The Interactive Fly
Zygotically transcribed genes
The role of cuticular pheromones in courtship conditioning of Drosophila males
Distinct memory traces for two visual features in the Drosophila brain
Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation
Analysis of a spatial orientation memory in Drosophila
Imaging of an early memory trace in the Drosophila mushroom body
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.
Courtship conditioning is an associative learning paradigm in Drosophila melanogaster, wherein male courtship behavior is modified by experience with unreceptive, previously mated females. While the training experience with mated females involves multiple sensory and behavioral interactions, it is hypothesized that female cuticular hydrocarbons function as a specific chemosensory conditioned stimulus in this learning paradigm. The effects of training with mated females were determined in courtship tests with either wild-type virgin females as courtship targets, or with target flies of different genotypes that express distinct cuticular hydrocarbon (CH) profiles. Results of tests with female targets that lacked the normal CH profile, and with male targets that expressed typically female CH profiles, indicate that components of this CH profile are both necessary and sufficient cues to elicit the effects of conditioning. Results with additional targets indicate that the female-specific 7,11-dienes, which induce naive males to court, are not essential components of the conditioned stimulus. Rather, the learned response is significantly correlated with the levels of 9-pentacosene (9-P), a compound found in both males and females of many Drosophila strains and species. Adding 9-P to target flies showed that it stimulates courting males to attempt to copulate, and confirmed its role as a component of the conditioned stimulus by demonstrating dose-dependent increases in the expression of the learned response. Thus, 9-P can contribute significantly to the conditioned suppression of male courtship toward targets that express this pheromone (Siwicki, 2005).
Based on the reasoning that trained males would exhibit a learned response
only in courtship tests with targets that expressed a conditioned stimulus,
flies with different combinations of putative female aphrodisiac cues were
used as test targets, and assessed for their ability to elicit a learned
response from Canton-S (CS) males. After 1 h of experience courting a previously mated CS
female, the effects of this experience were measured in courtship assays with
different test targets. For example, two variants on the CH profile were flies bearing pGAL4 insert in the desaturase 1 gene on Chromosome III (desat11573) and hs-tra virgin females, which are severely depleted of all CHs. Differences in the extent and duration of conditioned
courtship suppression in tests with different targets were attributed to
differences in the testing conditions, that is, in the combinations of
aphrodisiac and anti-aphrodisiac cues expressed by the test targets (Siwicki, 2005).
Target behavior was eliminated as a source of the observed differences in
male courtship suppression because the targets were immobilized by
decapitation. Nor did the presence of female abdominal anatomy correlate with
a target's ability to elicit a learned response: Targets that were
anatomically female and male elicited strong learned responses, while the
target genotypes that elicited the weakest learned responses
(desat11573 virgins and hs-tra virgins) were
anatomically female.
Therefore, the abdominal anatomy of a courtship target is not an essential
element of the conditioned stimulus. This conclusion also is supported by the
fact that visual stimuli are not required for courtship conditioning (Siwicki, 2005).
The courtship activity [mean cortship index (CI)] of naive males was not well correlated
with the hydrocarbon profiles of the different test targets, confirming the
results of prior studies showing that female CHs are sufficient, but not
necessary, to stimulate naive male courtship. In
particular, naive males courted hydrocarbon-depleted hs-tra virgins
and males depleted of unsaturated CHs as actively as they courted targets with
typically female CH profiles. This is consistent with the view that naive
males respond to a combination of aphrodisiac and anti-aphrodisiac pheromones
on a decapitated target fly. In contrast, the conditioned suppression of
courtship resulting from experience with mated females was closely correlated
with the hydrocarbon profiles of the test targets. Results of testing with
hydrocarbon-depleted female targets indicated that CHs are essential stimuli
for the conditioned suppression of courtship after experience with mated
females. Tests with
feminized males (pGAL4/+; UAS-tra/+) that expressed typically female CH
profiles revealed that this profile is sufficient both to stimulate naive male
courtship and to elicit conditioned suppression of courtship in trained male
subjects. Together,
these results strongly support the hypothesis that components of the female CH
profile function as the conditioned stimulus in courtship conditioning (Siwicki, 2005).
This raises the question of whether specific compounds or the overall
female profile are responsible for the learned response. The data indicate
that a target's ability to elicit a learned response is most strongly
correlated with the levels of a single monoene, 9-pentacosene (9-P).
Specifically, Learning Indexes (LIs) based on tests either 5 min or 60 min after training were significantly correlated with target levels of a single unsaturated
hydrocarbon, 9-pentacosene (9-P). Surprisingly, the LIs were not significantly correlated with levels of any principal compound previously known to possess aphrodisiac properties, that is, the 7,11-dienes or 7-P (Siwicki, 2005).
Interpretation of this result requires consideration of previous evidence
concerning the aphrodisiac properties of individual CHs. Two strategies have
been used to assess the aphrodisiac and anti-aphrodisiac potencies of
individual compounds in the cuticular profile. By applying synthetic compounds
to CH-stripped dummy targets, previous studies have found 7,11-HD to be the
most bioactive, stimulating aggressive male courtship with a threshold of
~100 ng/fly. This was confirmed by genetic
manipulations to produce live targets with distinctive CH profiles; even low levels of 7,11-ND (10-15 ng) strongly
potentiate the aphrodisiac effects of 7,11-HD. Both strategies produced
evidence for the anti-aphrodisiac properties of 7-T and the aphrodisiac
effects of 7-P. Prior evidence regarding the putative aphrodisiac
properties of 9-P is limited to a positive correlation with the frequency of
copulation attempts. When applied to hydrocarbon-stripped targets, 9-P failed to
stimulate male courtship (Siwicki, 2005 and references therein).
In the present study, one clear effect of adding exogenous, pure
(Z)-9-pentacosene to courtship chambers was to increase the fraction of CS
males that attempted to copulate with CS virgin females, and with CH-depleted
virgins of the hs-tra and desat1 genotypes. This result strongly
supports the hypothesis that the main aphrodisiac effect of 9-P is to increase
the likelihood of copulation attempts. Interestingly,
9-P did not increase the mean CI in most of these test groups: Only naive
males tested with desat1 females had a higher mean CI in the presence
of 9-P. This selective effect on the mean CI of naive males with desat1 females, but not trained males, as well as the dose-dependent effects of 9-P on the
Learning Index, clearly establish a role for 9-P as a component of the conditioned stimulus. With most targets, however, 9-P did not stimulate overall courtship activity; rather, it increased the probability for courting males to attempt
copulations. Moreover, because this effect was elicited by the lower dose of
9-P for naive males, and only at the higher dose for trained males, it suggests that a specific effect of training with mated females is to reduce the responsiveness of trained males to the aphrodisiac effects of this pheromone (Siwicki, 2005).
These results indicate a dual role for 9-P: (1) increasing the probability
that courting males will attempt to copulate, and (2) contributing to the
difference between naive and trained males in overall courtship. Yet,
depending on the target genotype, different doses of 9-P induce different
effects on the global amount of courtship (CI) and on the frequency of
copulation attempts. For example, 9-P only increases the CI of naive males
when present at 2 µg on desat1 females, but both 0.2-µg and
2-µg doses tended to increase the probability to attempt copulation when
combined with any of the four targets. This difference can be explained
because the duration of a copulation attempt is very brief compared to the
wing vibration and licking behaviors that make up most of the CI measure. The
selective effect of 9-P on CI with desat1 females suggests that its
effect could be potentiated by other CHs that are present in these targets but
absent in hs-tra females and immature males. At least two relatively
abundant CHs fit this criterion: n-tricosane and
2-methyl-tetracosane. In contrast, it is likely that 9-P has
minimal effects on CIs with CS females because all the CHs necessary to induce
high levels of courtship are already present, and the exogenous 9-P does not
increase the overall sex appeal of these targets. It was also with the three
CH-depleted genotypes, but not with CS females, that dose-dependent effects of
9-P were found on LI5' (LIs for groups tested 5 min after training), a measure of the relative suppression of overall
courtship in trained males, whereas 0.2 µg of 9-P was sufficient to elicit
the maximum effect of training on the probability to attempt copulation. It is
worth noting that the 0.2-µg dose of 9-P (applied to a filter paper under
the target fly) is very similar to the biological dose found on CS females
(50-70 ng). Thus, the results demonstrate that CS males can detect and
respond to physiologically relevant levels of this pheromone (Siwicki, 2005).
It is likely that 9-P is one component of a combination of CHs that
function as the conditioned stimulus. This is supported by the fact that 9-P
was not sufficient to elicit effects of training on courtship of CH-depleted
hs-tra females. It is also supported by
the modest, albeit significant, correlation between the LIs and 9-P levels in
different test targets. When possible synergistic effects of other unsaturated CHs
with 9-P were examined by running multiple regressions, there were no cases in
which the levels of 9-P plus another CH predicted the LI-values significantly
better than did 9-P alone. Thus, the identities of the putative interacting
components of the conditioned stimulus remain to be determined (Siwicki, 2005).
The surprising result of this study is that this particular compound
emerged as a conditioned stimulus. The levels of 7,11-dienes in a courtship
target were not correlated with the expression of the males' learned response, indicating that conditioning does not dramatically alter the potency of these highly abundant, female-specific pheromones. In contrast, 9-P is a relatively minor component of the Canton-S CH profile, and is expressed by both mature males and
females (Siwicki, 2005).
Having identified a role for 9-P as a chemosensory stimulus that is
particularly susceptible to conditioning during courtship of mated females,
the results may shed new light on the question of whether courtship
conditioning ability is relevant to the behavior of males in the wild. While
it is likely to be maladaptive for males to suppress their courtship toward
conspecific virgin females, it has been proposed that courtship
toward mated females might be decreased to a greater degree, thus improving
male selectivity for virgins. Since 9-P is not very specific (it is found in
cuticular extracts of both males and females of several Drosophila
species), it is possible that experience-dependent modification of
the responsiveness to this widely expressed hydrocarbon might provide a male
with the ability to learn to discriminate appropriate from inappropriate
courtship targets. For example, females of Drosophila affinis were
reported to express much higher levels of 9-P than D. melanogaster
females. These two species are sympatric in some areas of North America, and they have been observed courting in interspecific groups gathered on food sources.
These observations suggest that the ability of CS males to learn to be less
responsive to 9-P might allow them to learn to avoid courting D.
affinis females. In an environment where misdirected courtship of
heterospecific females is likely to occur, there would presumably be some
advantage for males that could learn to become less sensitive to particular
pheromones that are more abundant in heterospecific females than in
con-specifics (Siwicki, 2005).
D. melanogaster males also court immature males and females, which
express distinctly different blends of CHs than mature flies. Experience
courting immature males results in habituation to the aphrodisiac effects of
specific 31C monoenes in the immature male CH blend. The CH
profiles of immature males and females are similar to each other in that they
are comprised mostly of longer 27C-37C compounds and contain only trace
amounts of compounds smaller than 27C. It is
difficult to predict how experience courting a mated female, and the
corresponding decrease in aphrodisiac potency of 9-P, might affect a male's
subsequent courtship of immature flies with little or no 9-P and an abundance
of other CHs with unknown behavioral effects. This difficulty is compounded by
the possibility that some longer-chain CHs of immature flies may be detected
and processed through some of the same sensory pathways as mature CH
pheromones, a possibility that is reinforced by recent evidence that many
gustatory receptor neurons express combinations of receptors. At
present, therefore, it is not possible to interpret the effects of
mated-female training on male courtship of immature males and females in terms
of specific modifications of responses to specific CHs (Siwicki, 2005).
The present results suggest an additional level of complexity in the
functional organization of the neural systems that control and modulate male
courtship. Some CHs (7,11-dienes and 7-P) stimulate naive males to court
actively; and others (7-T) inhibit naive courtship, suggesting that there are
at least two distinct gustatory pathways by which contact pheromones influence
courtship control centers. Males repeatedly sample all of these pheromones during
training with mated females, yet the experience-dependent changes in
subsequent courtship behavior are strongly correlated only with a target
level of 9-P. This suggests that the sensorimotor circuits by which
7,11-dienes and 7-P stimulate courtship may be less modifiable than pathways
activated by 9-P. It follows from this hypothesis that these pheromones may be
detected and processed by distinct neural pathways, a prediction that can be
tested by manipulating the genes and cells involved in pheromone detection (Siwicki, 2005).
This study provides direct support for the hypothesis that the
conditioned stimulus in courtship conditioning of D. melanogaster
males is a chemical, rather than behavioral or anatomical, cue provided by the
female. The
results indicate that components of the female cuticular hydrocarbon profile
function as the conditioned stimulus in courtship conditioning. In particular,
the most relevant stimulus is 9-pentacosene, an unsaturated hydrocarbon found
on the cuticle of both males and females. Together with previous evidence
concerning the relative potencies of various cuticular substances at eliciting
male courtship, the present results suggest that naive males respond to a
combination of aphrodisiacs and anti-aphrodisiacs on a target fly, while
conditioned males are less responsive than naives to the aphrodisiac effects
of 9-P. Identifying the sensory pathways responsible for detection of this
chemical conditioned stimulus will allow for a more definitive analysis of the
neural mechanisms responsible for this form of associative learning (Siwicki, 2005).
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 suboesophageal 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 suboesophageal 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).
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).
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).
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).
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