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• W2036887396 abstract "Gap junctions play an important role in the regulation of neuronal metabolism and homeostasis by serving as connections that enable small molecules to pass between cells and synchronize activity between cells [1McCracken C.B. Roberts D.C. Neuronal gap junctions: Expression, function, and implications for behavior.Int. Rev. Neurobiol. 2006; 73: 125-151Crossref PubMed Scopus (25) Google Scholar, 2Nagy J.I. Dudek F.E. Rash J.E. Update on connexins and gap junctions in neurons and glia in the mammalian nervous system.Brain Res. Brain Res. Rev. 2004; 47: 191-215Crossref PubMed Scopus (286) Google Scholar, 3Kielian T. Glial connexins and gap junctions in CNS inflammation and disease.J. Neurochem. 2008; 106: 1000-1016Crossref PubMed Scopus (109) Google Scholar]. Although recent studies have linked gap junctions to memory formation [4Frisch C. De Souza-Silva M.A. Söhl G. Güldenagel M. Willecke K. Huston J.P. Dere E. Stimulus complexity dependent memory impairment and changes in motor performance after deletion of the neuronal gap junction protein connexin36 in mice.Behav. Brain Res. 2005; 157: 177-185Crossref PubMed Scopus (89) Google Scholar, 5Juszczak G.R. Swiergiel A.H. Properties of gap junction blockers and their behavioural, cognitive and electrophysiological effects: Animal and human studies.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2009; 33: 181-198Crossref PubMed Scopus (162) Google Scholar], it remains unclear how they contribute to this process [1McCracken C.B. Roberts D.C. Neuronal gap junctions: Expression, function, and implications for behavior.Int. Rev. Neurobiol. 2006; 73: 125-151Crossref PubMed Scopus (25) Google Scholar, 5Juszczak G.R. Swiergiel A.H. Properties of gap junction blockers and their behavioural, cognitive and electrophysiological effects: Animal and human studies.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2009; 33: 181-198Crossref PubMed Scopus (162) Google Scholar]. Gap junctions are hexameric hemichannels formed from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates [6Paul D.L. Molecular cloning of cDNA for rat liver gap junction protein.J. Cell Biol. 1986; 103: 123-134Crossref PubMed Scopus (512) Google Scholar, 7Phelan P. Starich T.A. Innexins get into the gap.Bioessays. 2001; 23: 388-396Crossref PubMed Scopus (188) Google Scholar]. Here we show that two modulatory neurons, the anterior paired lateral (APL) neuron and the dorsal paired medial (DPM) neuron, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNA interference-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons, respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM. Gap junctions play an important role in the regulation of neuronal metabolism and homeostasis by serving as connections that enable small molecules to pass between cells and synchronize activity between cells [1McCracken C.B. Roberts D.C. Neuronal gap junctions: Expression, function, and implications for behavior.Int. Rev. Neurobiol. 2006; 73: 125-151Crossref PubMed Scopus (25) Google Scholar, 2Nagy J.I. Dudek F.E. Rash J.E. Update on connexins and gap junctions in neurons and glia in the mammalian nervous system.Brain Res. Brain Res. Rev. 2004; 47: 191-215Crossref PubMed Scopus (286) Google Scholar, 3Kielian T. Glial connexins and gap junctions in CNS inflammation and disease.J. Neurochem. 2008; 106: 1000-1016Crossref PubMed Scopus (109) Google Scholar]. Although recent studies have linked gap junctions to memory formation [4Frisch C. De Souza-Silva M.A. Söhl G. Güldenagel M. Willecke K. Huston J.P. Dere E. Stimulus complexity dependent memory impairment and changes in motor performance after deletion of the neuronal gap junction protein connexin36 in mice.Behav. Brain Res. 2005; 157: 177-185Crossref PubMed Scopus (89) Google Scholar, 5Juszczak G.R. Swiergiel A.H. Properties of gap junction blockers and their behavioural, cognitive and electrophysiological effects: Animal and human studies.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2009; 33: 181-198Crossref PubMed Scopus (162) Google Scholar], it remains unclear how they contribute to this process [1McCracken C.B. Roberts D.C. Neuronal gap junctions: Expression, function, and implications for behavior.Int. Rev. Neurobiol. 2006; 73: 125-151Crossref PubMed Scopus (25) Google Scholar, 5Juszczak G.R. Swiergiel A.H. Properties of gap junction blockers and their behavioural, cognitive and electrophysiological effects: Animal and human studies.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2009; 33: 181-198Crossref PubMed Scopus (162) Google Scholar]. Gap junctions are hexameric hemichannels formed from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates [6Paul D.L. Molecular cloning of cDNA for rat liver gap junction protein.J. Cell Biol. 1986; 103: 123-134Crossref PubMed Scopus (512) Google Scholar, 7Phelan P. Starich T.A. Innexins get into the gap.Bioessays. 2001; 23: 388-396Crossref PubMed Scopus (188) Google Scholar]. Here we show that two modulatory neurons, the anterior paired lateral (APL) neuron and the dorsal paired medial (DPM) neuron, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNA interference-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons, respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM. The APL and DPM neurons are connected with heterotypic gap junctions within the MB These gap junctions are essential for ASM formation, rather than acquisition or ARM A recurrent neural network in the MB may underlie the aversive olfactory ASM Drosophila can be trained to form a Pavlovian association between an odor and electric shock. Flies naturally avoid electric shock, which serves as the unconditioned stimulus (US). When this shock is paired with delivery of a particular odor, the conditioned stimulus (CS), flies learn this association and subsequently avoid the odor [8Tully T. Quinn W.G. Classical conditioning and retention in normal and mutant Drosophila melanogaster.J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 1985; 157: 263-277Crossref PubMed Scopus (834) Google Scholar]. Coincident US and CS input is registered in the mushroom body (MB), a brain area composed of approximately 2500 Kenyon cells (KCs) [9Davis R.L. Olfactory memory formation in Drosophila: From molecular to systems neuroscience.Annu. Rev. Neurosci. 2005; 28: 275-302Crossref PubMed Scopus (441) Google Scholar, 10Margulies C. Tully T. Dubnau J. Deconstructing memory in Drosophila.Curr. Biol. 2005; 15: R700-R713Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 11Wu C.L. Chiang A.S. Genes and circuits for olfactory-associated long-term memory in Drosophila.J. Neurogenet. 2008; 22: 1-28Crossref Scopus (5) Google Scholar]. The dendrites of the KCs form the cap or calyx of the MB, and their axons project anteriorly to form the stalk or peduncle before those axons terminate in one or more lobes, termed αβ, α′β′, and γ lobes. KCs receive olfactory input from the projection neurons (PNs) of the antennal lobe, which contact KC dendrites in the calyx. KCs respond very selectively to odor, which is thought to be a useful property for forming accurate memories [12Turner G.C. Bazhenov M. Laurent G. Olfactory representations by Drosophila mushroom body neurons.J. Neurophysiol. 2008; 99: 734-746Crossref PubMed Scopus (236) Google Scholar]. Dorsal paired medial (DPM) neurons express the amnesiac (amn) gene, which encodes a neuropeptide transmitter. DPM neurons innervate all of the lobes of the MB and part of the peduncle but do not send processes anywhere else, including the calyx [13Waddell S. Armstrong J.D. Kitamoto T. Kaiser K. Quinn W.G. The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.Cell. 2000; 103: 805-813Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar] (see also Figure S1C available online). This anatomical arrangement suggests that DPM and KCs are recurrently connected; that is to say, DPM likely receives direct KC input and releases its neuropeptide transmitter back to the KC axons [14Keene A.C. Waddell S. Drosophila olfactory memory: Single genes to complex neural circuits.Nat. Rev. Neurosci. 2007; 8: 341-354Crossref PubMed Scopus (286) Google Scholar]. Consistent with the notion that the DPM neuron pools inputs from many KCs, the neuron responds both to shock and, as expected, to a wide range of different odors [15Yu D. Keene A.C. Srivatsan A. Waddell S. Davis R.L. Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning.Cell. 2005; 123: 945-957Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar] (see also Figures S1A and S1B). The anterior paired lateral (APL) neuron has a morphology that partly overlaps with DPM. Its cell body is located in the lateral protocerebrum, where it gives rise to a primary neurite that projects medially and bifurcates to innervate the entire MB, one branch entering the calyx and another branch entering the vertical lobe [16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar] (see also Figure S1D). The APL neuron also responds to the US shock [16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar], which is mediated through two clusters of dopaminergic neurons acting on KCs at the MB lobes [17Claridge-Chang A. Roorda R.D. Vrontou E. Sjulson L. Li H. Hirsh J. Miesenböck G. Writing memories with light-addressable reinforcement circuitry.Cell. 2009; 139: 405-415Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 18Krashes M.J. DasGupta S. Vreede A. White B. Armstrong J.D. Waddell S. A neural circuit mechanism integrating motivational state with memory expression in Drosophila.Cell. 2009; 139: 416-427Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 19Aso Y. Siwanowicz I. Bräcker L. Ito K. Kitamoto T. Tanimoto H. Specific dopaminergic neurons for the formation of labile aversive memory.Curr. Biol. 2010; 20: 1445-1451Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]. Using functional imaging to monitor intracellular calcium changes, we found that the APL neuron is also an odor generalist responsive to at least eight tested odorants (Figures S1A and S1B). The APL neuron has been reported to suppress associative olfactory learning by releasing the inhibitory neurotransmitter GABA; training attenuates its response to the conditioned odor [16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar]. On the other hand, the DPM neuron and its amnesiac gene product are dispensable during the acquisition phase of learning but are critical for a delayed memory trace and a robust intermediate-term memory [13Waddell S. Armstrong J.D. Kitamoto T. Kaiser K. Quinn W.G. The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.Cell. 2000; 103: 805-813Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 15Yu D. Keene A.C. Srivatsan A. Waddell S. Davis R.L. Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning.Cell. 2005; 123: 945-957Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Keene A.C. Stratmann M. Keller A. Perrat P.N. Vosshall L.B. Waddell S. Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output.Neuron. 2004; 44: 521-533Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 21Keene A.C. Krashes M.J. Leung B. Bernard J.A. Waddell S. Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation.Curr. Biol. 2006; 16: 1524-1530Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. The overlapping projections of DPM and APL neurons, along with their similar odor response properties, prompted us to examine whether information might be exchanged between these two neurons. Whole-cell patch-clamp recordings from the DPM cell body were used to dye fill the DPM neuron. We observed that, in addition to labeling the DPM cell body and its processes in the lobes, staining extended through the entire peduncle and calyx to the APL cell body (Figure 1A ; Figures S1C and S1D). Dye coupling is indicative of gap-junctional connections between neurons [22Stewart W.W. Lucifer dyes—highly fluorescent dyes for biological tracing.Nature. 1981; 292: 17-21Crossref PubMed Scopus (407) Google Scholar]. To further resolve the site of DPM-APL contact, we labeled the APL and DPM neurons with two different colors of fluorescent proteins using the dual binary systems of GAL4 and LexA (Figure 1B; Figures S1F and S1G). We found that although both neurons innervate all lobes of the MB, they contact preferentially in the α′β′ lobes (Figure 1C). Next, we asked which types of gap junctions exist between APL and DPM neurons and what role they play in memory formation. There are eight innexin-encoding loci in the Drosophila genome [23Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. et al.The genome sequence of Drosophila melanogaster.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4550) Google Scholar]. Taking advantage of a Drosophila RNA interference (RNAi) library [24Dietzl G. Chen D. Schnorrer F. Su K.C. Barinova Y. Fellner M. Gasser B. Kinsey K. Oppel S. Scheiblauer S. et al.A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.Nature. 2007; 448: 151-156Crossref PubMed Scopus (1845) Google Scholar] under control of the GAL4 expression system, we knocked down each of the eight innexin genes (Table S1) in both the APL and DPM neurons using a combination of two GAL4 drivers, GH146-GAL4 and C316-GAL4. The effectiveness of each inxRNAi was validated via quantitative PCR (Figure S2A). We then tested flies for memory retention 3 hr after associative conditioning, a phase of memory referred to as intermediate-term [25Berry J. Krause W.C. Davis R.L. Olfactory memory traces in Drosophila.Prog. Brain Res. 2008; 169: 293-304Crossref PubMed Scopus (49) Google Scholar]. We found that the inx6 or inx7 products in APL and DPM neurons are necessary for normal 3 hr memory (Figure 2A ; Figure S2B). We confirmed the specificity of knockdown using quantitative immunostaining to show that inx6RNAi or inx7RNAi expression in both GH146-GAL4 and C316-GAL4 neurons effectively decreases the levels of the respective proteins within the MB lobes without affecting their levels in other brain regions (Figure 2B). Immunocytochemistry also indicated that INX6 and INX7 are widely expressed in the fly brain but are not always colocalized. In the central complex, strong immunopositive signals were observed for INX6 and INX7 in the fan-shaped body and ellipsoid body, respectively. Within the MB, INX6 and INX7 colocalized preferentially in the α′β′ lobes and the anterior half of peduncle (Figures 2C and 2D), whereas the posterior half of the peduncle was immunonegative (data not shown). The preferential localization of INX6 and INX7 subunits to the α′β′ lobes, together with the fact that the anatomical overlap between APL and DPM neurons is most extensive in the α′β′ lobes, strongly suggests that this is the site of gap-junctional coupling between these two neurons. Because both APL and DPM neurons play critical but distinct roles in associative olfactory learning and memory [13Waddell S. Armstrong J.D. Kitamoto T. Kaiser K. Quinn W.G. The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.Cell. 2000; 103: 805-813Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar, 20Keene A.C. Stratmann M. Keller A. Perrat P.N. Vosshall L.B. Waddell S. Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output.Neuron. 2004; 44: 521-533Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 21Keene A.C. Krashes M.J. Leung B. Bernard J.A. Waddell S. Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation.Curr. Biol. 2006; 16: 1524-1530Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar], we next examined the role of the APL-DPM gap junctions at various stages of memory formation and retention. Surprisingly, we found that RNAi-mediated knockdown of either INX6 or INX7 in APL and DPM neurons impairs only intermediate-term memory, whereas initial learning is normal (Figures 3A and 3B ). Intermediate-term memory in flies comprises two distinct components, anesthesia-sensitive and anesthesia-resistant memory (ASM and ARM), each accounting for about half of the total retention level when tested 3 hr after one session of associative conditioning [10Margulies C. Tully T. Dubnau J. Deconstructing memory in Drosophila.Curr. Biol. 2005; 15: R700-R713Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 26Tamura T. Chiang A.S. Ito N. Liu H.P. Horiuchi J. Tully T. Saitoe M. Aging specifically impairs amnesiac-dependent memory in Drosophila.Neuron. 2003; 40: 1003-1011Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27Stough S. Shobe J.L. Carew T.J. Intermediate-term processes in memory formation.Curr. Opin. Neurobiol. 2006; 16: 672-678Crossref PubMed Scopus (54) Google Scholar]. Using cold-shock anesthetization at 2 hr after training to abolish ASM [28Li W. Tully T. Kalderon D. Effects of a conditional Drosophila PKA mutant on olfactory learning and memory.Learn. Mem. 1996; 2: 320-333Crossref PubMed Scopus (64) Google Scholar, 29Tully T. Boynton S. Brandes C. Dura J.M. Mihalek R. Preat T. Villella A. Genetic dissection of memory formation in Drosophila melanogaster.Cold Spring Harb. Symp. Quant. Biol. 1990; 55: 203-211Crossref PubMed Scopus (75) Google Scholar, 30Schwaerzel M. Jaeckel A. Mueller U. Signaling at A-kinase anchoring proteins organizes anesthesia-sensitive memory in Drosophila.J. Neurosci. 2007; 27: 1229-1233Crossref PubMed Scopus (32) Google Scholar, 31Folkers E. Drain P. Quinn W.G. Radish, a Drosophila mutant deficient in consolidated memory.Proc. Natl. Acad. Sci. USA. 1993; 90: 8123-8127Crossref PubMed Scopus (110) Google Scholar, 32Wu C.L. Xia S. Fu T.F. Wang H. Chen Y.H. Leong D. Chiang A.S. Tully T. Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body.Nat. Neurosci. 2007; 10: 1578-1586Crossref PubMed Scopus (134) Google Scholar], we found that ARM was normal in flies with knockdown of INX6 (Figure 3A) or INX7 (Figure 3B) proteins in both APL and DPM neurons. This result was verified with an independent inx6RNAi line (v8638) targeting a distinct sequence with a different insertion site (Table S1) to eliminate the possibility of an off-target effect (Figure S3). These experiments utilized chronic knockdown of INX6 and INX7 in the APL and DPM neurons. The gross anatomy of APL and DPM neurons in inxRNAi flies was not different from that in control flies (Figure 3C), suggesting that their development was not affected by the chronic knockdown. Nevertheless, considering the critical roles of INX6 and INX7 in neural development [33Stebbings L.A. Todman M.G. Phillips R. Greer C.E. Tam J. Phelan P. Jacobs K. Bacon J.P. Davies J.A. Gap junctions in Drosophila: Developmental expression of the entire innexin gene family.Mech. Dev. 2002; 113: 197-205Crossref PubMed Scopus (108) Google Scholar, 34Ostrowski K. Bauer R. Hoch M. The Drosophila innexin 7 gap junction protein is required for development of the embryonic nervous system.Cell Commun. Adhes. 2008; 15: 155-167Crossref PubMed Scopus (16) Google Scholar], we used an inducible knockdown strategy to reduce INX6 and INX7 expression specifically in adult flies. By inactivating the temperature-sensitive repressor GAL80, we selectively induced RNAi knockdown with a temperature shift 5 days before associative conditioning. Inducible knockdown of INX6 or INX7 still significantly impaired 3 hr memory (Figures 3D and 3E). Thus, the gap-junctional communication between APL and DPM neurons is critical for the formation of ASM but not ARM. Although the C316-GAL4 driver is expressed in DPM neurons, the expression pattern includes many other cells (Figure 4A ; Figure S1E), among them a portion of the MB KCs (Figure 4B). We used 2721-GAL4 as a more specific DPM neuron driver (Figure 4C) that does not express in KCs (Figure 4D) to show that INX6 is required in the DPM neuron but not the APL neuron (Figure 4E). INX7 was required in the APL neuron but not the DPM neuron for the formation of ASM (Figure 4H); learning remained normal in all cases (Figure S4). These results suggest that these gap junctions are heterotypic, a possible indication that they do form asymmetric gap junctions. We also refined the expression pattern of GH146-GAL4, which is expressed in many olfactory PNs in addition to APL neurons [35Stocker R.F. Heimbeck G. Gendre N. de Belle J.S. Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons.J. Neurobiol. 1997; 32: 443-456Crossref PubMed Scopus (265) Google Scholar] (Figure 4F). By combining GH146-GAL4 with Cha-GAL80 [36Kitamoto T. Conditional disruption of synaptic transmission induces male-male courtship behavior in Drosophila.Proc. Natl. Acad. Sci. USA. 2002; 99: 13232-13237Crossref PubMed Scopus (83) Google Scholar], which expresses a transcriptional repressor in many neurons, we could reduce expression to undetectable levels in APL neurons, leaving visible expression in only a subset of PNs (Figure 4G). Knockdown of INX7 in these remaining PNs did not affect 3 hr memory formation (Figure 4I), suggesting that knockdown in APL neurons is required for the memory defect. The key finding of our study is that two MB modulatory neurons, the APL and DPM neurons, form heterotypic gap junctions that are specifically required for anesthesia-sensitive intermediate-term memory of aversive olfactory conditioning in Drosophila. This conclusion is supported by three independent lines of evidence. First, APL and DPM neurons are dye coupled (Figure 1), a diagnostic feature of cells connected via gap junctions. Second, anatomically the two neurons intersect preferentially in the α′β′ lobes and anterior peduncle, which coincides with INX6 antibody and INX7 antibody immunostaining results (Figure 1; Figure 2). Third, intermediate-term ASM is impaired by inducible knockdown of either INX6 in DPM neurons or INX7 in APL neurons, but not the converse (Figure 3; Figure 4). This suggests that the APL-DPM gap junctions are heterotypic and raises the possibility that the connections are in some way asymmetric. Interestingly, both APL and DPM neurons respond to electric shock and to multiple odorants [15Yu D. Keene A.C. Srivatsan A. Waddell S. Davis R.L. Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning.Cell. 2005; 123: 945-957Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar] (Figures S1A and S1B). The overlapping anatomy of these two neurons, together with INX immunostaining results, suggests that sensory information about the US, CS, or both can be transferred between APL and DPM neurons through gap junctions in the α′β′ lobes. Although it remains to be determined which of the two dye-coupled partners receives the olfactory signals first, the APL neuron is the more likely candidate because it has calyx-wide dendritic arborizations where it could receive PN input (Figure 1; Figure S1). Surprisingly, disrupting this heterotypic gap-junctional communication does not perturb normal learning (Figure 3; Figure S4), despite the observation that diminishing GABA biosynthesis in APL neurons enhances olfactory learning [16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar]. This suggests that the coupling between APL and DPM neurons is partially independent from the role of GABAergic transmission of APL neurons. There are several possible explanations for this separation. Because the APL neuron innervates the MB through two branches (Figure S1D), one entering the calyx and the other entering the vertical lobe, the first possible explanation is that each APL branch plays a functionally distinct role. Learning may recruit the calyceal branch of the APL neuron, controlling inhibition onto KCs [12Turner G.C. Bazhenov M. Laurent G. Olfactory representations by Drosophila mushroom body neurons.J. Neurophysiol. 2008; 99: 734-746Crossref PubMed Scopus (236) Google Scholar, 16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar]. Later, the lobe branch of the APL neuron and the gap-junctional connection with the DPM neuron may come into play, mediating the formation of ASM in α′β′ lobes. Alternatively, the heterotypic composition of the gap junctions may mean that they favor diffusion in one direction, from APL to DPM, keeping GABA release of the APL neuron unaffected by activity in the DPM neuron. This type of rectification gated by heterotypic gap junctions has been demonstrated in the Drosophila giant fiber system [37Phelan P. Goulding L.A. Tam J.L. Allen M.J. Dawber R.J. Davies J.A. Bacon J.P. Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system.Curr. Biol. 2008; 18: 1955-1960Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar]. The latter hypothesis is also supported by the fact that blocking neurotransmission from the DPM neuron impairs intermediate-term memory without affecting learning [21Keene A.C. Krashes M.J. Leung B. Bernard J.A. Waddell S. Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation.Curr. Biol. 2006; 16: 1524-1530Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. Evidence on both the molecular and the cellular level indicates that a microcircuit of KCs and DPM neurons is involved in formation of ASM. At the molecular level, the amn-encoded neuropeptide, which is predominantly expressed in DPM neurons, plays an essential role in intermediate-term memory [13Waddell S. Armstrong J.D. Kitamoto T. Kaiser K. Quinn W.G. The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.Cell. 2000; 103: 805-813Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar]. It was later shown that amn mutants are specifically defective in ASM, the same phase of memory that is affected in age-related memory [26Tamura T. Chiang A.S. Ito N. Liu H.P. Horiuchi J. Tully T. Saitoe M. Aging specifically impairs amnesiac-dependent memory in Drosophila.Neuron. 2003; 40: 1003-1011Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar]. In the MB KCs, components of the cAMP-PKA signaling pathway play specific roles in ASM [30Schwaerzel M. Jaeckel A. Mueller U. Signaling at A-kinase anchoring proteins organizes anesthesia-sensitive memory in Drosophila.J. Neurosci. 2007; 27: 1229-1233Crossref PubMed Scopus (32) Google Scholar]. The PKA catalytic subunit DCO is required for an early phase of ASM, whereas PKA-anchoring proteins are associated with defects in a later phase of ASM [30Schwaerzel M. Jaeckel A. Mueller U. Signaling at A-kinase anchoring proteins organizes anesthesia-sensitive memory in Drosophila.J. Neurosci. 2007; 27: 1229-1233Crossref PubMed Scopus (32) Google Scholar, 38Yamazaki D. Horiuchi J. Miyashita T. Saitoe M. Acute inhibition of PKA activity at old ages ameliorates age-related memory impairment in Drosophila.J. Neurosci. 2010; 30: 15573-15577Crossref PubMed Scopus (20) Google Scholar]. Furthermore, knocking down NMDA receptors in KCs specifically abolishes ASM [32Wu C.L. Xia S. Fu T.F. Wang H. Chen Y.H. Leong D. Chiang A.S. Tully T. Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body.Nat. Neurosci. 2007; 10: 1578-1586Crossref PubMed Scopus (134) Google Scholar]. Thus, a variety of molecular changes that reside in either the KCs or DPM can affect ASM. At the cellular level, blocking synaptic release with shibire shows that transmission from α′β′ KCs is specifically required during consolidation but not retrieval [39Krashes M.J. Keene A.C. Leung B. Armstrong J.D. Waddell S. Sequential use of mushroom body neuron subsets during drosophila odor memory processing.Neuron. 2007; 53: 103-115Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar]. Similar experiments have established that transmission from DPM neurons is also specifically required during the consolidation period [15Yu D. Keene A.C. Srivatsan A. Waddell S. Davis R.L. Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning.Cell. 2005; 123: 945-957Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Keene A.C. Stratmann M. Keller A. Perrat P.N. Vosshall L.B. Waddell S. Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output.Neuron. 2004; 44: 521-533Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 21Keene A.C. Krashes M.J. Leung B. Bernard J.A. Waddell S. Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation.Curr. Biol. 2006; 16: 1524-1530Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. In contrast, transmission from αβ KCs is required during retrieval but not consolidation [14Keene A.C. Waddell S. Drosophila olfactory memory: Single genes to complex neural circuits.Nat. Rev. Neurosci. 2007; 8: 341-354Crossref PubMed Scopus (286) Google Scholar, 39Krashes M.J. Keene A.C. Leung B. Armstrong J.D. Waddell S. Sequential use of mushroom body neuron subsets during drosophila odor memory processing.Neuron. 2007; 53: 103-115Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar], indicating that KCs must act at different times during the learning and memory process. The observation that shibire blockade during the consolidation period impairs the memory process suggests that persistent activity of some form is required in α′β′ KC and DPM cells. Specifically, these data have led to a mnemonic model in which recurrent activity in an α′β′ KC-DPM loop is required to stabilize memories formed in the αβ KCs ([14Keene A.C. Waddell S. Drosophila olfactory memory: Single genes to complex neural circuits.Nat. Rev. Neurosci. 2007; 8: 341-354Crossref PubMed Scopus (286) Google Scholar]; see also [40Pitman J.L. Huetteroth W. Burke C.J. Krashes M.J. Lai S.-L. Lee T. Waddell S. A pair of inhibitory neurons are required to sustain labile memory in the Drosophila mushroom body.Curr. Biol. 2011; 21 (this issue): 855-861Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar] in this issue of Current Biology). Our data now link APL with the DPM-KC loop in intermediate-term ASM formation. Recent work has shown that activation of dopaminergic PPL1 neurons innervating the vertically projecting α and α′ lobes and heel is sufficient to signal reinforcement for aversive odor memory [17Claridge-Chang A. Roorda R.D. Vrontou E. Sjulson L. Li H. Hirsh J. Miesenböck G. Writing memories with light-addressable reinforcement circuitry.Cell. 2009; 139: 405-415Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar] whereas another subset of dopaminergic MB-M3 neurons innervating the tips of the horizontal (β and β′) lobes is specifically required for intermediate-term ASM [19Aso Y. Siwanowicz I. Bräcker L. Ito K. Kitamoto T. Tanimoto H. Specific dopaminergic neurons for the formation of labile aversive memory.Curr. Biol. 2010; 20: 1445-1451Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]. Also, functional imaging studies monitoring changes in response to trained odor have revealed that memory traces occur ∼5 min after conditioning in APL neurons [16Liu X. Davis R.L. The GABAergic anterior paired lateral neuron suppresses and is suppressed by olfactory learning.Nat. Neurosci. 2009; 12: 53-59Crossref PubMed Scopus (137) Google Scholar], ∼30 min in DPM neurons [15Yu D. Keene A.C. Srivatsan A. Waddell S. Davis R.L. Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning.Cell. 2005; 123: 945-957Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar], and ∼60 min in α′β′ KCs [41Wang Y. Mamiya A. Chiang A.S. Zhong Y. Imaging of an early memory trace in the Drosophila mushroom body.J. Neurosci. 2008; 28: 4368-4376Crossref PubMed Scopus (96) Google Scholar], whereas changes in αβ KCs are first detected 9 hr after learning [42Yu D. Akalal D.B. Davis R.L. Drosophila alpha/beta mushroom body neurons form a branch-specific, long-term cellular memory trace after spaced olfactory conditioning.Neuron. 2006; 52: 845-855Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. Taking these data together with our findings here that heterotypic gap junctions exist between the APL and DPM neurons, we propose a refined mnemonic model wherein reverberatory activity in the APL-DPM-α′β′ KC network evokes delayed memory traces in the α′β′ KCs and DPM neuron to stabilize memories formed in the αβ KCs. This reverberatory activity is likely triggered by the arrival of coincident CS-US information from olfactory PNs at the calyx [43Masse N.Y. Turner G.C. Jefferis G.S. Olfactory information processing in Drosophila.Curr. Biol. 2009; 19: R700-R713Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar] and shock-responsive dopaminergic PPL1 neurons at the MB lobes [17Claridge-Chang A. Roorda R.D. Vrontou E. Sjulson L. Li H. Hirsh J. Miesenböck G. Writing memories with light-addressable reinforcement circuitry.Cell. 2009; 139: 405-415Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar]. The evidence that output from both DPM and α′β′ KCs is required during the consolidation period suggests that some type of persistent neural activity arising in these neurons supports ASM formation. Our results show that gap junctions between DPM and APL are required for ASM formation and therefore likely contribute to this ongoing neural activity. What is the possible nature of the persistent activity, and how could gap junctions contribute to the process? Persistent activity can take the form of ongoing spiking activity, but it may also reflect some more subtle aspect of neural activity. For example, presynaptic residual calcium with slow clearance kinetics has been proposed as a buffer to sustain persistent activity in a recurrent circuit as working memory [44Wang X.J. Synaptic reverberation underlying mnemonic persistent activity.Trends Neurosci. 2001; 24: 455-463Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 45Mongillo G. Barak O. Tsodyks M. Synaptic theory of working memory.Science. 2008; 319: 1543-1546Crossref PubMed Scopus (612) Google Scholar]. However, the period of ASM consolidation is much longer than the time window that working memory operates within. Prolonging the time frame when some transient markers of past activities are sustained may require more complex circuitry with a self-sustaining feedback loop. Because DPM neurons likely release both the amn neuropeptide and the excitatory neurotransmitter acetylcholine [20Keene A.C. Stratmann M. Keller A. Perrat P.N. Vosshall L.B. Waddell S. Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output.Neuron. 2004; 44: 521-533Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar], DPM and α′β′ KCs could form a reciprocally connected excitatory recurrent loop. One limitation of a feedback loop driven by purely excitatory elements is that it is potentially susceptible to runaway excitation. The gap-junctional connection between DPM and APL could guarantee that excitation from DPM is balanced by a similar magnitude of inhibition from APL. Alternatively, chemical synapses of these two types of neurons may act independently, and additional components may be required to sustain this prolonged reverberatory activity. For example, the dopaminergic MB-M3 neurons specifically required for intermediate-term ASM [19Aso Y. Siwanowicz I. Bräcker L. Ito K. Kitamoto T. Tanimoto H. Specific dopaminergic neurons for the formation of labile aversive memory.Curr. Biol. 2010; 20: 1445-1451Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar] might be another component in the recurrent network. In conclusion, we provide both anatomical and functional evidence showing that neuronal gap junctions are required for ASM formation in a circuit that involves MB, APL, and DPM neurons. If ASM is indeed mediated by reverberatory activity within this recurrent network, gap-junctional coupling could enable APL and DPM neurons to respond synchronously throughout this period, ensuring that inhibition from APL together with neuromodulation from DPM neurons are both engaged during memory consolidation. Fly stocks were raised on standard cornmeal food at 25°C and 70% relative humidity on a 12:12 hr light:dark cycle. The lines FRTg13,GH146-GAL4,UAS-mCD8::GFP and FRTg13,tubP-GAL80 were gifts from Tzumin Lee (Howard Hughes Medical Institute, Janelia Farm, VA). The line UAS-G-CaMP-1.6 was a gift from Dierk Reiff [46Reiff D.F. Ihring A. Guerrero G. Isacoff E.Y. Joesch M. Nakai J. Borst A. In vivo performance of genetically encoded indicators of neural activity in flies.J. Neurosci. 2005; 25: 4766-4778Crossref PubMed Scopus (165) Google Scholar]. 2721 (w∗;P{GawB}5015) and 7017 (w∗;P{tubP-GAL80ts}2/TM2) were obtained from Bloomington Drosophila Stock Center. All of the RNAi lines and UAS-Dcr-2 (Table S1) were obtained from the Vienna Drosophila RNAi Center [24Dietzl G. Chen D. Schnorrer F. Su K.C. Barinova Y. Fellner M. Gasser B. Kinsey K. Oppel S. Scheiblauer S. et al.A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.Nature. 2007; 448: 151-156Crossref PubMed Scopus (1845) Google Scholar]. MB247-DsRed was a gift from André Fiala [47Riemensperger T. Völler T. Stock P. Buchner E. Fiala A. Punishment prediction by dopaminergic neurons in Drosophila.Curr. Biol. 2005; 15: 1953-1960Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar]. The transgenic fly line UAS-mKO was generated by standard techniques [48Rubin G.M. Spradling A.C. Genetic transformation of Drosophila with transposable element vectors.Science. 1982; 218: 348-353Crossref PubMed Scopus (2283) Google Scholar] in an Canton-S w1118 (iso1CJ) background. L0111-LexA::GAD was generated by P element transposition of a donor fly strain carrying P{lexA-GAD.C} on an X chromosome [49Lai S.L. Lee T. Genetic mosaic with dual binary transcriptional systems in Drosophila.Nat. Neurosci. 2006; 9: 703-709Crossref PubMed Scopus (313) Google Scholar]. We thank Tzumin Lee for the LexA system, André Fiala for MB247-DsRed, Dierk Reiff for UAS-G-CaMP-1.6, and the Bloomington Drosophila Stock Center and Drosophila Genomics Resource Center for fly stocks. We also thank Michael Hoch for the rabbit anti-INX7 antiserum and the Developmental Studies Hybridoma Bank for the 4F3 anti-Discs large antibody developed by Corey Goodman. We are very grateful to Shouzhen Xia for critical reading of the manuscript. This work was supported by a Distinguished Scholar Research Grant to A.-S.C. and a Distinguished Postdoctoral Research Grant to C.-L.W. from the National Science Council of Taiwan. Download .pdf (.63 MB) Help with pdf files Document S1. Four Figures, One Table, and Supplemental Experimental Procedures A Pair of Inhibitory Neurons Are Required to Sustain Labile Memory in the Drosophila Mushroom BodyPitman et al.Current BiologyApril 28, 2011In BriefLabile memory is thought to be held in the brain as persistent neural network activity [1–4]. However, it is not known how biologically relevant memory circuits are organized and operate. Labile and persistent appetitive memory in Drosophila requires output after training from the α′β′ subset of mushroom body (MB) neurons and from a pair of modulatory dorsal paired medial (DPM) neurons [5–9]. DPM neurons innervate the entire MB lobe region and appear to be pre- and postsynaptic to the MB [7, 8], consistent with a recurrent network model. Full-Text PDF Open Archive" @default.
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• W2036887396 title "Heterotypic Gap Junctions between Two Neurons in the Drosophila Brain Are Critical for Memory" @default.
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