output from αβs neurons among three subsets of αβ neurons is required for aversive memory retrieval. Whether the αβs neurons are the only aversive ARM substrate of RADISH remains to be identified (Yang, 2016).

    APL and DPM neurons are two pairs of modulatory neurons broadly innervating the ipsilateral MB, although the DPM neuron's fiber is lacking in the posterior part of pedunculus and the calyx. Broad, extensive fiber and non-spiking feature allow these two pairs of neurons to have multiple functional roles through different types of neurotransmission. The APL neuron has been shown to receive odor information from the MB neurons and provide GABAergic feedback inhibition as the Drosophila equivalent of a group of the honeybee GABAergic feedback neurons. This feedback inhibition has been proposed to maintain sparse, decorrelated odor coding by suppressing the neuronal activity of MB neurons, which can be somewhat linked to the mutual suppression relation with conditioned odor and the facilitation of reversal learning. Interestingly, Pitman (2011) proposed that the feedback inhibition from APL neurons sustains the labile appetitive ASM based on shibire manipulation. Since shibire manipulation can impact small vesicle release, and APL neurons have been demonstrated to co-release at least GABA and octopamine, it might worth conducting GABA-specific manipulation in APL neurons to confirm the role in appetitive ASM. For aversive olfactory memory, acute RNAi-mediated knockdown of Glutamic acid decarboxylase in APL neurons had no effect on 3-h memory. Instead, the octopamine synthesis enzyme mutant, TβhnM18, knockdown of Tβh in APL neurons, the octopamine receptor mutant, PBac{WH}octβ2Rf05679, and knockdown of octβ2R in α'β' neurons all phenocopied the 3-h ARM impairment caused by shibire-mediated neurotransmission blockade in APL neurons. Together with the serotonergic DPM-αβ neurons circuit , a model that is favored that two sets of triple-layered parallel circuits, octopaminergic APL-α'β'-MBON-β'2mp and serotonergic DPM-αβ-MBON-β2β'2a, additively contribute to 3-h aversive ARM (Yang, 2016).

    Although the data showed that 3-h ARM consolidation requires recurrent output from α'β'ap neurons but not from α'β'm neurons, RNAi-mediated knockdown of octβ2R in α'β'ap or α'β'm neurons impaired ARM, suggesting that Octβ2R functions for normal ARM expression in the entire population of α'β' neurons. On the other hand, neuronal activity during memory consolidation is naturally more quiescent than that during memory retrieval, and the shibire-mediated neurotransmission blockade requires an exhaustion of already-docked vesicles. Together with the unfavorable performance for experiments blocking the output from α'β'm neurons during consolidation, the possibility cannot be excluded that output from α'β'm neurons is also required for ARM during consolidation. Alternatively, octopamine signaling may also be involved in ARM retrieval (Yang, 2016).

    Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

    Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs (Hirano, 2016).

    This study has identified Bx and Smr as LTM maintenance genes and has characterize a shift in transcription between CREB/CRTC-dependent maintenance (1-4 days) to Bx-dependent maintenance (4-7 days). In addition, a biological consequence of this shift was identified in defining a time window during which LTM can be modified, β-Spec was identified as being required for memory extinction (Hirano, 2016).

    LTM maintenance mechanisms change dynamically during storage. In particular, CRTC, which is not required during memory formation, becomes necessary during 4-day LTM maintenance and then becomes dispensable again. Consistent with this, CRTC translocates from the cytoplasm to the nucleus of MB neurons during 4-day LTM maintenance and returns to the cytoplasm within 7 days. On the other hand, Bx expression is increased at both phases, suggesting that transcriptional regulation of memory maintenance genes may change between these two phases. Supporting this idea, it was found that Bx expression requires CRTC during 4-day LTM maintenance but becomes independent of CRTC 7 days after training. It is proposed that CREB/CRTC activity induces Bx expression, which subsequently activates a feedback loop where Bx maintains its own expression and that of other memory maintenance genes (Hirano, 2016).

    Although it is proposed that the shifts in transcriptional regulation that were observed occur temporally in the same cells, the possibility cannot be discounted that LTM lasting 7 days is maintained in different cells from LTM lasting 4 days. MB Kenyon cells can be separated into different cell types, which exert differential effects on learning, short-term memory and LTM. Thus, it is possible that LTM itself consists of different types of memory that can be separated anatomically. In this case, CRTC in one cell type may exert non-direct effects on another cell type to activate downstream genes including Bx and Smr. However, as that CRTC binds to the Bx gene locus to promote Bx expression and both CRTC and Bx are required in the same α/β subtype of Kenyon cells, it is likely that the shift from CRTC-dependent to Bx-dependent transcription occurs within the α/β neurons (Hirano, 2016).

    Currently, it is proposed that the alterations in histone acetylation and transcription that were uncovered are required for memory maintenance. However, it is noted that decreases in memory after formation could be caused by defects in retrieval and maintenance. Thus, it remains formally possible that the epigenetic and transcriptional changes reported in this study are required for recall, but not maintenance. However, this is unlikely, as inhibition of CRTC from 4 to 7 days after memory formation does not affect 7 day memory, whereas inhibition from 1 to 4 days does. This suggests that at least one function of CRTC is to maintain memory for later recall (Hirano, 2016).

    Consistent with a previous study in mice, which suggests distinct transcriptional regulations in LTM formation and maintenance (Halder, 2016), the data indicate that memory formation and maintenance are distinct processes. Although the HAT, CBP, is required for formation but dispensable for maintenance, other HATs, GCN5 and Tip60, are required for maintenance but dispensable for formation. Through ChIP-seq analyses, those downstream genes, Smr and Bx, were identified as LTM maintenance genes and these are not required for LTM formation. Collectively, these results suggest differential requirements of histone modifications between LTM formation and maintenance. Although other histone modifiers besides GCN5 and Tip60 were identified in the screen, knockdown of these histone modifiers did not affect LTM maintenance. There are ~50 histone modifiers encoded in the fly genome, raising the possibility that the lack of phenotype in some knockdown lines is due to compensation by other modifiers (Hirano, 2016).

    The results indicate some correlation of increase in CRTC binding with histone acetylation and gene expression. Interestingly, DNA methylation shows higher correlation to gene expression in comparison with histone acetylation in mice. Notably, flies lack several key DNA methylases and lack detectable DNA methylation patterns. Hence, histone acetylation rather than DNA methylation may have a higher correlation with transcription in flies. Reduction in histone acetylation was detected, overlapping with increase in CRTC binding. Those reductions could be due to CRTC interacting with a repressor isoform of CREB, CREB2b or other transcriptional repressor that binds near CREB/CRTC sites. These interactions would decrease histone acetylation and gene expression, and may be related to LTM maintenance. Although this study focused on the upregulation of gene expression through CREB/CRTC, downregulation of gene expression by transcriptional repressors may also be important in understanding the transcriptional regulation in LTM maintenance. The results demonstrate the importance of HATs for LTM maintenance; however, the data do not conclude that histone acetylation is a determinant for gene expression, but rather it might be a passive mark of gene expression. HATs also target non-histone proteins and also interact with various proteins, both of which could support gene expression in LTM maintenance (Hirano, 2016).

    Similar to traumatic fear memory in rodents, this study found that aversive LTM in flies can be extinguished by exposing them to an extinction protocol specifically during 4-day LTM maintenance. These observations suggest the time-limited activation of molecules that allows LTM extinction only during the early storage. Supporting this concept, it was found that CRTC is activated during the extinguishable phase of LTM maintenance and prolonging CRTC activity extends the time window for extinction. Thus, CRTC is the time-limited activated factor determining the time window for LTM extinction in flies. In cultured rodent hippocampal neurons, CRTC nuclear translocation is not sustained, suggesting that other transcription factors may function in mammals to restrict LTM extinction (Hirano, 2016).

    This work demonstrates that LTM formation and maintenance are distinct, and involve a shifting array of transcription factors, coactivators and HATs. A key factor in this shift is CRTC, which shows a sustained but time-limited translocation to the nucleus after spaced training. Thus, MB neurons recruit different transcriptional programmes that enable LTM to be formed, maintained and extinguished (Hirano, 2016).

    Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae

    Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

    Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

    Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

    Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

    For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

    In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

    Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

    In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

    KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca2+ activation (Widmann, 2016).

    Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn97 mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn97 mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn97 mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut1, rut2080 and dnc1 mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

    Basic reversal-learning capacity in flies suggests rudiments of complex cognition

    The most basic models of learning are reinforcement learning models (for instance, classical and operant conditioning) that posit a constant learning rate; however many animals change their learning rates with experience. This process is sometimes studied by reversing an existing association between cues and rewards, and measuring the rate of relearning. Augmented reversal-learning, where learning rates increase with practice, can be an important component of behavioral flexibility; and may provide insight into higher cognition. Previous studies of reversal-learning in Drosophila have not measured learning rates, but have tended to focus on measuring gross deficits in reversal-learning, as the ratio of two timepoints. These studies have uncovered a diversity of mechanisms underlying reversal-learning, but natural genetic variation in this trait has yet to be assessed. A reversal-learning regime was conducted on a diverse panel of Drosophila melanogaster genotypes. Highly significant genetic variation was found in their baseline ability to learn. It was also found that they have a consistent, and strong (1.3x), increase in their learning speed with reversal. No evidence was found, however, that there was genetic variation in their ability to increase their learning rates with experience. This may suggest that Drosophila have a hitherto unrecognized ability to integrate acquired information, and improve their decision making; but that their mechanisms for doing so are under strong constraints (Foley, 2017).


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    list of genes

    Zygotically transcribed genes

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