Reward signal in a recurrent circuit drives appetitive long-term memory formation
Environmentally relevant information , such as poison or food , can trigger formation of long-term memory ( LTM ) in a single association event ( Garcia et al . , 1955; Alexander et al . , 1984 ) . Because LTM formation is energetically costly ( Mery and Kawecki , 2005; Placais and Preat , 2013 ) , it is advantageous for animals to refer to surrounding conditions when selecting what information to retain as stable LTM . Appetitive olfactory learning in Drosophila melanogaster provides an opportunity to uncover the mechanisms underlying such integration of information , as a single presentation of nutritious sugar and an odor drives robust short-term memory ( STM ) and LTM , while non-nutritious sugar induces only STM ( Krashes and Waddell , 2008; Colomb et al . , 2009; Burke and Waddell , 2011 ) . In Drosophila , sugar reward is mediated by a group of dopamine neurons called the protocerebral anterior medial ( PAM ) cluster ( Burke et al . , 2012; Liu et al . , 2012 ) . Artificial activation of the PAM cluster neurons in the presence of odor drives robust appetitive STM and LTM formation . Recent studies subdivided the PAM cluster neurons into STM- and LTM-inducing cell types and identified several cell types responsible for LTM formation , including PAM-α1 ( Huetteroth et al . , 2015; Yamagata et al . , 2015 ) . Interestingly , the induced LTM is undetectable immediately after conditioning but gradually develops with time ( Das et al . , 2014; Huetteroth et al . , 2015; Yamagata et al . , 2015 ) . Dopamine reward signals are conveyed by PAM cluster neurons to a brain structure called mushroom body ( MB ) , where the convergence of the reward and the odor is assumed to take place ( Schwaerzel et al . , 2003; Krashes et al . , 2007; Trannoy et al . , 2011; Cervantes-Sandoval et al . , 2013; Huang et al . , 2013 ) . The MB represents odors using a sparse subset of ∼2000 intrinsic neurons , Kenyon cells ( KCs ) ( Turner et al . , 2008; Aso et al . , 2009; Honegger et al . , 2011; Campbell et al . , 2013 ) . KCs send parallel fibers to form lobe structures , where multiple MB-output neuron ( MBON ) types elaborate spatially segregated dendritic arbors ( Aso et al . , 2014a ) . The KC-MBON synapses are presumed to be modified in the course of memory formation by the reinforcing dopamine neurons ( Tomchik and Davis , 2009; Gervasi et al . , 2010; Sejourne et al . , 2011; Cervantes-Sandoval et al . , 2013; Placais et al . , 2013; Boto et al . , 2014 ) . While there has been progress on the molecular and cellular mechanisms underlying LTM formation resulting from repetitive conditioning ( Pagani et al . , 2009; Placais et al . , 2012; Philips et al . , 2013 ) , how a single presentation of a reward can trigger LTM remains to be understood . Here , we identify and behaviorally characterize a recurrent reward circuit that is essential for appetitive LTM formation and consolidation . The recurrent circuit consists of PAM-α1 dopamine neurons and a single MBON type , MBON-α1 . This recurrent dopamine reward circuit may provide insights into a general mechanism for LTM formation .
PAM-α1 delivers reward signals essential for appetitive LTM formation ( Yamagata et al . , 2015 ) . To understand the neuronal circuit that regulates rewards for LTM formation , we first sought to anatomically identify the input neurons to PAM-α1 . The presynaptic terminals of PAM-α1 are localized to the basal compartment of the MB α lobe ( α1 ) ( Liu et al . , 2012 ) , whereas the dendrites stretch horizontally across the superior medial , intermediate , and lateral protocerebra ( SMP , SIP , and SLP , respectively ) ( Aso et al . , 2014a; Yamagata et al . , 2015 ) ( Figure 1A , B , H ) . By segmenting the dendrites of PAM-α1 in confocal images ( Aso et al . , 2014a ) , we queried a database of GAL4 expression patterns ( Jenett et al . , 2012 ) to computationally search for candidate upstream neurons . Strikingly , one of the input candidates we identified was the output neurons from the α1 compartment of the MB ( MBON-α1 ) ( Figure 1C , D ) . Transgenic expression of a presynaptic marker ( Robinson et al . , 2002 ) by MB310C-GAL4 revealed that the presynaptic terminals of MBON-α1 lie in close apposition to the dendrites of PAM-α1 ( Figure 1E , I ) . The terminals of MBON-α1 were immunoreactive for the vesicular glutamate transporter ( Figure 1F ) ( Daniels et al . , 2008 ) but not for markers for GABAergic or cholinergic neurons ( glutamic acid decarboxylase [Jackson et al . , 1990] or choline acetyltransferase [Takagawa and Salvaterra , 1996] , respectively ) ( Figure 1—figure supplement 1 ) , consistent with MBON-α1 being glutamatergic ( Aso et al . , 2014a ) . Differential labeling of the individual MBON-α1 neurons in the same brain ( Nern et al . , 2015 ) revealed that MBON-α1 comprises two cells with very similar morphology ( Figure 1G ) . 10 . 7554/eLife . 10719 . 003Figure 1 . MBON-α1 receives inputs from MB-α1 and projects to the dendrites of PAM-α1 . ( A–D ) Anatomy of PAM-α1 ( A , B ) and MBON-α1 ( C , D ) . Dotted line indicates α/β lobes in the mushroom body ( MB ) ( A , C ) or SMP , SIP , and SLP ( superior medial , intermediate , and lateral protocerebra , respectively ) ( B , D ) . Arrows indicate α1 and arrowheads the SIP and SLP ( A , C ) . PAM-α1 and MBON-α1 are visualized by pJFRC2-10xUAS-mCD8GFP in VK00005 and MB299B-GAL4 ( A , B ) or MB310C-GAL4 ( C , D ) , respectively . ( E ) Presynaptic terminals of MBON-α1 are highly localized in SIP and SLP . MB310C-GAL4 is used to drive a general membrane marker ( green ) and a presynaptic marker ( magenta ) in MBON-α1 . ( F ) Double labeling of the membrane of MBON-α1 ( green ) and anti-vesicular glutamate transporter ( dVGluT , magenta ) ( F1 ) , membrane staining ( F2 ) , and anti-dVGluT staining ( F3 ) . Arrowheads highlight the overlap . ( G ) Two individual MBON-α1 neurons visualized by multi-color flip-out with different colors ( red and cyan ) . ( H , I ) Schematics of PAM-α1 and MBON-α1 , respectively . The α/β lobe of the MB is outlined with light orange . Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 00310 . 7554/eLife . 10719 . 004Figure 1—figure supplement 1 . MBON-α1 is neither GABAergic nor cholinergic . Double labeling of the membrane of MBON-α1 ( green ) and anti-glutamic acid decarboxylase ( GAD1 ) ( A , magenta ) or choline acetyltransferase ( ChAT ) ( B , magenta ) . Arrowheads highlight some of the processes , where no obvious overlaps are observed . Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 004 To precisely determine the relative arrangement of PAM-α1 and MBON-α1 neurites , we performed a double-labeling experiment using R72D01-LexA , which labels PAM-α1 , and the split-GAL4 driver MB310C-GAL4 , which labels MBON-α1 ( Aso et al . , 2014a ) . Confocal images revealed that the processes of MBON-α1 and PAM-α1 substantially intermingled both in the SIP and SLP , and in the α1 compartment of the MB—the input and output sites of PAM-α1 , respectively ( Figure 2A–E , Figure 2—figure supplement 1 , Video 1 ) . To observe these processes in SIP and SLP in even greater detail , we employed a microscope with a super resolution detection system and found bouton-like structures of MBON-α1 attached to the dendrites of PAM-α1 ( Figure 2B–D ) . Furthermore , a GFP reconstitution across synaptic partners ( GRASP ) ( Feinberg et al . , 2008; Gordon and Scott , 2009 ) experiment using R72D01-LexA and MB310C-GAL4 revealed strong GRASP signals in SIP , SLP , and the α1 compartment ( Figure 2F ) . This further supports the close juxtaposition between PAM-α1 and MBON-α1 . 10 . 7554/eLife . 10719 . 005Figure 2 . Processes of PAM-α1 and MBON-α1 intermingle each other . ( A ) Double labeling of PAM-α1 and MBON-α1 . PAM-α1 ( magenta ) and MBON-α1 ( green ) are visualized using R72D01-LexA and MB310C-GAL4 , respectively . ( B–D ) Magnified substack images in SIP and SLP , obtained by a super-resolution detection system . ( E ) Magnified substack image in the α1 compartment in the MB . ( F ) GFP reconstitution across synaptic partner ( GRASP ) signals in SIP , SLP , and MB α1 support the contacts between PAM-α1 and MBON-α1 . Scale bars , 10 µm ( A , B , E , F ) , 1 µm ( C , D ) . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 00510 . 7554/eLife . 10719 . 006Figure 2—figure supplement 1 . Stereotyped projections of PAM-α1 and MBON-α1 . ( A , C ) Two additional examples of the double labeling confirm stereotypy of the projections . ( B , D ) Magnified substacks for the insets in ( A ) and ( C ) , respectively . Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 00610 . 7554/eLife . 10719 . 007Video 1 . Volume-rendered image of processes of PAM-α1 ( magenta ) and MBON-α1 ( green ) in SIP and SLP . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 007 If the MB feedback modulates PAM-α1 , the output of MBON-α1 might be required for the acquisition of LTM of the sugar reward . We examined the requirement of PAM-α1 and MBON-α1 by expressing a temperature-sensitive dominant negative dynamin , Shibirets1 ( Kitamoto , 2001 ) using the split-GAL4 drivers MB299B-GAL4 and MB310C-GAL4 ( Figure 3B , F ) . MB299B-GAL4 has strong expression in PAM-α1 ( Aso et al . , 2014a; Yamagata et al . , 2015 ) ( Figure 3B ) . Consistent with our previous results ( Yamagata et al . , 2015 ) , the blockade of PAM-α1 during conditioning did not significantly affect STM ( Figure 3C ) , but led to a severe impairment of sucrose-rewarded LTM ( Figure 3D ) . The unimpaired performance in appetitive STM demonstrates that flies of these genotypes have normal sugar and odor perception at the restrictive temperature . Intriguingly , we found a similar preferential impairment of LTM upon the blockade of MBON-α1 , using MB310C-GAL4 driver ( Figure 3H ) . The same blockade did not significantly impair STM ( Figure 3G ) . We confirmed the preferential requirement of MBON-α1 for LTM using another driver , MB323B-GAL4 ( Figure 3—figure supplement 1A ) , and obtained similar results ( Figure 3G , H ) ; neither MB310C/UAS-shi nor MB323B/UAS-shi flies showed a significant LTM impairment when trained and tested at the permissive temperature ( Figure 3—figure supplement 1B ) . 10 . 7554/eLife . 10719 . 008Figure 3 . Feedback from MBON-α1 is required for appetitive LTM formation . ( A ) Diagram of the experiment . ( B ) Expression pattern of MB299B-GAL4 . ( C ) Blockade of PAM-α1 does not impair STM significantly . 5-min appetitive memory was measured ( n = 10 , 10 , 12 ) . c: conditioning , t: test . ( D ) Blockade of PAM-α1 during conditioning impairs LTM . 24-hr appetitive LTM was measured ( n = 9 , 12 , 12 ) . ( E ) Diagram of experiment . ( F ) Expression pattern of MB310C-GAL4 . ( G ) Blockade of MBON-α1 does not impair STM significantly ( n = 6 , 7 , 10 , 6 , 10 ) . ( H ) Blockade of MBON-α1 during conditioning impairs LTM . MB323B-GAL4 is a second driver line that expresses in MBON-α1 ( see figure supplement; n = 23 , 24 , 13 , 14 , 24 ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , ***: p < 0 . 001 , n . s . : p > 0 . 05 . Scale bars , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 00810 . 7554/eLife . 10719 . 009Figure 3—figure supplement 1 . ( A ) Expression pattern of MB323B-GAL4 . Scale bar , 20 µm . ( B ) The LTM performance of MB310C/UAS-shi and MB323B/UAS-shi flies at the permissive temperature is not affected ( n = 17 , 16 , 8 , 9 , 11 ) . n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 009 We next examined whether glutamatergic input to PAM-α1 is required for LTM formation by down-regulating the expression of glutamate receptors using MB299B-GAL4 . For efficient and cell-type-specific manipulation , we used UAS-RNAi fly strains based on the shRNA technique ( Ni et al . , 2011 ) . Intriguingly , knocking down either dNR-1 or dNR-2 , which together form the functional NMDA receptor ( Xia et al . , 2005 ) , impaired LTM ( Figure 4C ) but not STM ( Figure 4B ) . This preferential requirement in LTM is similar to the blockade of MBON-α1 and PAM-α1 , suggesting the direct glutamatergic feedback . 10 . 7554/eLife . 10719 . 010Figure 4 . NMDA receptors in PAM-α1 are required for appetitive LTM . ( A ) The subunits of NMDA receptor are down-regulated in PAM-α1 . ( B , C ) Knocking down NMDA receptor subunits does not impair 5-min memory ( B: n = 16 , 8 , 13 , 12 , 13 ) but impairs 24-hr memory significantly ( C: n = 18 , 8 , 22 , 20 , 29 ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , **: p < 0 . 01 , n . s . : p > 0 . 05 . Scale bars , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 010 Given that thermogenetic stimulation of PAM-α1 with a temperature-sensitive cation channel dTrpA1 ( Hamada et al . , 2008 ) can provide the reward signal for LTM formation ( Yamagata et al . , 2015 ) , we asked if activation of MBON-α1 would also be capable of inducing appetitive LTM ( Figure 5A ) . Indeed , we found appetitive LTM upon thermo-activation of MBON-α1 in the presence of an odor ( Figure 5B ) . These results , together with the consistent requirements of MBON-α1 and NMDAR-signaling in PAM-α1 , are most easily explained if the glutamatergic feedback regulation from MBON-α1 augments dopamine reward signaling by PAM-α1 . 10 . 7554/eLife . 10719 . 011Figure 5 . MBON-α1 signals reward for appetitive LTM . ( A ) Activation of MBON-α1 was paired with odor presentations and 24-hr memory was measured . ( B ) Activation of MBON-α1 induces appetitive LTM formation ( n = 16 , 24 , 24 ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , n . s . : p > 0 . 05 . Scale bars , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 011 Several lines of evidence suggest that D1-like dopamine receptor ( DopR1 ) is required in KCs for appetitive memory ( Kim et al . , 2007; Liu et al . , 2012; Boto et al . , 2014 ) . However , previous studies focused on only STM . To identify the receiving site of dopamine for LTM formation , we knocked down DopR1 , either in α/β KCs or in MBON-α1 , the potential targets of dopamine from PAM-α1 , and measured appetitive LTM . To target the expression specifically to the α/β KCs , we employed two driver lines , MB008B-GAL4 ( Aso et al . , 2014a; Vogt et al . , 2014 ) ( Figure 6—figure supplement 1A ) and c739-GAL4 ( Yang et al . , 1995 ) . Interestingly , knocking down DopR1 in the α/β KCs , using c739-GAL4 or MB008B-GAL4 , resulted in a severe impairment of appetitive LTM ( Figure 6A ) while leaving STM unaffected ( Figure 6—figure supplement 1B ) . These results are consistent with the observation that the coincidence detector adenylate cyclase encoded by rutabaga , thought to act downstream of the dopamine receptor ( Tomchik and Davis , 2009; Gervasi et al . , 2010; Boto et al . , 2014 ) , functions for LTM in the α/β KCs ( Trannoy et al . , 2011 ) . On the other hand , knocking down DopR1 in MBON-α1 did not significantly impair either appetitive LTM or STM ( Figure 6A and Figure 6—figure supplement 1B ) . 10 . 7554/eLife . 10719 . 012Figure 6 . α/β KCs receive dopamine signals through DopR1 for LTM formation . ( A ) Knocking down DopR1 in α/β Kenyon cells ( KCs ) but not in MBON-α1 impairs appetitive LTM ( n = 17 , 10 , 11 , 11 , 18 , 12 , 7 , 12 , 27 ) . ( B ) KC expression of DopR1 in the dumb2 mutant background fully rescues the LTM impairment ( n = 12 , 10 , 7 , 13 ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , ***: p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 01210 . 7554/eLife . 10719 . 013Figure 6—figure supplement 1 . ( A ) Expression pattern of MB008B-GAL4 . Scale bar , 20 µm . ( B ) Knocking down DopR1 in α/β KCs or MBON-α1 does not impair appetitive STM , suggesting normal sugar or odor perception of these flies ( n = 7 , 7 , 12 , 10 , 10 , 10 , 9 , 9 , 22 ) . Bar graphs are mean ± s . e . m . n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 013 To further localize the role of DopR1 in KCs , we performed a rescue experiment , using a piggyBac insertion mutant , dumb2 ( Liu et al . , 2012; Qin et al . , 2012 ) . dumb2 mutant flies showed severely impaired appetitive LTM . Targeting wild-type DopR1 gene expression in KCs fully rescued the defects ( Figure 6B ) . Together , although we cannot exclude the possible role of other dopamine receptors in MBON-α1 , we propose that DopR1-mediated memory trace is formed in α/β KCs not in MBON-α1 . Previous studies have highlighted the role of the output from α/β KCs for memory retrieval ( Krashes et al . , 2007; Trannoy et al . , 2011; Cervantes-Sandoval et al . , 2013; Perisse et al . , 2013 ) . Given the requirement of MBON-α1 for the acquisition of appetitive LTM , the output of the α/β KCs during conditioning may be important for LTM formation . To test this possibility , we blocked the output of the α/β KCs with Shits1 . Strikingly , the blockade of the α/β KCs during conditioning significantly impaired appetitive LTM , but not STM ( Figure 7A , B ) , similar to the blockade of PAM-α1 and MBON-α1 ( Figure 3 ) . When the flies were trained and tested at the permissive temperature , no significant impairment of appetitive LTM was observed ( Figure 7—figure supplement 1 ) . The requirement for α/β KC synaptic signaling during LTM formation supports our model that MBON-α1 provides information about the memory trace , formed in the α/β KCs , to the reward signaling PAM-α1 neurons . 10 . 7554/eLife . 10719 . 014Figure 7 . Output of α/β KCs has an essential role in the acquisition of appetitive LTM . ( A ) Blockade of α/β KCs does not impair STM significantly ( n = 8 , 7 , 7 , 8 , 9 ) . ( B ) Blockade of α/β KCs during conditioning impairs LTM ( n = 7 , 7 , 18 , 19 , 20 ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 01410 . 7554/eLife . 10719 . 015Figure 7—figure supplement 1 . Training and testing c739/UAS-shi and MB008B/UAS-shi flies at the permissive temperature do not impair appetitive LTM ( n = 7 , 6 , 8 , 9 , 6 ) . Bar graphs are mean ± s . e . m . n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 015 If the nascent memory trace in α/β KCs is communicated by MBON-α1 during conditioning , MBON-α1 might also convey the read-out of LTM . Intriguingly , when MBON-α1 was blocked only during the test phase of LTM , we found a severe impairment of conditioned odor approach ( Figure 8A ) . In contrast , the blockade of PAM-α1 during LTM retrieval did not have a significant effect ( Figure 8B ) . These results imply that MBON-α1 plays a dual role in both formation and retrieval of LTM; different post-synaptic targets of MBON-α1 would be used in these two roles , with the recurrent PAM-α1-MBON-α1 circuit selectively involved in LTM formation . 10 . 7554/eLife . 10719 . 016Figure 8 . Appetitive LTM is read out through MBON-α1 . ( A ) Blocking MBON-α1 during test impairs appetitive LTM retrieval ( n = 20 , 21 , 11 , 13 , 20 ) . ( B ) Blocking PAM-α1 during test does not significantly impair LTM retrieval ( n = 7 , 7 , 10 ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , **: p < 0 . 01 , n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 016 The glutamatergic input from MBON-α1 might provide an excitatory feedback on the PAM-α1 neurons via the NMDA receptor thereby prolonging the dopamine release after the cessation of sugar presentation . If this model were correct , the post-training blockade of the feedback circuit might be expected to impair appetitive LTM . Consistent with this idea , blocking any component of the feedback circuit for 1 hr immediately following conditioning resulted in a significant impairment of appetitive LTM ( Figure 9A , C , E ) , whereas the same blockade 22 hr after training did not ( Figure 9B , D , F ) . These results strongly suggest that the recurrent circuit has an ongoing activity , which plays a key role in early phase of appetitive LTM consolidation . 10 . 7554/eLife . 10719 . 017Figure 9 . Dopamine release from PAM-α1 is required for appetitive LTM consolidation . ( A , C , E ) Blocking PAM-α1 ( A ) , MBON-α1 ( C ) , or α/β−KC ( E ) for 1 hr immediately after conditioning impairs appetitive LTM ( n = 16 , 15 , 22 ( A ) ; n = 15 , 16 , 14 , 20 , 34 ( C ) ; n = 9 , 8 , 10 , 7 , 15 ( E ) ) . ( B , D , F ) Blocking PAM-α1 ( B ) , MBON-α1 ( D ) , or α/β−KC ( F ) for 1 hr , 22 hr after conditioning does not impair appetitive LTM ( n = 15 , 15 , 14 ( B ) ; n = 16 , 15 , 13 , 13 , 23 ( D ) ; n = 12 , 12 , 15 , 12 , 15 ( F ) ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , ***: p < 0 . 001 , n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 017 Prolonged dopamine release in the absence of odor may also modulate KCs that do not respond to the rewarded odor . If so , the odor specificity of appetitive LTM might be compromised compared to STM . Thus , we measured the generalization profile of STM and LTM by testing with the graded mixtures of ‘contaminating’ odors ( Figure 10 ) . Odor mixtures were created by systematically varying the relative contents of the contaminant ( trained odor: contaminant = 100:0 , 80:20 , 60:40 , 40:60 , 20:80 , or 0:100 ) : if the memory is specific to the trained odor , the performance quickly decreases with the increasing contaminant ratio . Strikingly , the performance decline is shallower in LTM compared to STM ( Figure 10C , D ) . We confirmed these results with another set of odorants , suggesting that compromised stimulus specificity in LTM is general ( Figure 10—figure supplement 1 ) . Considering that the only difference between these groups is the retention time , there may be a trade-off between consolidation and specificity , possibly due to the prolonged dopamine release in the MB following memory acquisition . 10 . 7554/eLife . 10719 . 018Figure 10 . LTM specificity is compromised . ( A ) Chemical structures of odorants . ( B ) Design of the experiment . For one group , presentation of 4-methylcyclohexanol was paired with sugar reward . The reciprocal group received sugar without odor . In the test situation , flies of each group were allowed to choose between the air and the mixture of the trained odor and the ‘contaminant’ ( 2-methylcyclohexanol ) . ( C , D ) The performance index declined more sharply with the increasing contamination ratio in STM ( C; n = 16 for each ) than in LTM ( D; n = 24 for each ) . Each group was compared to the group that was tested without the contaminant ( left most bar ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , **: p < 0 . 01 , ***: p < 0 . 001 , n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 01810 . 7554/eLife . 10719 . 019Figure 10—figure supplement 1 . LTM is less specific with another pair of odors . ( A ) Chemical structures of odorants . ( B , C ) The performance declined more sharply in STM ( B: n = 20 for each ) than in LTM ( C: n = 20 for each ) also with this odor pair . Each group was compared to the group that was tested without the contaminant ( left most bar ) . Bar graphs are mean ± s . e . m . *: p < 0 . 05 , n . s . : p > 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 019
Prior studies on the role of MBONs in learning and memory have focused on memory retrieval ( Sejourne et al . , 2011; Pai et al . , 2013; Placais et al . , 2013; Aso et al . , 2014b ) . Here , we extend the known role of MBONs and show that MB output through MBON-α1 is required not only for the retrieval , but also for the acquisition and early consolidation of long-term appetitive olfactory memory . This unanticipated aspect of the MB output for LTM formation appears to function through a recurrent loop ( Figure 11 ) : MBON-α1 dendrites and the terminals of PAM-α1 occupy the same MB compartment ( Aso et al . , 2014a ) ; the DopR1 dopamine receptor is required in the KCs of that compartment ( Figure 6 ) ; MBON-α1 in turn sends a portion of its outputs to the dendrites of PAM-α1 ( Figures 1 , 2 ) closing the loop; synaptic signaling by all three cell types—α/β KCs , MBON-α1 , and PAM-α1—is required during LTM formation and consolidation ( Figures 3 , 7 , 9 ) . These findings strongly support our model that feedback signals from α/β KCs , mediated by MBON-α1 , play a critical role in controlling the rewarding dopamine signals from PAM-α1 ( Figure 11 ) . Inhibitory recurrent circuits in the MBs have been implicated in appetitive learning of honeybees ( Grunewald , 1999a , 1999b ) . Our present study reveals a new circuit motif , a feedback circuit that is required for dopamine neurons to provide reinforcement during appetitive LTM formation . 10 . 7554/eLife . 10719 . 020Figure 11 . Recurrent reward circuit drives appetitive LTM formation . Feedback circuit model of appetitive LTM formation . Neuronal signal of a sugar reward mediated by PAM-α1 converges with olfactory information in the α/β KCs . The coincidental signal is read out through MBON-α1 to give glutamatergic feedback onto PAM-α1 for the gain control of the dopamine release . Appetitive LTM is read out through MBON-α1 to activate other downstream targets . DOI: http://dx . doi . org/10 . 7554/eLife . 10719 . 020 Dopamine has been thought to simply relay rewarding or punitive stimuli ( Gerber et al . , 2004; Waddell , 2013 ) . However , our results call into question this prevailing circuit model . More specifically , PAM-α1 might integrate simultaneous inputs from sugar perception and the nascent memory trace to augment and sustain the dopamine release to gate LTM formation ( Figure 11 ) . The requirement of the NMDA receptor in PAM-α1 ( Figure 4 ) further supports this model . Such integration by PAM-α1 might enhance the reward gain selectively in relevant sensory environments . In other words , the reinforcing signal for LTM formation may depend on the presence of other sensory stimuli ( e . g . , odor ) . As LTM formation is energetically costly ( Mery and Kawecki , 2005; Placais and Preat , 2013; Musso et al . , 2015 ) , this gain control of the reinforcing signal might serve to restrict LTM formation to ecologically relevant situations . Although the requirement of NMDA receptor in PAM-α1 implies that MBON-α1 provides excitatory input , the synaptic connectivity between MBON-α1 and PAM-α1 awaits physiological demonstration , given functional inhibitory glutamate receptor signaling ( Liu and Wilson , 2013 ) . In addition to the role in memory acquisition , the PAM-α1 output is required during consolidation ( Figure 9A , B ) . The dopamine release is likely to be sustained by the ongoing activity of the recurrent circuit ( Figure 9C–F ) . A recent study reported a consistent finding that the output from α/β KCs during consolidation has an essential role for appetitive and aversive LTM ( Huang et al . , 2012 ) in addition to their role in retrieval ( Krashes et al . , 2007; Trannoy et al . , 2011; Cervantes-Sandoval et al . , 2013; Xie et al . , 2013 ) . Recent studies revealed a gradual development of appetitive LTM after conditioning ( Das et al . , 2014; Huetteroth et al . , 2015; Yamagata et al . , 2015 ) . Ongoing activity in the recurrent loop we propose here may provide a circuit mechanism for the gradual LTM development . In addition , previous studies reported that LTM consolidation in Drosophila ( Placais et al . , 2012; Musso et al . , 2015 ) or in rats ( Rossato et al . , 2009 ) requires post-training dopamine inputs . Our generalization experiments show that appetitive LTM is less specific to the trained odor , compared to STM ( Figure 10 ) . This result fits well into previous studies in mice and bees , demonstrating that long-lasting memories are more generalized ( Stach and Giurfa , 2005; Wiltgen and Silva , 2007 ) . More generalizable LTM makes sense from an ecological viewpoint: it is worth taking a chance for survival even if the smell is not exactly the same as learned . In the fly brain , distinct types of dopamine neurons signal reward for STM and LTM , and these memories are independently formed in different MB lobes ( Trannoy et al . , 2011; Huetteroth et al . , 2015; Yamagata et al . , 2015 ) . Octopamine has been widely accepted as a positive reinforcement signal in insects ( Hammer , 1993; Hammer and Menzel , 1998; Schwaerzel et al . , 2003; Schroll et al . , 2006; Perry and Barron , 2013 ) . In Drosophila , it is considered to provide inputs to dopamine neurons that induce STM formation ( Burke et al . , 2012; Das et al . , 2014; Huetteroth et al . , 2015 ) . Recent studies in mammals reached a similar conclusion that memories with different temporal dynamics are mutually conflicting and formed in distinct areas , to which different classes of dopamine neurons project ( Liljenstrom , 2003; Abraham and Robins , 2005; Kim and Hikosaka , 2013; Kim et al . , 2014 ) . Independent processing of STM and LTM might be a conserved strategy among animals . In addition to the function in the recurrent reward circuit , MBON-α1 mediates retrieval of the LTM trace in the MB ( Figure 8 ) . MBON-α1 samples from the MB-α1 and terminates in the SIP and the SLP . Another type of MBONs also mediates appetitive LTM retrieval: MBON-α3 ( also known as MB-V3 ) ( Placais et al . , 2013 ) . In contrast to MBON-α1 , blocking MBON-α3 during conditioning does not impair appetitive LTM acquisition ( Placais et al . , 2013 ) . MBON-α3 arborizes dendrites in the tip of the α lobe and projects to the SMP and the SIP where the terminals of MBON-α1 and MBON-α3 are intermingled ( Aso et al . , 2014a ) . Therefore , appetitive LTM formed in the α/β KCs is read out through at least two different classes of MBONs . These MBONs converge in the SIP ( Aso et al . , 2014a ) and might activate shared downstream targets to guide conditioned odor approach . The recent comprehensive connectivity map of the afferent and efferent neurons of the Drosophila MB revealed many potential feedback connections outside the MB ( Aso et al . , 2014a ) . Considering the central role of the MB in sensory integration and memory-guided behavior ( Aso et al . , 2014b ) , recurrent computation between learned information and reinforcement signals might be a fundamental principle of memory processing . In both vertebrates and invertebrates , the activity of reinforcement neurons is modified upon associative learning ( Hammer , 1993; Mirenowicz and Schultz , 1994; Riemensperger et al . , 2005; Cohen et al . , 2012; Schultz , 2013 ) . Our results delineated such a feedback circuit , which was predicted by a physiological study in Drosophila ( Riemensperger et al . , 2005 ) . In this circuit , MBON-α1 mediates monosynaptic feedback to the dopamine neurons . In contrast , there is no monosynaptic feedback to the dopamine neurons found in mammals , rather anatomical studies suggest such a feedback to be polysynaptic ( Lisman and Grace , 2005; Watabe-Uchida et al . , 2012 ) . Further comparative studies should assess how the recurrent circuit motif of reinforcement is implemented in different animal brains .
Canton-S was used as a wild-type strain . Generation and basic characterization of the split-GAL4 drivers ( MB008B-GAL4 , MB299B-GAL4 , MB310C-GAL4 , and MB323B-GAL4 ) are described in Aso et al . ( 2014a ) . R72D01-LexA was constructed using the methods described in Pfeiffer et al . ( 2010 ) and inserted into attP40 . c739-GAL4 is an enhancer-trap GAL4 driver described in Yang et al . ( 1995 ) . Flies were raised at 60% relative humidity at 24°C . Flies for the NMDAR knock down ( Figure 4 ) were incubated at 30°C for 4–5 days prior to the experiment to facilitate the GAL4 activity . The synaptic blockade experiments ( Figures 3 , 7-9 ) used F1 progeny of crosses between females of w;;UAS-shits1 ( single copy of UAS-shits1 from Thomas Preat laboratory , CNRS , France ) or w and males of the GAL4 drivers . The thermoactivation experiment ( Figure 5 ) used F1 progeny of crosses between females of w; UAS-dTrpA1 ( Hamada et al . , 2008 ) or w and males of MB310C-GAL4 or w . The receptor knock down experiments ( Figures 4 , 6 ) used F1 progeny of crosses between females of UAS-Nmdar1RNAi ( P ( TRiP . HMS02200 ) attP40; Bloomington stock center #41667 ) , UAS-Nmdar2RNAi ( P ( TRiP . HMS02012 ) attP2; Bloomington stock center #40846 ) , UAS-DopR1RNAi ( P ( TRiP . HMC02344 ) attP2; Bloomington stock center #55239 ) ( Ni et al . , 2011 ) or CS and males of the GAL4 drivers . The dumb2 rescue experiments ( Figure 6B ) used F1 progeny of crosses between females of UAS-DopR1;;dumb2 or CS and males of MB247-GAL4; dumb2 or dumb2 . CS flies were used as wild type ( Figures 6B , 10 ) . For immunohistochemistry , the following reporter strains were crossed to the respective GAL4 or LexA drivers: pJFRC2-10xUAS-mCD8GFP in VK00005 ( Figure 1A–D ) , pJFRC200-10XUAS-IVS-myr::smGFP-HA in attP18 ( Figure 2A–D; Figure 2—figure supplement 1; Figure 3B , F; Figure 6—figure supplement 1A ) and pJFRC216-13XLexAop2-IVS-myr::smGFP-V5 in su ( Hw ) attP8 ( Figure 2A-E; Figure 2—figure supplement 1 ) , pJFRC206-5xUAS-IVS-myr-smGFP-FLAG in VK00005 ( Figure 1E , F; Figure 1—figure supplement 1; Figure 3—figure supplement 1A ) , pJFRC51-3xUAS-Syt-smHA in su ( Hw ) attP1 ( presynaptic marker; Figure 1E ) , UAS-CD4::spGFP1-10 and LexAop-CD4::spGFP11 ( Figure 2F ) . pJFRC2 is described in Pfeiffer et al . ( 2010 ) . pJFRC200 and pJFRC206 are described in Viswanathan et al . ( 2015 ) and pJFRC216 in Nern et al . ( 2015 ) . pJFRC51 was generated by standard methods described in Pfeiffer et al . ( 2010 ) . UAS-CD4::spGFP1-10 and LexAop-CD4::spGFP11 are described in Gordon and Scott ( 2009 ) . Multicolor flp-out ( MCFO; Figure 1G ) is a stochastic method that labels individual cells in different colors using a set of three UAS-STOP-epitope constructs that each expresses a different epitope when the STOP cassette is removed . The STOP cassettes in these constructs are each flanked by FRT sites that are removed in a stochastic way by limited expression of flp recombinase . Reagents for MCFO are described in Nern et al . ( 2015 ) . Dissection and immunohistochemistry of fly brains were done as previously described ( Aso et al . , 2014a ) . The following antibodies were used: rabbit-anti-GFP ( 1:1000; A11122; Invitrogen ) , mouse-anti-GFP ( 1:200; UC Davis/NIH Neuromab Facility; clone N86/38; for GRASP ) , mouse anti-nc82 ( 1:33 . 3; Developmental Studies Hybridoma Bank , Univ . Iowa , Iowa City , IA , United States ) ( Hofbauer et al . , 2009 ) , rat-anti-nCad ( 1:200; DN-Ex #8; Developmental Studies Hybridoma Bank , University of Iowa ) , rabbit anti-HA ( 1:300; Cell Signaling Technology , MA , United States ) , rat anti-FLAG ( 1:200; Novus Biologicals , Littleton , CO , United States ) , mouse anti-Drosophila ChAT ( ChAT4B1; 1: 100; Developmental Studies Hybridoma Bank , Univ . Iowa ) ( Takagawa and Salvaterra , 1996 ) , rabbit anti-Drosophila GAD1 ( 1:1000; a gift from Dr FR Jackson ) ( Featherstone et al . , 2000 ) , rabbit anti-DvGluT ( 1:5000; a gift from Dr A DiAntonio ) ( Daniels et al . , 2008 ) as primary antibodies , and cross-adsorbed secondary antibodies to IgG ( H+L ) : AlexaFluor-488 donkey anti-mouse ( 1:400; Jackson Labs ) , Cy3 donkey anti-rabbit ( 1:500; Jackson Labs ) , AlexaFluor-647 donkey anti-rat ( 1:300; Jackson Labs ) , AlexaFluor-488 goat anti-rabbit ( 1:800; A11034; Invitrogen , Carlsbad , CA , United States ) , and AlexaFluor-568 goat anti-mouse ( 1:400; A11031; Invitrogen ) . Confocal images were obtained by using LSM710 ( Zeiss , Oberkochen , Germany ) ( Figures 1A–F , 2A , E , 3B , F , Figure 1—figure supplement 1 , Figure 2—figure supplement 1 , Figure 3—figure supplement 1 , Figure 6—figure supplement 1 ) and FV1200 ( Olympus , Tokyo , Japan ) ( Figure 2F ) and processed by using ImageJ . The magnified images in SIP and SLP ( Figure 2B–D ) were obtained by using LSM880 ( Zeiss ) with Airyscan and were deconvoluted using the Fiji plugin provided by Bob Dougherty ( http://fiji . sc/Deconvolution ) . Airyscan is equipped with 32 GaAsP detectors that collect light from one illumination spot . It can resolve 140 nm ( lateral ) or 400 nm ( axial ) at 488-nm wavelength . For the 3D rendering video , confocal images from LSM710 were deconvoluted using the Fiji plugin and then were subjected to 3D image rendering software ( Volocity: from PerkinElmer , Waltham , MA , United States ) . The conditioning and testing protocol was as described previously with minor modifications ( Liu et al . , 2012; Aso et al . , 2014b; Yamagata et al . , 2015 ) . Briefly , a group of approximately 50 flies in a training tube alternately received octan-3-ol ( 3-OCT; Merck ) and 4-methylcyclohexanol ( 4-MCH; Sigma–Aldrich , St . Louis , MO , United States ) for 1 min in a constant air stream with or without dried sucrose paper . 3-OCT and 4-MCH were diluted 1% and 2% , respectively , in paraffin oil ( Sigma–Aldrich ) , placed in a cup with a diameter of 14 mm and presented to flies . For thermoactivation experiments ( Figure 5 ) , 3-OCT and 4-MCH were diluted 10% in paraffin oil , placed in a cup with a diameter of 3 mm and 5 , respectively , and presented to flies . For the generalization experiments ( Figure 10 ) , 1% 1-octen-3-ol ( Sigma–Aldrich ) and 2% 2-methylcyclohexanol ( Sigma–Aldrich ) were also utilized . These diluted odors were mixed with the respective ratios . The restrictive temperature for the experiments with UAS-shits1 was 32°C and the permissive temperature was 24°C , measured with Venta-Hygrometer ( Venta Luftwäscher GmbH , Weingarten , Germany ) . At the test , the trained flies were allowed to choose between MCH and OCT for 2 min in a custom-made transparent Plexiglas T-maze , illuminated by infrared LEDs ( Osram 720-SFH487P , Mouser electronics , Mansfield , TX , United States ) , and the walls were covered with Fluon ( Insect-a-Slip , PTFE30 , BioQuip Products , Inc . , Rancho Dominguez , CA , United States ) and the distribution of flies was imaged by cameras ( FFMV-03M2M , Point Grey , Richmond , Canada ) . Captured images were sent to the computer and the fly number in each tube was counted by a custom-made ImageJ macro . The preference index was calculated by taking the mean indices of the last 60 s in the 2-min choice and the learning index was calculated by taking the mean of preference indices of the two reciprocally trained groups . Half of the trained groups received reinforcement together with the first presented odor and the other half with the second odor to cancel the effect of the order of reinforcement . For thermoactivation with dTrpA1 ( Figure 5 ) , flies were trained by being transferred from a background temperature of 24°C to a prewarmed tube in a climate box ( 30°C ) and presented with the training odorant for 1 min . Flies were aged 3–10 days after eclosion and were starved so that the mortality rate reaches 5–15% at the test . Between the training and the test , flies were kept without food . Conditioning was performed in dim red light and testing was in darkness . Statistical analyses were performed with StatPlus and Prism5 ( GraphPad , La Jolla , CA , United States ) . Data did not violate the assumption of normal distribution ( Shapiro–Wilk test ) and homogeneity of variance ( Bartlett test ) . Therefore , the data were analyzed with parametric statistics: one-sample t-test or one-way analysis of variance followed by the planned pairwise multiple comparisons ( Bonferroni ) . The significance level of statistical tests was set to 0 . 05 .
Dopamine signals reward in animal brains . A single presentation of a sugar reward to Drosophila activates distinct subsets of dopamine neurons that independently induce short- and long-term olfactory memories ( STM and LTM , respectively ) . In this study , we show that a recurrent reward circuit underlies the formation and consolidation of LTM . This feedback circuit is composed of a single class of reward-signaling dopamine neurons ( PAM-α1 ) projecting to a restricted region of the mushroom body ( MB ) , and a specific MB output cell type , MBON-α1 , whose dendrites arborize that same MB compartment . Both MBON-α1 and PAM-α1 neurons are required during the acquisition and consolidation of appetitive LTM . MBON-α1 additionally mediates the retrieval of LTM , which is dependent on the dopamine receptor signaling in the MB α/β neurons . Our results suggest that a reward signal transforms a nascent memory trace into a stable LTM using a feedback circuit at the cost of memory specificity .
An animal that finds particularly nutritious and palatable food will often develop a long-lasting memory—even if they experience that event only once . One example of this is the ability of the fruit fly Drosophila to form a long-term association between a sugar reward and a specific odor that was present when they received the reward . The consumption of sugar triggers the release of a chemical called dopamine on specific compartments of a brain structure called the mushroom body . Dopamine then acts to modify the connection between cells called “Kenyon cells” , which encode specific odors , and the neurons that send signals out from the mushroom body ( called MBONs ) . The result is the formation of a memory that links the odor with the reward . However , little is known about how this process differs for long-term vs . short-term memories , and how it can occur when the fly has experienced the odor and reward together on only a single occasion . To find out , Ichinose et al . combined behavioral testing of fruit flies with genetics . The results confirmed that the dopamine neurons and the MBONs that project to a single compartment of the mushroom body , called α1 , are both required for the formation of long-term odor-reward memories , but not their short-term equivalents . These neurons are called PAM-α1 and MBON-α1 , respectively . Unexpectedly , anatomical data revealed that PAM-α1 dopamine neurons receive input from MBON-α1; that is , long-term memory formation involves a feedback circuit: from PAM-α1 to Kenyon cells , then to MBON-α1 and back to PAM-α1 . Blocking feedback from the MBON-α1 onto the PAM-α1 neurons shortly after odor-reward training disrupted long-term memory formation . Conversely , blocking feedback at a later stage did not . This suggests that prolonged activation of PAM-α1 by MBON-α1 helps to strengthen newly established memories , converting them into memories that will last for a long time . The discovery of a specific circuit that supports long-term , but not short-term , memory formation in fruit flies is consistent with evidence of distinct mechanisms underlying these processes in mammals . Further work is now required to determine whether feedback circuits similar to those in fruit flies also contribute to reward-based learning in other animals .