These data indicate that increases in calcium influx in response to presentation of a conditioned odor occur specifically in the αbranch of the α/β MB neurons only after spaced forward conditioning. Neither 1x forward, 5x massed forward conditioning, nor any variation of backward conditioning is sufficient to promote the post-conditioning increase in calcium influx in this axon branch in response to the CS+. The memory trace was detected as a significant increase in G-CaMP fluorescence in response to the CS+ odor from the three-dimensional region of interest of the brain imaged in flies that received spaced forward conditioning compared to the response from the same area in naïve flies or in flies receiving other types of conditioning that were ineffective at producing long-term behavioral memory. Because the comparison was made for a specific region of interest between flies conditioned by various protocols, we believe that the failure to observe a memory trace in the β branch indicates that the physiological changes producing the memory trace in the αbranch do not occur in the β branch. Nevertheless, we cannot rule out the possibility that some unknown technical constraint, possibly related to the orientation of fibers relative to the angle of light detection, obscured the detection of an authentic trace in the β branch. Furthermore, we find that the formation of the cellular memory trace using functional optical cellular imaging is disrupted by feeding flies an inhibitor of protein synthesis, by the absence of the amn gene product, or by expressing a repressor of the transcription factor, Creb, in the α/β MB neurons. Thus, the evidence together indicates that the calcium-based memory trace discovered from these studies is localized in the α/β MB neurons, formed only in one axon branch of these neurons, formed only after spaced forward conditioning, and is dependent on amn+ activity, normal protein synthesis and Creb activity during training.
The strong correlation between the conditions necessary for the formation of the branch-specific cellular memory trace described here and those that underlie long-term behavioral memory provide an extremely strong argument that the newly discovered memory trace may guide behavior long after the conditioning event. Long-term behavioral memory in Drosophila
, like the observed cellular memory trace, is induced by spaced forward conditioning, is disrupted by inhibiting protein synthesis, and requires the activity of dCreb and as shown here, the function of the amn
gene. Moreover, behavioral studies of mutant animals with brain structural defects have hinted at a possible tie between long-term behavioral memory and the vertical lobes of the MB neurons. Pascual and Preat (2001
) reported that long-term memory induced with spaced forward conditioning is abolished in ala
) flies that are missing the α/α ’ branches of the MB neurons but not the β/β ’ branches, although the low penetrance of the ala
mutant weakened this conclusion. Nevertheless, this observation is consistent with the possibility that long-term memory induced by spaced forward training either forms in the α/α ’ branches of the MB neurons or that these branches are required for the circuit-based retrieval of long-term memory formed after spaced forward conditioning. The observation of a long-term cellular memory trace in the αbranch of the α/β MB neurons supports that idea that the memories are formed and stored in this branch of this specific class of MB neuron.
Why is it that a memory trace representing early memories was not discovered during these studies, given that the MBs have been widely implicated in all temporal phases of memory? For instance, ablation of the MBs during development by hydroxyurea poisoning impairs the memory of adult flies immediately after training (de Belle and Heisenberg, 1994
), suggesting the importance of the MBs for short-term memory expression. The expression of a wild-type rutabaga
transgene specifically in the MB of adult flies rescues the memory impairment of rutabaga
mutants that is observed immediately after olfactory conditioning (McGuire et al., 2003
; Mao et al., 2004
), indicating that normal rutabaga
function in the MBs is required for the expression of early memory. Expression of Uas-Shits
specifically in MB neurons impairs olfactory memory retrieval at 0.5, 3, and 24 hr after conditioning (McGuire et al., 2001
; Dubnau et al., 2001
; Isabel et al., 2004
), strongly indicating that early memories either form in the MBs or are expressed through these neurons.
There exist at least two explanations for why we detected a trace that is specific to long-term memories. First, it is possible that memory traces that underlie early memories are formed in neurons other than the α/β MB neurons. For instance, early memories may be subserved by the short-term memory trace detected in the antennal lobes (Yu et al., 2004
) and/or by memory traces that form in other types of MB neurons such as the α’/β’ or γMB neurons. Second, memory traces produced by cellular mechanisms other than increased calcium influx likely exist and these may subserve early memory. In either case, the expression of both short- and long-term memory appears to be via the output synapses of the MB neurons (McGuire et al., 2001
; Dubnau et al., 2001
; Isabel et al., 2004
), irrespective of where various memory traces may form upstream of these synapses within the olfactory nervous system.
Two other discovered olfactory memory traces - the immediate and short-lived antennal lobe PN memory trace and the medium-term memory trace formed in DPM neurons - occur prior to the long-term memory trace that forms in the MB neurons. This raises the general question of whether the various memory traces are dependent upon one another in a time series for their formation. We have performed an initial query into this question by determining whether the long-term MB trace forms in amn
mutants, which fail to form the medium-term memory trace in the DPM neurons (Yu et al., 2005
). As shown by the data, amn
mutants fail to form long-term behavioral memory measured at 24 hr and they fail to form the long-term memory trace in the αbranch as detected in control flies. These results are therefore consistent with the model that the formation of the medium-term trace in DPM processes that appear to innervate the αbranch of the MB neurons is required for formation of the long-term memory trace in these axon branches. Perhaps the increased calcium influx and synaptic release that occurs in response to an odor CS+ after training from the DPM processes innervating the αlobe are responsible for guiding or permitting the formation of the long-term memory trace in the putative DPM targets - the αbranches of the MB neurons. Further tests of issues regarding the interdependence of the various cellular memory traces require the development of new gene promoter systems that are independent of the Gal4:Uas system to permit expressing the neuronal reporters and transgenes that block synaptic transmission, for instance, in different sets of neurons. Although the mechanisms underlying the branch-specific modifications that result in calcium influx in response to the CS+ after spaced forward conditioning remain to be elucidated, the discovery of multiple memory traces that form at different times after training for different durations in various parts of the olfactory nervous system leads to the general proposition that behavioral memory is guided from multiple and discrete memory trace elements rather than from a single, continuously decaying memory trace.