Long-Term Enhancement of Synaptic Transmission Between

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J Physiol591.1 (2013) pp 287302 287

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cience Long-term enhancement of synaptic transmission between

antennal lobe and mushroom body in cultured Drosophilabrain

Kohei Ueno1, Shintaro Naganos1, Yukinori Hirano1,2, Junjiro Horiuchi3 and Minoru Saitoe1

1Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 1568506, Japan2PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 3320012, Japan3Tokyo Metropolitan University, 1-1 Minami-osawa, Hachiouji, Tokyo, 1920397, Japan

Key points

During olfactory aversive conditioning in Drosophila, odour and shock information aredelivered to the mushroom bodies (MBs) through projection neurons in the antennal lobes(ALs) and ascending fibres of the ventral nerve cord (AFV), respectively.

Using an isolated cultured brain expressing a Ca2+

indicator in the MBs, we demonstrated thatthe simultaneous stimulation of the ALs and AFV establishes long-term enhancement (LTE)in AL-induced Ca2+ responses.

The physiological properties of LTE, including associativity, input specificity and persistence,are highly reminiscent of those of olfactory memory.

Similar to olfactory aversive memory, LTE requires the activation of nicotinic acetylcholinereceptors that mediate the AL-evoked Ca2+ response, NMDA receptors that mediate theAFV-induced Ca2+ response, and D1 dopamine receptors during the simultaneous stimulationof the ALs and AFV.

Considering thephysiologicalandgenetic analogies,we proposethatLTE at theALMB synapsecan be a relevant cellular model for olfactory memory.

Abstract In Drosophila, the mushroom body (MB) is a critical brain structure for olfactoryassociative learning. During aversive conditioning,the MBs are thought to associateodour signals,conveyed by projection neurons (PNs) from the antennal lobe (AL), with shock signals conveyedthrough ascending fibres of the ventral nerve cord (AFV). Although synaptic transmissionbetween AL and MB might play a crucial role for olfactory associative learning, its physiologicalproperties have not been examined directly. Using a cultured Drosophilabrain expressing a Ca2+

indicator in the MBs, we investigated synaptic transmission and plasticity at the ALMB synapse.Following stimulation with a glass micro-electrode, AL-induced Ca2+ responses in the MBs weremediated through Drosophilanicotinic acetylcholine receptors (dnAChRs), while AFV-inducedCa2+ responses were mediated through DrosophilaNMDA receptors (dNRs). ALMB synaptic

transmission was enhanced more than 2 h after the simultaneous associative-stimulation of ALand AFV, and such long-term enhancement (LTE) was specifically formed at the ALMB synapsesbut not at the AFVMB synapses. ALMB LTE was not induced by intense stimulation of theAL alone, and the LTE decays within 60 min after subsequent repetitive AL stimulation. Thesephenotypesof associativity, inputspecificityand persistence of ALMB LTE are highly reminiscentof olfactory memory. Furthermore, similar to olfactory aversive memory, ALMB LTE formationrequired activation of the DrosophilaD1 dopamine receptor, DopR, along with dnAChR anddNR

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288 K. Ueno and others J Physiol591.1

during associative stimulations. These physiological and genetic analogies indicate that ALMBLTE might be a relevant cellular model for olfactory memory.

(Received 12 August 2012; accepted after revision 28 September 2012; first published online 1 October 2012)

Corresponding authors K. Ueno and M. Saitoe: Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa,

Setagaya-ku, Tokyo, 1568506, Japan. Emails: [email protected], [email protected]

Abbreviations AFV, ascending fibres of the ventral nerve cord; AL, antennal lobe; AN, antennae; CS, conditioned

stimulus;DA, dopamine; dnAChR, Drosophilanicotinicacetylcholine receptor;dNR, DrosophilaNMDA receptor; DopR,

DrosophilaD1 dopamine receptor; GPCR, G-protein coupled receptor; LH, lateral horn; LTE, long-term enhancement;

LTP, long-term potentiation; MB, mushroom body;MP, maxillary palp;ORN, olfactory receptorneuron;PN, projection

neuron; US, unconditioned stimulus; VNC, ventral nerve cord.

Introduction

After olfactory aversive conditioning, Drosophilaselectively increases avoidance toward the conditionedodour (conditioned stimulus, CS), which was previouslypresented with foot-shock (unconditioned stimulus,US). Because chemical ablation of the mushroom bodies

(MBs) completely prevents olfactory learning (de Belle& Heisenberg, 1994) and blocking of the synapticoutput from MBs disrupts retrieval (Dubnau et al. 2001;McGuire et al. 2001), the MBs are considered as a criticalneuronal structure for integrating odour and foot-shockinformation. Consistent with this model, recent in vivoimaging studies have demonstrated the formation ofmemory traces in the MBs after conditioning; Ca2+

responses in the MBs to an odour, which were previouslypaired with foot-shock, are increased for more than 1 h(Wang et al. 2008; Tan et al. 2010). However, the synapticmechanisms involved in neural plasticity in the MBsremain unknown.

In Drosophila, odour information is delivered to theMBs through projection neurons (PNs) in the antennallobe (AL) (Marin et al. 2002; Wong et al. 2002), whereasfoot-shock information from the body is delivered to theMBs via the ascending fibres of the ventral nerve cord(AFV) (Fig. 1A). Although in vivoimaging has suggestedthat the physiological properties of the ALMB synapsesarepotentiallyimportantforneuronalplasticityintheMB,current in vivoimaging methods are unsuited to analysingALMB synaptic transmission by stimulating AL directly.Furthermore, considerable brainmovement duringinvivorecording reduces the signal-to-noise ratio, thereby hind-

ering the kinetic analysis of MB responses.To overcome these difficulties and analyse ALMBsynaptic transmission and plasticity, we employed invitro imaging (Wang et al. 2008; Tomchik & Davis,2009) using an isolated cultured Drosophila brain todirectly stimulate the AL and AFV without brainmovement during recording of the Ca2+ responsesin the MBs. Using this in vitro imaging system inconjunction with high-speed scanning confocal micro-scopy, we observed that cholinergic ALMB synaptictransmission was enhanced for more than 2 h after the

simultaneous stimulation of the AL and AFV. Strikingly,the physiological properties and genetic requirementsof this long-term enhancement (LTE) at the ALMBsynapse are highly reminiscent of the characteristics ofolfactory memory. We propose that ALMB LTE mightbe a reasonable cellular model for learning and memorysimilar to long-term potentiation(LTP) at the mammalian

hippocampal synapses.

Methods

Fly stocks

All fly stocks were maintained at 25 2C and 60 10%humidity under a 12/12 h lightdark cycle. All transgenicflies and mutants were outcrossed to ourwild-type controlline w(CS10) (Dura et al. 1993; Tamura et al. 2003). Weused female flies for imaging analyses, and both male andfemale flies for behavioural tests.

Imaging analysis

Brains with attached ventral nerve cords (VNCs) (Fig. 1B)were dissected in 0 mM Ca2+ HL3 medium (in mM, NaCl,70; sucrose,115; KCl,5; MgCl2, 20; NaHCO3,10;trehalose,5; Hepes, 5; pH 7.3) (Stewart et al. 1994). The isolatedbrains were immobilized by placing their optic lobesbetween two nylon fibre bundles attached to the platinumgrid and were placed in a bath chamber (Fig. 1C). We cuttheVNC at thecervical connective. The ALs andAFVwereelectrically stimulated using glass micro-electrodes with

a stimulator (SEN-7103; Nihon Kohden, Tokyo, Japan)and an isolator (SS-104J for AL, SS202J for AFV, NihonKohden). During the experiments, fresh HL3 medium (inmM, NaCl, 70; sucrose, 115; KCl, 5; MgCl2, 20; CaCl2, 1.8;NaHCO3, 10; trehalose, 5; Hepes, 5; pH 7.3) was infusedinto the chamber using a peristaltic pump (2 ml min1,MiniPuls3, Gilson, Inc., Middleton, WI, USA).

Images were captured using a high-speed scanningconfocal microscope system (A1R, Nikon Corp., Tokyo,Japan) with a 20 water-immersion lens (numericalaperture 0.5; Nikon Corp.). In this imaging system,


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J Physiol591.1 Synaptic plasticity in cultured Drosophila brain 289

512 512 pixel images canbe capturedat 30 Hzfrequency.Before the experiments, the offset value was set toobtain a background intensity of approximately 0. TheF value was calculated for each pixel in the region ofinterest using NIS-elements software (NIS-Elements Ar;Nikon Corp.).

To record AL- and AFV-induced Ca2+ responses, we

stimulated the AL or AFV with three trains of 30 pulses(100 Hz, 1.0 ms pulse duration, intensity 12 thresholdcurrent) with an inter-train interval of 10 s. We obtainedinitial fluorescence values (F0) by averaging the F valuesobtained in the five sequential frames before stimulationonset. We averaged the three fluorescent traces obtainedafter stimulation and calculated (F F0)/F0 to obtainF/F0. To evaluate the relative Ca

2+ responses inducedthrough the associative stimulation of the AL andAFV or intensive AL stimulation, the F/F0 obtainedafter associative or intensive stimulation was dividedby the F/F0 obtained prior to associative or intensive

stimulation.

Electrical stimulation protocols

For associative stimulation, the AL and AFV weresimultaneously stimulated with 12 trains at an inter-val of 5 s, and the average of the first three responseswas considered as the relative Ca2+ response duringthe associative stimulation. For unpaired stimulation,AL stimuli were induced at 3 min after the end ofthe AFV stimuli. In intensive AL-alone stimulation,the pulse duration for AL stimulation was extended

from 1.0 to 1.5 ms to obtain a Ca2+

response duringintensive stimulation comparable with that elicited duringassociative stimulation.

Behavioural tests

The procedure for measuring olfactory memory hasbeen previously described (Tully & Quinn, 1985). Briefly,two aversive odours (OCT or MCH) were sequentiallydelivered to approximately 100 flies for 1 min at an inter-val of 45 s between each odour exposure. When the flieswere exposed to the first treatment, CS odour (either OCT

or MCH), they were also subjected to 1.5 s pulses of 60 VDC electric shocks every 5 s. To test olfactory memory, theflies were placed at the choice point of a T-maze whereboth odours were delivered and were allowed to choosebetween theodours. After 1.5 min, memory wascalculatedas a performance index, such that a 50:50 distribution (nomemory) yielded a performance index of zero and a 0:100distributionawayfromtheCSyieldedaperformanceindexof 100.

For extinction, the flies were sequentially exposed tothe CS odours for 1 min at 5 min intervals in the training

chamber after conditioning to expose the conditioned fliesto the CS odours 11 times before examining 1 h memory.

LexA/LexAop system in DopR rescue flies

To construct the pMBp-LexA construct, which contains

the LexA gene under the control of an MBpromoter/enhancer, a DNA fragment containing 247 bpof the 5 flanking sequence from the Mef2 gene (whichis expressed in the MBs) and the hsp70Bb minimalpromoter was subcloned into pCasper-W-LexA::GAD(Diegelmann et al. 2008) using the Gateway system (LifeTechnologies Corp., Carlbad, CA, USA) according to themanufacturers instructions. To obtain LexAop-G-CaMP2for expression of G-CaMP2 from a LexA driver, G-CaMP2cDNA amplified from pN1-G-CaMP2 was subclonedinto pCasper-lexAop-W (Diegelmann et al. 2008). Thetwo constructs were injected into embryos from ourstandard w(CS10) strain to obtain w;MBp-LexA andw;LexAop-G-CaMP2transgenic flies.

In the DopRf02676 mutant, DopRexpression is inhibitedby insertion of a piggyBac construct in the first intronof the DopR gene (Kim et al. 2007). Because thepiggyBac construct carries a UAS sequence (Thibaultet al. 2004), DopR expression can be induced from anin-frame ATG in the second exon using a GAL4 driver(Kim et al. 2007). Using this system, we generatedw;MBp-LexA,LexAop-G-CaMP2/+;DopRf02676/DopRf02676

and w;MBp-LexA/c309,LexAop-G-CaMP2;DopRf02676/DopRf02676 flies.

Statistics

All data are expressed as mean SEM. Students t test forpaired data was used to evaluate the statistical significancebetween two data sets. Formultiple comparisons, one-wayANOVA was used, followed by Bonferroni post hocanalyses. The time constant for decay kinetics (see Fig. 3I)was calculated by fitting a single exponential curve, usingPrism (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Imaging analyses of ALMB synaptic transmission

We isolated Drosophila brains expressing the Ca2+

indicator G-CaMP in the MBs using a GAL4/UAS binarysystem (Brand & Perrimon, 1993) and immobilized theisolated brains in the recording bath chamber (Fig. 1Band C). To measure synaptic transmission between PNsand MB neurons, we stimulated the AL using a glassmicro-electrode (Fig. 1C).

The MBs contain approximately 5000 intrinsic neuronscalled Kenyon cells. Kenyon cells receive the presynaptic





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terminals of the PNs at the calyx, dendritic regions ofthe MBs, and project terminals to vertical and horizontallobes.Kenyoncellsareclassifiedas/,/ andneuronsusing functional and anatomical criteria (Ito et al. 1997;Crittenden et al. 1998). While AL-induced Ca2+ responseswere consistently observed in the distal ends of the verticallobes (/), these responses were weak and infrequent in

the horizontal lobes (//) (Fig. 2A and B) and in thecalyx (Fig. 2C and D).

In contrast, previous imaging studies havedemonstrated that odour and electrical stimulationto antennae (AN) induces significant Ca2+ responsesnot only in vertical lobes but also in horizontal lobesand the calyx (Martin et al. 2007; Wang et al. 2008).Notably, while our external recording solution contains20 mM Mg2+, these previous studies employed lowerconcentrations of Mg2+ (4 mM). Therefore, we suspectthat the robust Ca2+ responses in the horizontal lobeobserved in other studies are due to the low concentration

of Mg2+

.To test this possibility, we examined AL-induced

responses in 4 mM Mg2+ recording solution. As suspected,we observed a significant increase in responses in thehorizontal lobes and in the calyx in recording solutioncontaining 4 mM Mg2+ (Fig. 2E). This result suggests that

Ca2+ responses in the horizontal lobe and in the calyx aremore sensitive to external Mg2+ than Ca2+ responses inthe vertical lobes.

Consistent with anatomical studies indicating that PNsof each AL send their axons to the ipsilateral MB (Marinet al. 2002; Wong et al. 2002), stimulation of an ALinduced Ca2+ responses in the ipsilateral, but not the

contralateral, MB (Fig. 2F and G). In contrast to ALstimuli, AFV stimulation induced Ca2+ responses in theMBs of both brain hemispheres (Fig. 2H and I). Weemployed 20 mM Mg2+ in the recording solution in thisstudy, as this concentration lies within the physiologicallyrelevant range (see Discussion), and we primarily focusedon Ca2+ responses in the vertical lobes in subsequentexperiments.

LTE of ALMB synaptic transmission

In our in vitro imaging system, we observed the LTEof AL-induced Ca2+ responses in the MB after thesimultaneous stimulation of AL and AFV for 1 min(Fig. 3AC). In contrast to associative AL and AFVstimulation, unpaired AL and AFV stimulation did notenhance the Ca2+ responses (Fig. 3A, B and D). In this

Figure 1. Preparation of isolated cultured Drosophila brains for imaging

A, odour signalling pathway in Drosophila. An odour signal perceived through the dendrites of the ORNs in the AN

is transmitted to PNs in the AL and subsequently to MB or LH neurons that project inhibitory GABAergic terminals

onto MB neurons. The foot-shock signal from the body is delivered through the AFV. B, a c309;UAS-G-CaMP

fly brain with the VNC. The VNC was cut at the cervical connection (arrowhead) for stimulating AFV. G-CaMP

expression was observed throughout the , , , and lobes of the MBs. OL, optic lobe; SEG, sub-oesophageal

ganglion. C, schematic diagram of the recording setup. Glass micro-electrodes were placed on the AL, and the

cut ends of the AFV were suctioned for stimulation.





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Figure 2. Ca2+ responses in the MBs

A, Ca2+ responses induced by AL stimulation in the vertical (arrow) and horizontal (arrowhead) lobes of the

MB. While robust Ca2+ responses were observed in the distal end of the vertical lobe, the significant responses

were not observed in the horizontal lobe (33 of 38 brains). B, nine traces of AL-induced Ca2+ responses in the

vertical (upper) and horizontal (lower) lobes of the MB. C, the calyx exhibited weak AL-induced Ca2+ responses. D,

summary of Ca2+ responses in the vertical lobes (black line) and the calyx (grey line) of the MB (P< 0.05 between

the vertical lobe and the calyx; n = 6). E, AL-induced Ca2+ responses were increased in the horizontal lobes (upper)

and in the calyx (middle) with decreasing extracellular Mg2+ concentration from 20 to 4 mM (P< 0.05, n = 5).

F and G, AL stimulation produced Ca2+ responses in the ipsilateral but not in the contralateral MB lobe. Hand I,

AFV stimulation produced Ca2+ responses in both sides of MB lobes (n = 5). All data in this study are shown as

mean SEM.





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study, we primarily used the c309 Gal4 driver to measurethe Ca2+ response in the MBs because we observed themost significant LTE with this MB driver. For example,although the OK107 MB driver generated higher UAStransgene expression than c309 (Aso et al. 2010), theLTE observed with OK107 was lower than that observed

with c309; the relative Ca2+ responses at 15 min after theassociativestimuli were 1.48 with OK107 (see Fig. 4H)and2.40 with c309 (Fig. 3C).

These results suggest that the associative AL and AFVstimulation produces the LTE of ALMB synaptic trans-mission. However, PNs in the AL also convey olfactory

Figure 3. LTE of AL-induced Ca2+

responses in the MBA, stimulation protocol. In associative stimulation, AL and AFV were stimulated simultaneously with 12 stimulus

trains (AL + AFV). In unpaired stimulation, AL stimulation was induced after the end of AFV stimulation. B,

typical changes in G-CaMP fluorescence at the distal end of the MB vertical lobes (/ lobes), and the traces

of AL-induced Ca2+ responses in the MBs before and after associated or unpaired stimulation. The arrowhead

indicates stimulation onset. C and D, summary of AL-induced Ca2+ responses in the MBs measured before (pre)

and after associative (C) and unpaired (D) stimulation. The shaded column in C indicates Ca2+ responses during

associative stimulation (AL + AFV) (P< 0.05 compared with pre responses). E, averaged traces of AL-induced

Ca2+ responses before and after treatment with 50 M picrotoxin for 15 min. F, averaged traces of AL-induced

Ca2+ responses before and 5 min after associative AL + AFV stimulation. G and H, peak Ca2+ responses were

normalized to 1.0 to evaluate the effects of picrotoxin (G) and associative stimulations (H) on decay kinetics. I,

picrotoxin treatment reduced the decay kinetics (1/, where represents the time constant) of the Ca2+ response,

while associative stimulations did not (P< 0.05 by t test). n = 68 for all data.





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Figure 4. LTE occurred in both and lobes but not in the

calyx

A, G-CaMP was expressed in each and lobe by the c305a and

c739 GAL4 drivers, respectively. B and C, typical traces of

AL-induced Ca2+ responses in the (B) and lobes (C) before (pre)

and 5 min after associative AL and AFV stimulation. D and E,

AL-induced Ca2+ responses were enhanced after associative

stimulation in both (D), and (E) lobes (P< 0.05 compared with

pre responses, and P< 0.05 comparison between 5 and 30 min).

signalling to lateral horn (LH) neurons (Marin et al.2002; Wong et al. 2002), which in turn project theirGABAergic terminals onto MB neurons (Perez-Oriveet al. 2002; Yasuyama et al. 2002; Fig. 1A). Therefore,it is possible that the enhanced Ca2+ responses in theMBs reflect the inhibition of LH neurons rather thanincreases in excitatory transmission between the AL and

the MB.To test this possibility, we recorded AL-induced

Ca2+ responses in the brain following treatment withpicrotoxin, a GABA receptor blocker (Su & ODowd,2003). Picrotoxin treatment increased the amplitude ofthe AL-induced Ca2+ response (Fig. 3E) but significantlydecreased the decay kinetics of the Ca2+ response(Fig. 3G and I). However, after associative AL and AFVstimulation, the AL-induced Ca2+ response amplitudesincreased (Fig. 3F) without any changes in the decaykinetics (Fig. 3H and I). These results support theidea that LTE in the MBs results from an increase in

excitatory synaptic transmission between PNs and MBneurons.

The vertical lobes in Drosophila consist of and

lobes (Fig. 4A; Ito et al. 1997). Although previous studiesdemonstrate that the associative stimulation of the ANand VNC enhances AN-induced Ca2+ responses in

but not lobes (Wang et al. 2008), we observed that theassociative AL and AFV stimulation enhances AL-inducedCa2+ responsesin bothlobes. To confirm the enhancementin both the and the lobes, we employed the and

lobe-specific GAL4 drivers c739 and c305a, respectively(Yang et al. 1995; Krashes et al. 2007).

As shown in Fig. 4A and B, LTE was significantlyinduced in both lobes after associative AL and AFVstimulations. Notably, the PNs in the AL received olfactoryinformation from the olfactory receptor neurons (ORNs)in the maxillary palps (MPs) in addition to the AN(Rajashekar & Shamprasad, 2004), and AN stimuli evokedmuch higher Ca2+ responses in the lobe than in the lobe (Wang et al. 2008). Because we stimulated the entireAL, AL-induced Ca2+ responses and LTE in the lobemight reflect the activation of PNs that receive input fromORNs in the MPs. Consistent withthe results of a previousstudy (Wang et al. 2008), we did not observe substantialLTE at the calyx (Fig. 4F and G), although pre-synaptic

PNs form synapses with post-synaptic MB neurons at thecalyx (Fig. 1A).

F and G, typical traces of AL-induced Ca2+ responses in the vertical

lobe (F) and the calyx (G) before (pre) and after associative AL and

AFV stimulation. Both traces were obtained from identical

OK107-GAL4/UAS-G-CaMPbrains. Hand I, LTE was observed after

associative stimulation in the vertical lobe (H) but not in the calyx (I)

(P< 0.05 compared with pre responses). n = 67 for all data.





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Associativity, input specificity and persistence of LTE

LTP in the mammalian hippocampus and post-tetanicpotentiation in the Drosophila neuromuscular junction(Zhong & Wu, 1991) can be induced through theintensive stimulation of fibres of a single input pathway.Therefore, we examined whether the association of AL

and AFV stimuli is essential for LTE formation or whetherintense AL stimulation alone is sufficient(Fig. 5A). Duringassociative stimulation, robust increases in Ca2+ responseswere observed in MB neurons (Fig. 5B and C). Toincrease Ca2+ responses in MB neurons during intense ALstimulationalone,weextendedthepulsedurationfrom1.0to 1.5 ms. Although this intense AL stimulation producedincreases in Ca2+ responses comparable with associativestimulation (Fig. 5BD), no subsequent enhancement ofAL-induced Ca2+ responses was observed (Fig. 5Band D).Thus, LTE at the ALMB synapse probably requires theassociation of AL and AFV stimuli.

The association of an odour with foot-shock selectivelyincreases avoidance to the odour but not to the shock(Fig. 5E). This observation prompted us to test the inputspecificity of LTE in the MBs. As shown in Fig. 5F,the simultaneous stimulation of AL and AFV enhancedAL-induced responses but not AFV-induced responses.This indicates that although stimulation of the AFV isrequired for LTE induction, LTE is selectively induced atALMB synapses but not at AFVMB synapses.

To further assess the physiological properties of LTEat the ALMB synapse, we examined the persistenceof this LTE. When we stimulated AL at 120 min afterthe induction of LTE, we still observed a significant

enhancement in the Ca2+

response (Fig. 6A, filled circles).However, when we stimulated AL repeatedly every 5 minafter the associative stimulations, LTE decayed rapidly anddisappeared within 1 h (Fig. 6B, filled circles). This decaywas not theresult of synaptic transmission fatigue betweenthe AL and MB or the desensitization of postsynapticresponses, as repeated AL stimulation every 5 min didnot decrease AL-induced Ca2+ responses (Fig. 6B, opencircles).

Notably, the persistence of LTE is highly reminiscentof the persistence of olfactory memory. Similar to LTE,the olfactory memory formed by olfactory conditioningis extinguished through subsequent repeated exposure tothe CS odours (Quinn et al. 1974; Qin & Dubnau, 2010).Indeed, the 1 h memory after olfactory conditioningwas extinguished through repetitive CS presentationsapplied every 5 min (Fig. 6C). This effect was not due todesensitization, as repeated exposure to the CS odoursevery 5 min for 1 h prior to olfactory conditioningdid not affect the 3 min memory, which representslearning ability and is lowered by desensitization tothe odour used in subsequent olfactory conditioning(Fig. 6D).

Figure 5. Associative stimulation of AL and AFV induces LTE in

the ALMB synaptic transmission

A, stimulation protocols for associative stimulations (associative) and

intense AL stimulations (AL alone). B, typical traces of AL-induced

Ca2+ responses before, during and after associative or AL-alone

stimulation. C and D, summary of AL-induced Ca2+ responses in the

MB before and after associative (C) or AL-alone (D) stimulation. The

shaded columns indicate Ca2+ responses during associative and

intense AL-alone stimulation (P< 0.05 compared with pre

responses). E, avoidance of flies to 30 and 60 V electric shocks. Flies

that had been previously trained to associate 3-octanol (OCT) or

4-methylcyclohexanol (MCH) with foot-shocks did not exhibit altered

shock avoidance. Avoidance tests were performed at 15 min after

conditioning. F, AL- and AFV-induced Ca2+ responses before (pre)

and 15 min after the associative stimulation (P< 0.05). NS, not

significant; n = 68 for all data.





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nAChR and NMDA receptors mediate Ca2+ responses

and LTE in the MBs

In previous studies, it was demonstrated that thecholinergic synaptic currents in MB neurons are mediatedthrough Drosophila nicotinic acetylcholine receptors(dnAChRs), which are widely expressed in Drosophila

brain including MBs (Jonas et al. 1994; Su & ODowd,2003). Consistent with this result, we observed thatAL-induced Ca2+ responses were suppressed in the pre-sence of mecamylamine, an nAChR antagonist (Kazama& Wilson, 2008; Fig. 7AC). However, the AFV-inducedCa2+ responses were not suppressed by mecamylamine(Fig. 7Aand B).

In olfactory aversive conditioning, foot-shockinformation, which is conveyed through the AFV, canbe substituted by artificial stimulation of dopaminergic

(DAergic) neurons (Claridge-Chang et al. 2009; Aso et al.2010). DA neurons innervate the MBs (Mao & Davis,2009), and the dopamine D1 receptor, DopR (Sugamoriet al. 1995), is required in the MBs for olfactory aversivememory formation (Kim et al. 2007). Therefore, we werecurious to test whether AFV-induced Ca2+ responsesare mediated by DopR, although it is classified as

G-protein coupled receptor (GPCR) (Sugamori et al.1995). However, AFV-induced Ca2+ responses were notsuppressed byDopRIn(3LR)234 mutations (Kim et al. 2007)or exposure to butaclamol, a DopR antagonist (Sugamoriet al. 1995; Fig. 7D and E). AL-induced Ca2+ responseswere also unaffected by DopRIn(3LR)234 mutations andbutaclamol (Fig. 7F and G). These results suggest thatneither AL- nor AFV-induced Ca2+ responses in the MBsare mediated through DopR.

Figure 6. LTE is extinguished through

repetitive post-associative AL stimulation

A, AL-induced Ca2+ responses in the MB

measured at 60 and 120 min after associative

stimulation (filled circles) were still significantly

higher than the control responses (open

circles), indicating that LTE persists for at least120 min. B, the enhanced Ca2+ responses

declined within 60 min through repetitive AL

stimuli, induced every 5 min, after associative

stimulation (filled circles). Ca2+ responses were

not diminished by repetitive AL stimuli in the

control MBs (open circles). C, extinction of

olfactory memory in trained flies through

repetitive presentation of the conditioned

odour. D, the extinction protocol administered

prior to olfactory conditioning did not affect

olfactory learning (P< 0.05 compared with

pre-associative responses). n = 68 for all data.





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Drosophila NMDA receptors (dNRs) are expressed inthe MBs and are involved in olfactory memory formation(Xia et al. 2005; Wu et al. 2007; Sinakevitch et al. 2010;Miyashita et al. 2012); therefore, we hypothesized thatdNRs might mediate the AFV-induced Ca2+ response.Consistent with this hypothesis, AFV-induced Ca2+

responses were suppressed in response to exposure to the

NR antagonist MK801 (Fig. 7HJ). These results indicatethat while AL-induced Ca2+ responses are mediatedthrough dnAChRs, AFV-induced Ca2+ responses aremediated through dNRs.

To test whether dnAChRs and dNRs are required forLTE formation, each receptor antagonist was appliedduring associative stimulations (Fig. 7K). As shown inFig. 7LQ, each antagonist suppressed LTE formation.Because AL-induced Ca2+ responses are mediatedthrough dnAChRs and the effects of mecamylaminelast approximately 10 min after washout (Fig. 7C),mecamylamine treatment reduced AL-induced Ca2+

responses at 5 min after associative stimulation (Fig. 7P).

DopR in the MBs is required for PAE formation

As reported previously (Kim et al. 2003, 2007), DopRis preferentially expressed in MB and required forolfactory aversive memory formation (Fig. 8A), althoughit mediates neither AL nor AFV-induced Ca2+ responses.To address whether DopRs are necessary for inducing LTE,we examined LTE in DopR mutants. Consistent with thebehavioural data, LTE was not produced in DopRmutants(Fig. 8B and C). Furthermore, LTE formation in controlbrainswassuppressedinresponsetobutaclamoltreatment

(Fig. 8D and E) during associative stimulations (seeFig. 7K), indicating that DopR is physiologically requiredfor LTE formation. Interestingly, Ca2+ responses duringthe associative stimulation were enhanced above controllevels as a result of the DopR mutation (Fig. 8F) and inresponse to butaclamol treatment (Fig. 8G), although AL-and AFV-induced Ca2+ responses were not different fromthose in the control (Fig. 7DG).

Considering that DopR mutations do not affectAFV-inducedCa2+ responsesintheMBs,DopRsexpressedin areas outside of MB neurons might be required forLTE in contrast to the DopR requirement for olfactoryaversive memory formation. To test this possibility, weused rescue experiments with DopRf02676 mutants (Kimet al. 2007). DopRf02676 is a null allele as a result of inter-

ference of transcription from its endogenous promoterby the insertion of piggyBac in the first intron. ThispiggyBac insertion acts as a carrier of rescue constructin the presence of GAL4, as the inserted piggyBac containsUAS, whichfunctions as an exogenous enhancer/promoterwhen bound by GAL4 to initiate transcription of thedownstream gene (Thibault et al. 2004). We used boththe GAL4/UAS (Brand & Perrimon, 1993) and theLexA/LexAop binary systems (Lai & Lee, 2006; Fig. 9Aand B) to generate transgenic flies expressing both theDopR transgene and G-CaMP2 (Tallini et al. 2006)independently in the MBs ofDopRf02676. Similar to hypo-

morphic DopRIn(3LR)234

mutants, LTE did not occur in theDopRf02676-null mutants (Fig. 9C and D). However, in arescue line expressing DopRsin the MBs, LTE was restoredto the control level (Fig. 9E). Notably, while LTE wasrestored in this transgenic line, the hyper-enhancementof Ca2+ responses during associative stimulation was notreduced to normal levels (Fig. 9F). These results suggestthat while DopR in the MBs is essential for LTE formation,the larger Ca2+ response during associative stimulationresults from defects in DopRexpressed in areas outside ofthe MBs.

Discussion

Using in vitro imaging, we directly measured ALMBsynaptic transmission and successively observed theinduction, persistence and decline of LTE at the ALMBsynapse. Previous studies have shown that odour-inducedCa2+ responses in the MBs were increased more than 1 hafter single-cycle olfactory aversive conditioning (Wang

Figure 7. dnAChR and dNR activities during associative stimulation are required for LTE

A, typical tracesof AL-and AFV-induced Ca2+ responses before and afterthe application of 100 M mecamylamine.

B, summary of the effects of mecamylamine on AL- and AFV-induced Ca 2+ responses (P< 0.05). C, after bath

application of 100 M mecamylamine, AL-induced Ca2+

responses were decreased and abolished within 9 min.Subsequently, the responses were restored at 10 min after washout (18 min). D and E, neither DopR mutations nor

the DopR antagonist (50 M butaclamol) altered AFV-induced Ca2+ responses. Typical traces of AFV-induced Ca2+

responses (D) and the summary ofthe effects of DopR inhibitions (E). Fand G, neither DopR mutationsnor theDopR

antagonist altered AL-induced Ca2+ responses. H, AFV-induced Ca2+ responses were suppressed using 100 M

MK801, while mecamylamine and the AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione)

had no effect (P< 0.05 compared with the response at 0 min). I and J, MK801 suppressed AFV-induced Ca2+

responses but not AL-induced Ca2+ responses. Typical AL- and AFV-induced Ca2+ traces (I), and averaged peak

responses in saline (filled columns) and after (open columns) application of 100 M MK801 (J) (P< 0.05). KQ,

both dnAChR and dNR activities during associative stimulation are required for LTE formation. K, experimental

design. While LTE was induced in control conditions (L, O), the application of mecamylamine (M, P) and MK801

(N, Q) during associative stimulation prevented LTE induction (P< 0.05 compared with pre-associative responses

(pre). n = 68 for all data.





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et al. 2008; Tan et al. 2010). However, the synaptic basisof such neural plasticity in the MBs is not understood.Odour information received through ORNs in the ANand MPs is transmitted to PNs in the AL, which in turnconvey signals to MB neurons. Although synaptic trans-mission from ORNs to PNs is also increased after olfactoryconditioning, this increase disappears within 10 min (Yu

et al. 2004). Thus, it has been suggested that memory tracemight be formed through the enhancement of ALMB

synaptic transmission. Consistent with this hypothesis,LTE at the ALMB synapse lasts more than 2 h.

ALMB LTE exhibits characteristic physiologicalproperties, including associativity, input specificity andpersistence. Importantly, these properties are alsoobserved in olfactory aversive memory. While ALMBLTE formation requires the correlated activity of AL

and AFV inputs, olfactory aversive memory formationrequires the correlated presentation of CS odour and

Figure 8. DopR activity during associative stimulation is required for LTE

A, 5 min memory was impairedin the DopR mutant, DopRIn(3LR)234 . B and C, DopR mutants are defectivefor ALMB

LTE formation. D and E, application of 50 M butaclamol during associative stimulation blocked LTE induction. F

and G, DopR mutations and butaclamol treatment further increased Ca2+ responses during associative stimulation

compared with the wild-type control and DMSO treatment (P< 0.05). n = 58 for all data.





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US foot-shock. In olfactory aversive conditioning, odourand foot-shock signals are associated in the MBs.In this study, we demonstrated that signals deliveredfrom the AL and AFV are associated in the MBs andproduce LTE at ALMB synapses. Trained flies increaseavoidance to CS odour but not to US foot-shock.Similarly, LTE is specifically formed at ALMB but not

AFVMB synapses. This input specificity of LTE mightexplain why avoidance to foot-shock is not increasedfollowing olfactory aversive conditioning. Furthermore,both ALMB LTE and olfactory memory were reducedafter subsequent repetitive AL stimulations and CS odourpresentations, respectively. This suggests that attenuatedALMB synaptic transmission might be involved in

Figure 9. DopRs expressed in the MBs are required for LTE formation

A, G-CaMP2 fluorescence observed in the MBs of an MBp-LexA;LexAop-G-CaMP2 fly. B, G-CaMP2 fluorescence

is observed in all , , , and lobes of an MBp-Lex;LexAop-G-CaMP2 brain. CE, ALMB LTE

(C) disrupted in DopR mutants (D) was restored through expression of the DopR+ transgene in the

MBs (E). AL-induced Ca2+ responses before (pre) and after associative stimulation were recorded in the

MBs from control MBp-Lex,LexAop-G-CaMP2/+ (C), MBp-Lex,LexAop-GCaMP2/+;DopRf02676/DopRf02676 (D)

and MBp-Lex,LexAop-GCaMP2/c309;DopRf02676/DopRf02676 flies (E) (P< 0.05 compared with pre-associative

stimulation). F, the larger increase in Ca2+ responses during associative stimulation in DopRf02676 (open column)

were not decreased in the rescue line (shaded column) (P< 0.05 compared with control brains). n = 67 for all

data.





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the memory extinction process. In addition to thesephysiological similarities, both ALMB LTE and olfactoryaversive memory require the activity of dNRs and DopRsduring association. These results demonstrate that LTE atthe ALMB synapse can be an appropriate cellular modelfor olfactory memory.

In this study, we employed 20mM Mg2+ in the

external recording solution because various groups haveshown that the Mg2+ concentration in the Drosophilahaemolymph ranges between 20 and 33 mM (Croghan &Lockwood, 1960; Begg & Cruickshank, 1963; Stewart et al.1994). In mammals, the Mg2+ concentration is higher inthe cerebrospinal fluid than in the plasma (McKee et al.2005). Furthermore, while 10 mM Mg2+ concentrationis not sufficient for the Mg2+ blockade of DrosophilaNMDA receptors, 20 mM Mg2+ is sufficient to producethis effect (Miyashita et al. 2012). We thus propose that the20 mM Mg2+ concentration used in our study lies withinthe physiologically relevant range. We employed a much

higher Mg2+

concentration (20 mM) than those used inprevious in vivo and in vitro imaging studies (


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Author contributions

K.U. designed and performed most of the experiments. S.N.

contributed to the imaging study, and Y.H. contributed to thegenetics and behaviour studies. M.S. supervised and wrote the

manuscript with K.U. and J.H.

Acknowledgements

We thank J. Dubnau (Cold Spring Harbor Laboratory)for DopR mutants, K. Scott (University of California,

Berkeley) for UAS-G-CaMP transgenic flies, S. Diegelmann

(University of Cambridge, UK) for pCasper-W-LexA::GAD and

pCasper-lexAop-W vectors and J. Nakai (Saitama University,

Japan) for G-CaMP2. We also thank Ms Fukuda and MsOfusa for stock maintenance and rearing flies. This research

was supported by a Grant-in-Aid for Scientific Research in

Innovative Areas Systems Molecular Ethology to M.S., and

MEXT/JSPS KAKENHI Grant (21700376 and 23700405) to K.U.

C2012 The Authors The Journal of Physiology


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