Author manuscript, published in "Nature Neuroscience (2011)"
DOI : 10.1038/nn.2846
Mushroom Body Efferent Neurons Responsible For
Aversive Olfactory Memory Retrieval In Drosophila
Julien Séjourné1, Pierre-Yves Plaçais1, Yoshinori Aso2,3, Igor Siwanowicz2, Séverine
Trannoy1, Vladimiros Thoma2, Stevanus R. Tedjakumala2, Gerald M. Rubin3, Paul Tchénio1,4,
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Kei Ito5, Guillaume Isabel1, Hiromu Tanimoto2,6 and Thomas Preat1,6
1
Genes and Dynamics of Memory Systems, Neurobiology Unit, CNRS, ESPCI, 75005 Paris,
France.
2
Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, D-82152 Martinsried, Germany.
3
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia
20147, USA.
4
Nanooptique et Physiologie Intégrée, Université Paris-Sud, CNRS UPR 3321, Batiment 505,
Campus d'Orsay, 91400 Orsay, France.
5
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo
113-0032, Japan.
6
These authors contributed equally to this work.
1
Aversive olfactory memory is formed in the Drosophila mushroom bodies (MB).
Memory retrieval requires MB output, but it remains unknown how a memory trace in
the MB drives conditioned avoidance of a learned odour. To identify neurons involved
in olfactory memory retrieval, we performed an anatomical and functional screen of
defined sets of MB extrinsic neurons. Here we show that MB-V2 neurons are essential
for retrieval of both short- and long-lasting memory, but neither for memory formation
nor for memory consolidation. We further show that MB-V2 are cholinergic efferent
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neurons that project from the MB vertical lobes to the middle superiormedial
protocerebrum and the lateral horn (LH). Notably, the odour response of MB-V2
neurons is modified after conditioning. As the LH is implicated in innate responses to
repellent odorants, we propose that during memory retrieval, MB-V2 neurons reinforce
the olfactory pathway involved in innate odour avoidance.
Different odours induce innate approach or avoidance behaviours in Drosophila. Innate
odour responses can be modulated by experience, such as associative learning. After
simultaneous exposure to an electric shock and an odorant, flies form aversive memory and
show robust conditioned odour avoidance that lasts for hours to days, depending on the
training protocol1-3. The neural pathways for odour or shock processing and signal integration
in the fly brain have been intensely studied in recent years. Odour information is first
represented in the antennal lobes in the form of olfactory receptor neuron activity4. Projection
neurons then convey this information to higher order processing centres4: the mushroom
bodies (MB) and the lateral horn (LH). In contrast, aversive reinforcement signals in response
to electric shock are relayed to the MB via dopaminergic neurons5-7. The olfactory and
2
electric shock signals are integrated in the MB to form aversive olfactory memory1, 2. The MB
are however dispensable for innate avoidance of the repellent odours8, 9.
In adult Drosophila, the MB consist of approximately 2000 Kenyon cells per brain
hemisphere, which may be classified into three major types based on their axonal projection: γ
neurons, which form only a medial lobe, α/β neurons, whose axons branch to form a vertical
(α) and a medial (β) lobe, and α’/β’ neurons, which also form a vertical (α’) and a medial
(β’) lobe10. Functional brain imaging has revealed localised activation of cAMP/PKA
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signalling in the MB α lobe in response to simultaneous cholinergic and dopaminergic
stimulation11,12, that represent respectively the odorant and electric shock pathways.
Following associative conditioning, calcium imaging studies have shown that a short-term
memory trace is formed in the α’/β’ neurons13, and a long-term one in α lobes14. Previous
studies have shown that the output of the α/β neurons is necessary for the retrieval of all
phases of olfactory memory15, 16, but the neural circuits that translate the associative memory
trace in the MB into conditioned odour avoidance remain unknown.
To understand the neuronal basis of memory retrieval, we conducted a functional screen
based on collections of GAL4 driver lines for MB-extrinsic neurons17, 18 (data not shown). We
blocked the neurotransmission of GAL4-expressing cells during the retrieval of different
forms of short- and long-lasting memories using Shits [19], a dominant negative temperature
sensitive variant of Dynamin that can reversibly block synaptic vesicle endocytosis at
elevated temperatures. Because neurons responsible for memory retrieval should receive
synaptic inputs from the MB and project to other parts of the brain, we characterized the
polarity of MB extrinsic neurons by expressing a presynaptic marker20. We further
characterized the identified MB output neurons with regard to neurotransmitter and
physiological response properties using immunohistochemistry and functional calcium
imaging.
3
Results
MB-V2 neurons are efferent to the MB
In a preliminary screen, we functionally analyzed 58 enhancer-trap GAL4 lines showing
expression in putative MB extrinsic neurons for a defect in aversive memory retrieval (data
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not shown). We identified a particular set of MB extrinsic neurons, the MB-V2, as a candidate
for the memory retrieval pathway. MB-V2 neurons are labelled in the GAL4 driver lines
NP249218 (Fig. 1a-c and Supplementary Fig. 1a) and MZ16017, 18 (Supplementary Fig. 1d).
The MB-V2 neurons have their cell bodies posterior to the LH (Supplementary Fig. 1b,e),
and arborise in the vertical lobes of the MB, the lateral part of the middle superiormedial
protocerebrum (msmpr) just posterior to the vertical lobes, and the LH (Fig. 1a-c). Whereas
membrane-targeted markers (mCD8::GFP or myr::RFP) uniformly label MB-V2 processes in
all three target neuropils (Fig. 1a-c and Supplementary Fig. 2), the signals of the presynaptic
marker Syt::GFP (a fusion of eGFP and the synaptic vesicle protein Synaptotagmin20) were
highly enriched in the msmpr and LH but not detectable in the MB vertical lobes (Fig. 1d-f).
We observed similar results with other presynaptic markers (Syt::HA
21
and nSyb::GFP
17
)
(Fig. 1m-r and Supplementary Fig. 2d-i). Quantification of Syt::HA and mCD8::GFP signal
intensity in the MB-V2 terminals confirmed that Syt::HA is highly enriched in the msmpr and
the LH, but not on MB (Supplementary Fig. 2j). Thus, MB-V2 neurons are likely to relay
information from the MB vertical lobes to the msmpr and LH.
Flp-out clones22 of single MB-V2 neurons revealed two stereotyped neuron classes:
MB-V2α and MB-V2α’. MB-V2α neurons innervate across the shaft of the α lobe of the
ipsilateral MB and terminates in the msmpr and dorsal area of the LH of both hemispheres
4
(Fig. 1g-i), whereas MB-V2α’ neurons arborise in the tip of the α’ lobe of the ipsilateral MB
and project to the msmpr and the ventral area of the LH (Fig. 1j-l) of both sides. The
collaterals of MB-V2α’ also terminate in a brain area ventral medial to the LH. MB-V2α and
MB-V2α’ neurons are labelled in NP2492, whereas only the MB-V2α appears to be labelled
in MZ160 (Supplementary Videos 1 and 2). Neurons in the MB-V2 cluster are the only
GAL4-expressing cells that are clearly shared by NP2492 and MZ160.
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MB-V2 output is required for olfactory memory retrieval
To address the role of the MB-V2 neurons in innate odour responses, we first measured
avoidance of 4-methylcyclohexanol (MCH) and 3-octanol (OCT), while blocking the output
of MB-V2 neurons. Consistent with previous reports showing that MB are dispensable for
innate odorant avoidance8, 9, we found that the odour avoidance of both NP2492/UAS-shits
and MZ160/UAS-shits flies at restrictive temperature was normal (Table 1). We then
examined the effect of the transient blockade of these neurons during the test on 2-h memory
retrieval after a single cycle conditioning. We observed a strong memory impairment in both
NP2492/UAS-shits and MZ160/UAS-shits flies (Fig. 2a).
To assess whether MB-V2 output is also required for memory formation, we next
blocked these neurons transiently during training and consolidation, and tested the memory 2
hours later at the permissive temperature. Interestingly, blocking the MB-V2 during training
and consolidation did not significantly impair 2-h memory performance (Fig. 2b). Moreover,
no memory defect was observed when flies were conditioned and tested at permissive
temperature (Fig. 2c).
Memory directly after training was also reduced significantly when MB-V2 output was
blocked continuously (Supplementary Fig. 3a). As MB-V2 output is not required for
5
memory acquisition (Fig. 2a), this result suggests that immediate-memory retrieval also
depends on MB-V2 output.
Output of α/β neurons is required for appetitive memory retrieval23. In order to
determine whether MB-V2 output is required for all types of memory retrieval, we performed
appetitive conditioning, where electric shocks are replaced by sugar reward24. A sugar
response defect was observed in NP2492/UAS-shits flies tested at restrictive temperature
(Supplementary Fig. 3b1) that may correspond to a motivation defect, and therefore
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appetitive memory could not be assessed in these flies. No sugar defect was observed in
MZ160/UAS-shits flies at restrictive temperature (Supplementary Fig. 3b2) and no appetitive
memory defect was detected in these flies when MB-V2 neurons were blocked during
retrieval (Supplementary Fig. 3c). These results show that MB-V2α output is dispensable
for appetitive memory retrieval.
The odorants are highly repellent to naïve flies at the concentration we typically use for
conditioning (e.g. Table 1). To address whether the requirement of MB-V2 for memory
retrieval depends on how aversive the odour is, we next used odorants that are neutral to naïve
flies for conditioning. We found that OCT and MCH are no longer aversive when diluted 300
times compared to the usual concentration (1.2 x 10-6 M and 1.08 x 10-6 M respectively, data
not shown). When conditioning and test were performed with the diluted odours, both
NP2492/UAS-shits and MZ160/UAS-shits flies showed a robust 2-h memory impairment
(Supplementary Fig. 2d), suggesting that MB-V2 output is required for aversive memory
retrieval irrespective of the repulsiveness of odorants.
GAL80 expression rescues the memory retrieval impairment
6
Neurons in the MB-V2 cluster are the only GAL4-expressing cells that are clearly
shared by NP2492 and MZ160, but each line shows expression in additional brain regions,
such as the fan-shaped body in NP2492 (Supplementary Fig. 1a and Supplementary Video
1) and the optic lobes in MZ160 (Supplementary Fig. 1c,g and Supplementary Video 2).
Even though the behavioural phenotype is common and very specific for the retrieval of
aversive memory (Fig. 2 and Supplementary Fig. 3), one could not formally exclude
possibilities that different sets of neurons outside MB-V2 happen to induce the same memory
phenotype. To confirm that blocking MB-V2 neurons is the cause of the memory impairment,
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we aimed to use the intersectional approach and searched for GAL80 lines that can
preferentially silence GAL4 activity in MB-V2. Cha3.3kb-GAL80
[25]
reduced the reporter
expression in MB-V2 in NP2492 and MZ160 (Fig. 3a-b, Supplementary Fig. 1a,c,d,f and
Supplementary Video 3). To improve the specificity of the intersectional manipulation to
MB-V2, we also replaced the GAL4 gene in NP2492-GAL4 with GAL80
26
(see Methods
section for details). The resulting line, NP2492-GAL80, appeared to largely recapitulate the
expression pattern of NP2492-GAL4, as it silenced the majority of reporter expression in
NP2492-GAL4 (Supplementary Fig. 1h). In combination with MZ160, NP2492-GAL80
significantly reduced the reporter expression in MB-V2 (Fig. 3c, Supplementary Fig. 1i,j
and Video 4).
Consistent with the anatomical data, impaired 2-h memory of MZ160/UAS-shits flies
was fully rescued by combination to Cha3.3kb-GAL80 (Fig. 3d) and NP2492-GAL80 (Fig. 3e).
As both Cha3.3kb-GAL80 and NP2492-GAL80 suppress most of the GAL4-expressing cells in
NP2492 driver (Supplementary Fig. 1c,h), no rescue experiments were performed with these
genotypes. These results strongly suggest that the memory retrieval defect observed in
MZ160/UAS-shits flies is due to MB-V2 blockade.
7
MB-V2 output is required to retrieve consolidated memories
Aversive odour memory in Drosophila consists of various phases1,3. Short-term
memory (STM) can be formed by a single cycle of training and is labile. Several cycles of
massed or spaced training (i.e. without or with rest intervals between conditioning cycles,
respectively) induce distinct forms of long-lasting memory3: anaesthesia-resistant memory
(ARM), and long-term memory (LTM), respectively15.
Memory 24 hours after both massed and spaced conditioning was strongly impaired in
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both genotypes when MB-V2 output was blocked during testing (Fig. 4a,b). Consistent with
the fact that MB-V2 output is not required during training or consolidation after a single
conditioning cycle (Fig. 2b), blockade of MB-V2 output during spaced training and the
following two hours does not affect 24-h memory (Fig. 4c). Taken together, these results
demonstrate that the retrieval of all phases of aversive olfactory memory, i.e. STM, LTM, and
ARM, requires the MB-V2 output whereas training and consolidation do not.
MB-V2 neurons are cholinergic
To characterize MB-V2 with regard to neurotransmitter, we examined colocalization of
MB-V2 and markers for various transmitters. Their cell bodies were not colocalized with
Drosophila
Vesicular
Glutamate
Transporter,
Tyrosine
Hydroxylase,
Glutamate
Decarboxylase 1 (DVGLUT, TH, GAD1 resp., Fig. 5a-c), as well as octopamine/tyramine or
serotonin (data not shown). We confirmed the results obtained with NP2492 with another
driver line, R71D08 (Supplementary Fig. 4), constructed as described in reference
[27]
(see
methods). The R71D08 driver strongly labels a smaller number of MB-V2 cluster neurons
(Supplementary Fig. 1k and Supplementary Video 5). Consistently with the behavioural
8
results obtained with NP2492 and MZ160 drivers, we observed a 2-h memory impairment
when MB-V2 output is blocked during the test using the R71D08 driver (Supplementary
Fig. 5a). Moreover, no memory defect was observed when R71D08/UAS-shits flies were
conditioned and tested at permissive temperature (Supplementary Fig. 5b) and the odour
avoidance of R71D08/UAS-Shits flies was normal at restrictive temperature (Supplementary
Fig. 5c).
We next examined Choline Acetyltransferase (ChAT)-immunoreactivity in the
presynaptic terminals of MB-V2, as the antibody does not significantly labels cell bodies28.
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We found that a fraction, but not all, of the terminals of MB-V2 neurons colocalized with
ChAT (Fig. 5d-i and Supplementary Fig. 6 and 7), indicating that at least some of the MBV2 neurons are cholinergic. We also confirmed the colocalization with ChAT with the
terminals of single MB-V2 neurons (Fig. 5j-o). The presynaptic terminals of MB-V2 were not
immunoreactive to DVGLUT or GAD1 (Supplementary Fig. 8 and 9), consistently with the
observations of the cell bodies (Fig. 5a-c and Supplementary Fig. 4). The conclusion that
MB-V2 are cholinergic is in line with the suppression of transgene expression in MB-V2 by
Cha3.3kb-GAL80 (Fig. 3b).
MB-V2 neurons show a reduced response to trained odorants
To understand the physiological role of the MB-V2 neurons during aversive olfactory
memory retrieval, we analyzed the MB-V2 response to odours using in vivo calcium imaging.
We expressed GCaMP 3.0, a calcium-sensitive fluorescent protein29 using NP2492 driver and
recorded the fluorescence of GCaMP3 from the MB-V2 neurons in the region of the MB
vertical lobes and msmpr in naïve flies (Fig. 6a). A strong calcium increase was detected in
response to benzaldehyde, 3-octanol, methylcyclohexanol and isoamylacetate (Fig. 6c-f)
9
compared to that obtained with pure paraffin oil (Fig. 6b and h). Responses to all odours were
similar in MB-V2 neurons (Fig. 6h) and decreased rapidly after the end of the stimulation
(OCT, MCH: Fig. 6g; isoamylacetate and benzaldehyde, data not shown). These results show
that the MB-V2 neurons respond to odours by increasing intracellular calcium concentration.
As the MB-V2 neurons respond to odorants, we asked whether the MB-V2 show
training-induced change of their response properties to the conditioned stimulus after aversive
training. NP2492/+; UAS-GCaMP3/+ flies showed a wild-type memory (Fig. 7a) suggesting
that expression of the GCaMP3 reporter in the MB-V2 neurons does not affect their
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physiology. Three hours after the flies had been trained with OCT as the conditioned stimulus
(paired training, Fig. 7b), the calcium response to OCT in MB-V2 neurons was significantly
decreased compared to MCH response (Fig. 7c,g). This decrease is the result of the temporal
pairing of the odorant and the shock stimuli, as no such decrease was observed (Fig. 7e,i)
when OCT and electric shocks were delivered 120 seconds apart (unpaired training, Fig. 7b).
Consistently, the relative response of OCT to MCH in paired flies was reduced by 45%
compared to unpaired flies (Fig. 7k). Similar results were obtained when MCH was used as
the conditioned odorant (Fig. 7d,f,h,j,l). Thus, the MB-V2 neurons transmit a memory trace
in the MB by decreasing their calcium response to the trained odorant.
DISCUSSION
Here, we described for the first time the function of a class of MB efferent neurons in
Drosophila, MB-V2. We found two distinct populations of MB-V2 neurons, MB-V2α and
MB-V2α' that innervate the shaft of the MB α lobe and the tip of the MB α' lobe,
respectively. The MB α lobe has been implicated as a site of olfactory learning that employs
an associative synergy of cholinergic and dopaminergic inputs to activate cAMP/PKA
10
signalling11, 12. In addition to the MB, both MB-V2α and MB-V2α' arborise in the neuropil
just posterior to the MB vertical lobes and in the lateral horn (LH), the other major secondary
olfactory centre. By transiently blocking synaptic transmission, we found that MB-V2 output
is required specifically to retrieve aversive olfactory memory.
Although the molecular pathways underlying STM, LTM, and ARM differ1, 2, 15, our
results suggest that a common neuronal network is used to translate the memory trace in the
Kenyon cells projecting to the MB vertical lobes into avoidance behaviour. Other MB output
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neurons may also be involved in short-lasting memory retrieval, as blockade of MB-V2
output, while fully abolishing consolidated memories, only partially impairs short-lasting
memories. Interestingly, MB-V2 neurons are not required for the retrieval of sugar-associated
memory and thus other neurons must provide the output for appetitive memory. Finally, no
significant behavioural differences were observed with the aversive protocol when both V2α
and α' (NP2492) or only V2α (MZ160) were blocked, and the role of MB-V2α' neurons
remains to be elucidated.
Functional optical imaging revealed that MB-V2 neurons respond to odorants and
decrease their response to the trained odorant. Thus, aversive olfactory conditioning represses
the activity of MB-V2 cholinergic neurons. How can an increased calcium response in the
MB lobes to the learned odour (CS+)13, 14 induce a decreased calcium response in the MB-V2
neurons? Inhibitory neurons that project onto MB-V2 may be recruited during conditioning.
Strikingly, the PE1 neuron in Apis mellifera relays information from the MB to the LH and
shows a reduced response to CS+ after appetitive training30. It has been proposed that this
reduced response could be due to inhibitory GABAergic inputs on PE131. The conservation
between bees and flies suggest a particularly important role of this circuit in both insects.
Alternatively, the decreased calcium response of MB-V2 to the CS+ may correspond to
synaptic depression. As shown in mammals, long-term potentiation or depression may recruit
11
differential molecular mechanisms, such as NMDA receptor activation32, 33 and retrograde
signalling by endocannabinoids32-34. Such depression may occur in the MB-efferent synapses
during memory acquisition35, although underlying mechanisms are still yet to be identified in
Drosophila. Interestingly, in Drosophila, the GABAergic APL neuron that projects onto the
MB shows a reduced response to the trained odour36. Therefore, the plasticity mechanism that
occurs at the MB synapse could be shared between APL and MB-V2 neurons.
How is the reduced calcium response to the conditioned odorant in MB-V2 neurons
translated into the avoidance behaviour? We found that MB-V2 project to the LH, a brain
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region that receives direct olfactory inputs and is involved in the innate avoidance of repellent
odorants8, 9. Thus, in Drosophila, two olfactory pathways converge toward the LH: one innate
odorant avoidance pathway that goes directly from antennal lobes to the LH, and an
associative learning-dependant one that requires integration of the negative value of shock by
the MB. We found the pre-synaptic cholinergic terminals of MB-V2 to be in close proximity
to GABAergic neurons in the LH (Supplementary Fig. 9). Thus, reduced calcium response
in MB-V2 neurons upon exposure to the conditioned odorant could lead to an enhanced
avoidance to the aversive odour by reducing GABAergic inhibitory input on projection or LH
neurons. Alternatively, it has been shown in mammals that activation of muscarinic receptors
by acetylcholine can directly induce LTD37. Thus, the reduced calcium response observed to
CS+ in MB-V2 could directly result in lower inhibition of olfactory signalling mediated by
the LH.
MB-V2 neurons also have pre-synaptic terminals in the msmpr area. As there are
interneurons that connect the dorsal LH and msmpr38, the learned odor may induce avoidance
behavior at the msmpr upon the stimulation of the LH interneurons via MB-V2. The
functional relationships between the LH and the msmpr, both targets of the MB-V2 neurons,
remains to be resolved.
12
Note: Supplementary information is available on the Nature Neuroscience website
ACKNOWLEDGMENTS We thank Joachim Urban and Gehrard M. Technau for the MZ160 line and the
members of the NP consortium for the NP2492 line. We thank Jean-Maurice Dura for the y1w1118;GAL80[y+],Sb
line and for helpful discussions. We thank Loren L. Looger for the UAS-GCaMP3 line. We thank T. Kitamoto
for the GAD1-GAL80 line. We thank Vincent Hakim, Isabelle Rivals and members of our laboratories for helpful
discussions. This work was supported by grants from the Japan Society for the Promotion of Science (K.I.), the
Agence Nationale pour la Recherche (T.P.), the Fondation Bettencourt-Schueler (T.P.), the Fondation pour la
Recherche Médicale (T.P.), the Emmy-Noether Program from Deutsche Forschungsgemeinschaft (H.T.), the
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Bernstein focus Learning from BMBF (H.T.) and the Max-Planck-Gesellschaft (H.T.). J.S. and G.I. were
supported by the Fondation pour la Recherche Médicale, P-Y.P was supported by grant from Région Ile-deFrance, Y.A. was supported by Deutscher Akademischer Austausch Dienst.
AUTHOR INFORMATION The authors declare no competing financial interests. Correspondence and
requests for materials should be addressed to T.P. (thomas.preat@espci.fr) or H.T. (hiromut@neuro.mpg.de).
AUTHOR CONTRIBUTIONS
YA and HT designed and IS, SRT and VT carried out anatomical experiments. IS, YA and HT analyzed the
microscopic data and assembled figures. KI and GMR provided Gal4 lines with their expression patterns. JS, GI,
TP designed and JS, ST and PYP carried out behaviour experiments. JS carried out the P-replacement
experiment. PYP, TP and PT designed in vivo imaging experiments and PYP carried out these experiments. JS,
PYP, IS, YA, HT and TP wrote the paper. TP and HT supervised the work.
Figure legends
13
Figure 1 MB-V2 neurons connect the MB to the lateral horn and middle
superiormedial protocerebrum.
Distribution of mCD8::GFP (white; a-c) and Syt::GFP (white; d-f) in the MB-V2
neurons driven by NP2492. Neuropils were labelled with an antibody against
synapsin (orange). MB vertical lobes are outlined (dashed lines in a, d, h and k).
a-c,
A membrane reporter mCD8::GFP is localised in the terminals of MB-V2
neurons in the α- and α’-lobes of the MB (a), msmpr (b), and LH (c).
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d-f, A presynaptic marker Syt::GFP is highly enriched in the terminals of MB-V2
neurons in the msmpr (e) and in the LH (f), but not in the MB vertical lobes (d).
g-l, Two subtypes of MB-V2 neurons – MB-V2α (g-i) and MB-V2α' – (j-l) are
identified by single-cell analyses. Schematic diagrams show the two types of MB-V2
neurons (blue) relative to the MB and LH (light green) (g, j). Dashed lines outline the
brain surface. MB-V2α neurons arborise in the MB α lobes (h) and project to the
msmpr and the dorsal part of the LH (i; n = 87). MB-V2α' neurons arborise in the α'
lobes (k) and project to the msmpr, the ventral part of the LH, and to a region
ventromedial to the LH (l; n = 44). .
m-r, The polarity of the single MB-V2α neuron visualized by a flp-out clone driving
mCD8::GFP and Syt::HA using NP2492. The terminals of a single MB-V2α neuron in
the LH (m-o) and the MB and msmpr (p-r) are magnified.
Scale bars: 20 µm.
Figure 2 Output of the MB-V2 neurons is specifically required for the retrieval
of short-lasting memory.
Temperature shift protocols are shown above each graph.
14
a, Blocking MB-V2 neuron output during retrieval impairs 2-h memory. (a1: F(2,39) =
4.937, p = 0.0123, ANOVA; b2: F(2,36) = 3.935 , p = 0.0185, ANOVA). NP2492/UASshits and MZ160/UAS-shits are significantly different from their respective genetic
controls. n ≥ 12.
b, Blocking MB-V2 neuron output during training and consolidation does not affect 2h memory. ANOVA revealed no significant difference among the groups with (a1)
NP2492 (F(2,32) = 1.337, p = 0.28) and (a2) MZ160 (F(2,33) = 1.796 , p = 0.18). n ≥ 10.
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c, Expression of Shits in MB-V2 neurons does not affect 2-h memory when flies are
conditioned and tested at permissive temperature. ANOVA revealed no significant
difference among the groups with (c1) NP2492 (F(2,27) = 1.508, p = 0.24) and (c2)
MZ160 (F(2,27) = 1.101 , p = 0.35). n ≥ 10.
Each graph displays mean performance indices ± SEM.
Figure 3 Suppressing MB-V2 neurons in MZ160 rescues the memory defect
a-c, Projection of the brain region including the MB lobes (light green).
In UAS-mCD8GFP; MZ160 (a), terminals of MB-V2 in the α lobe are visualized
(arrows), whereas the signal is strongly reduced in combination with Cha3.3kb-GAL80
(b) and NP2492-GAL80 (c). Scale bars: 20 μm.
d, Suppressing GAL4 expression in MB-V2 neurons with Cha3.3kb-GAL80 restores a
wild-type 2-h memory (F(4,45) = 3.305, p = 0.0186, ANOVA). MZ160/UAS-shits differs
significantly from UAS-shits, MZ160/+ and Cha3.3kb-GAL80/+; MZ160/UAS-shits.
Cha3.3kb-GAL80/+ ; MZ160/UAS-shits does not differ significantly from UAS-shits,
MZ160/+ and Cha3.3kb-GAL80/+ ; +/UAS-shits. n ≥ 10.
e, Suppressing GAL4 expression in MB-V2 neurons with NP2492-GAL80 restores a
wild-type 2-h memory (F(2,36) = 5.774, p = 0.0067, ANOVA). MZ160/UAS-shits differs
15
significantly
from
NP2492-GAL80/+;
MZ160/UAS-shits.
NP2492-GAL80/+;
MZ160/UAS-shits does not differ significantly from NP2492-GAL80/+; +/UAS-shits.
Only females were used in this experiment. n ≥ 12.
Each graph displays mean performance indices ± SEM.
Figure 4 Output of the MB-V2 neurons is required for retrieval of consolidated
long-lasting memories.
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Temperature shift protocols are shown above each graph.
a, Blocking MB-V2 neuron output during retrieval impairs 24-h memory after massed
conditioning (a1: F(2,39) = 4.822, p = 0.0134, ANOVA; a2: F(2,27) = 12.95, p = 0.0001,
ANOVA). NP2492/UAS-shits and MZ160/UAS-shits are significantly different from
their respective genetic controls. NP2492/UAS-shits and MZ160/UAS-shits do not
differ significantly from 0 (one-sample T-test, p = 0.44 and p = 0,72 resp.). n ≥ 10.
b, Blocking MB-V2 neuron output during retrieval impairs 24-h memory after spaced
conditioning (b1: F(2,30) = 5.469, p = 0.0094, ANOVA; b2: F(2,38) = 6.247, p = 0.0037,
ANOVA). NP2492/UAS-shits and MZ160/UAS-shits are significantly different from
their respective genetic controls. NP2492/UAS-shits does not differ significantly from
0 (one-sample T-test, p = 0.25). n ≥ 10.
c, Blocking MB-V2 neuron output during spaced training and 2 hours after training
does not affect 24-h memory. ANOVA revealed no significant difference among the
groups with (a1) NP2492 (F(2,27) = 0.1157, p = 0.89) and (a2) MZ160 (F(2,27) = 0.1936 ,
p = 0.83). n ≥ 10.
Each graph displays mean performance indices ± SEM.
Figure 5 MB-V2 neurons are cholinergic.
16
a-c, Cell bodies including the MB-V2 neurons in NP2492 do not overlap with the
markers for glutamate, (DVGLUT; a), dopamine (TH; b) and GABA (GAD1; c). See
also Fig. S6 for consistent results with R71D08.
d-o, Terminals of the MB-V2 neurons in the LH and msmpr colocalize with ChAT
(arrow), while the signal is occasionally undetectable in some terminals (arrowhead).
The processes of MB-V2 is visualized with mCD8::GFP driven by R71D08. Terminals
of a population of MB-V2 neurons (d-i) or a single MB-V2 α (j-o) are labelled with
mCD8::GFP. See also Fig. S5 for consistent results with NP2492.
peer-00653182, version 1 - 19 Dec 2011
Scale bars: 10 μm.
Figure 6 MB-V2 neurons respond to olfactory stimuli in naïve flies.
a, Illustrative mean greyscale image of a time series acquisition showing a horizontal
section of the right hemisphere of NP2492/+; UAS-GCaMP3/+ flies brain. MB-V2
neurons exhibited basal GCaMP3 fluorescence in their projections on the MB vertical
lobes (i), msmpr (ii), lateral horn (iii) and in cell bodies (iv). The area delimited by
dashed line includes MB and msmpr regions and was used to quantify odorant
responses, here and in Fig. 7.
b-f, Colour coded variations of fluorescence in the same fly, after 2s exposure to
benzaldehyde (c), octanol (d), methylcyclohexanol (e) and isoamylacetate (f). MB-V2
neurons responded with calcium increase to all four odorants. Strong activation was
observed in the MB and msmpr area, as delimited on (a). The calcium influx was
much lower when no odorant was diluted in the paraffin oil (f).
g, Time course, averaged across all animals, of octanol (red; n = 7),
methylcyclohexanol (blue; n = 7) and oil (black; n = 5) responses. The solid black bar
indicates the delivery of the stimulus. The peak responses for all odorants, calculated
17
as the temporal mean over the time window shaded in grey, are displayed in h
(traces for benzaldehyde and isoamylacetate are not shown).
h,
There were no significant differences among the mean responses to
isoamylacetate (I, n = 3), benzaldehyde (B, n = 3), octanol (O) and
methylcyclohexanol (M), while the response to pure oil was significantly lower (oneway ANOVA, F(4,220) = 26.56, p<0.01).
Figure 7 MB-V2 neurons show a reduced response to the trained odour after
peer-00653182, version 1 - 19 Dec 2011
conditioning.
a, Overexpression of the GCaMP3 driven by NP2492-GAL4 did not significantly alter
3 hours memory performance (n = 13; one-way ANOVA, F(2,36) = 2, p = 0.15).
b, Sketch illustrating delivery of odours (black line) and electric shocks (red spikes)
during the paired and unpaired training.
Items c,e,g,i,k and d,f,h,j,l display similar results of two sets of reciprocal
experiments, performed either on flies trained with OCT (left) or MCH (right) as the
CS+ odour (“CS+” in unpaired training). c-f: Mean greyscale images (scale bars: 15
µm) of MB-V2 projections on msmpr, and colour coded patterns of the responses
obtained to both odours in representative paired (c-d) and unpaired flies (e,-f). Paired
flies specifically show a lower activation after CS+ than after CS– (arrowheads).
Average time courses of odour responses are shown for paired (g-h) and unpaired (ij) flies. Black bars indicate the delivery of the stimulus. In paired flies, during the time
window shaded in gray, the time course of CS+ response is decreased compared to
the CS– response (g n = 7, p = 0.0035; h n = 6 p = 4.5x10-4) while CS+ and CS–
responses remain undistinguishable in unpaired flies (i n = 7 p = 0.27; j n = 6 p =
0.13). The CS+ to CS– relative response, quantified as the natural logarithm of the
18
CS+/CS– response, is lower in paired than in unpaired flies (k: p = 0.0024; l: p =
0.0043).
Table 1 Sensory acuity controls.
Response of naïve flies to the odorants used in the conditioning experiments at the restrictive
temperature (33°C). Temperature was shifted 30 min prior to the measurement of odorant
avoidance. Flies have 1 min to choose between the aversive odour or air bubbled through
peer-00653182, version 1 - 19 Dec 2011
mineral oil. For 3-octanol and 4-methycyclohexanol, one-way ANOVA revealed no
significant differences between the genotypes (wild-type, UAS-shits, GAL4 and UASshits/GAL4; p > 0.05). n ≥ 8 groups. Each value displays mean performance indices ± SEM.
Table 1
Genotype
Odour Avoidance
Octanol
Methylcyclohexanol
wild-type
0.54 ± 0.12
0.53 ± 0.10
UAS-shits
0.51 ± 0.08
0.70 ± 0.06
NP2492
0.45 ± 0.10
0.80 ± 0.04
NP2492/+ ; UAS-shits/+
0.49 ± 0.10
0.78 ± 0.07
wild-type
0.50 ± 0.11
0.56 ± 0.10
UAS-shits
0.49 ± 0.08
0.70 ± 0.08
MZ160
0.59 ± 0.09
0.59 ± 0.08
19
MZ160/UAS-shits
0.59 ± 0.06
0.59 ± 0.06
Methods
Stocks. Flies were raised at 18°C, 60% humidity in a 12:12 h light:dark cycle. All strains
were outcrossed to flies with a wild-type strain Canton-Special (Canton-S). The enhancer trap
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lines NP2492 [X] and MZ160 [III] were previously identified through a large-scale screen for
mushroom body extrinsic neurons17,
18
. UAS-shits1 [III] was used for behavioural
experiments19. The transformation vector for R71D08 was generated using the 3.2kb DNA
fragment of the division abnormally delayed locus that was amplified with PCR and cloned
into
the
transformation
vector
pBPGUw
[27]
(Primers
sequence:
GAAGTGCATGGCAAGGGAAGCAGAG and GCCAACCACTTATTTCTCGCCGTGT).
For experiments with NP2492 whose insertion is on the X chromosome, only females of
NP2492/+;UAS-shits/+ as well as the respective control genotypes were taken into account to
calculate the memory index. NP2492-GAL80 line was obtained by crossing y1, w1118 females
to y1, w1118, NP2492-GAL4 [w+]; ΔHop,CyO ; GAL80[y+],Sb males. Positive events were
detected by screening for y1, w1118, GAL80[y+] females on next generation and verified on
confocal microscopy by crossing to NP2492-GAL4; UAS-mCD8::GFP.
Olfactory conditioning. For aversive training, flies were conditioned by an odour paired with
electric shocks and subsequent exposure to a second odour in the absence of shock, as
previously described39. Conditioning was performed on samples of 25–35 flies aged between
2 and 3 days with 3-octanol (>95% purity; Fluka 74878, Sigma-Aldrich) and 4methylcyclohexanol (99% purity; Fluka 66360, Sigma-Aldrich) at 0.360 mM and 0.325 mM,
20
respectively. Odours were diluted in paraffin oil (VWR international, Sigma-Aldrich).
Memory tests were performed with a T-maze apparatus40. Flies could choose for 1 min
between 2 arms delivering each a distinct odor. An index was calculated as the difference
between the numbers of flies in each arm divided by the sum of flies in both arms. A
performance index (PI) results from the average of two reciprocal experiments. For appetitive
training, electric shocks were replaced by 1-minute sugar exposure, as previously described24.
Statistical analysis. We performed behavioural statistical analysis using GraphPad Prism 5.0
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(GraphPad Software, Inc.). Each graph displays mean performance indices ± SEM. One-way
ANOVA was performed on each set of data followed by pairwise planned comparisons
between relevant groups with a Student-Newman-Keuls test, except when specified. Wildtype flies were not taken in account for statistical analysis. For the data that violated the
assumption of normal distribution (Supplementary Fig. 2j), non-parametric statistics was
applied (Kruskal–Wallis test followed by Dunn’s multiple comparisons). Asterisks denote
significant differences with the post-hoc pair-wise comparisons (* P < 0.05, ** P < 0.01, ***
P < 0.001). NS: not significant.
Immunohistochemistry. We examined female F1 progenies (5–8 days after eclosion at
25°C) between males of corresponding Gal4 lines and females carrying a single copy of
UAS-mCD8::GFP [II]22, -syt::GFP [II]
[20]
, -syt::HA [X], -nSyb::GFP [III], -myr::RFP [III]
or their combination. For single-cell clonal analyses, y w hsp70-flp; UAS>CD2 y
>mCD8::GFP/CyO; TM2/TM6b
[41]
females were crossed to NP2492, MZ160 or R71D08
males. One day post-eclosion, adult flies received a heat shock at 37°C for 30–45 min to
remove the Flp-out cassette (CD2 y), and examined 5–7 days after heat shock. We analysed in
total 131 single cell clones (87 for MB-V2α and 44 for MB-V2α’) with the three drivers. For
21
single-cell analyses driving the membrane and presynaptic markers (i.e. mCD8::GFP and
Syt::HA), UAS-mCD8::GFP UAS-Syt::HA hsp70-flp females were crossed to NP2492;
Tub>GAL80> (II) males42. Late 3rd instar larvae/early pupae of the progeny were heatshocked at 34°C for 20 minutes to remove the Flp-out cassette, and dissected after 10–12 days
post-eclosion.
Brains were prepared by a standard immunolabeling procedure18, 43, using antibodies against
Drosophila GAD144,
45
(1:200), DVGLUT46 (1:10.000), ChAT47 (1:100), TH48 (1:1000),
Synapsin49 (1:100), GFP (rabbit polyclonal, 1:1000, Invitrogen or rat monoclonal, 1:200,
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Chromotek), HA (1:200, Covance) and DsRed (1:100, Clontech). Coronal optical sections of
whole-mount brains taken by confocal microscopy (Olympus FV1000) were analyzed with
Image-J (NIH). In most figures, stained fibres and counterstaining were presented in white
(RGB ratio; 255:255:255) and orange (255:85:0), respectively. Nomenclature of brain regions
follows Otsuna and Ito (2006)43.
In vivo calcium imaging of odour responses
Preparation of the flies: We targeted genetically the expression of the GCaMP3.0 calcium
reporter to the MB-V2 neurons by combining a UAS-GCaMP3 [II] construct29 with the
NP2492-GAL4 driver. Measurements on naïve animals were performed on 1–2 days flies
homozygous for both NP2492 and UAS-GCaMP3 transgenes. Female flies were caught
without anaesthesia, then glued and operated as previously described for in vivo imaging50.
The proboscis was also glued to the thorax to limit motion artefacts during image acquisition.
The recording chamber was then placed beneath the water immersion 20x objective (NA = 1;
Leica) of a TCS-SP5 laser-scanning confocal microscope (Leica), the brain of the fly being
thus observed from the top. Experiments were performed at 20°C, and the aperture on the top
22
of the fly head bathed in a continuously flowing perfusion of Drosophila Ringer’s solution
(130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 36 mM sucrose, 5 mM HEPESNaOH; pH 7.3; 305 mOsm) oxygenated by air bubbling.
For experiments on trained or unpaired flies, NP2492/+; UAS-GCaMP3/+ female flies with
more moderate levels of reporter expression were used, in order to minimize physiological
side effects of GCaMP3 expression. Consequently, GCaMP3–labelled areas and odourevoked activation patterns may slightly differ from experiments on naïve flies. One fly was
captured 2h30 after training and prepared with the same procedure, so that imaging actually
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took place approximately 3 hours after the end of the training protocol.
Odorant delivery: Each odorant was diluted 250-fold v/v in 100mL paraffin oil in a glass
bottle. Control bottles contained pure oil. A constant air stream, bubbling through pure oil,
was directed at the fly throughout the recording. Following a trigger, a set of solenoid-driven
valves redirected 20% of the air stream to the appropriate odour or control bottle for the
aimed duration. The odour stream (200 mL.min-1) rejoined the primary stream (800 mL.min-1)
23 cm from the end of the delivery tube, which measured 4 mm in diameter. The end of the
delivery tube was positioned ~7–8 mm from the fly’s antennae. All odorant concentrations are
therefore ~2000-fold diluted (5.3 x 10-4 M), not including the additional dilution factor
between the end of the tube and the antennae.
Data acquisition and analysis: GCaMP3 probe was excited by the 488 nm line of an argon
laser, scanning at a line rate of 400 Hz. Fluorescence-induced light emission was collected by
a photomultiplier in the 505–555 nm wavelength range. The pinhole was wide open, the
collected light originated from thick horizontal sections of either of the two hemispheres of
the brain.
Naïve flies were delivered each olfactory stimulus for 2 s, and odorant presentations were
spaced 3 min apart, in a random order. On each trained fly, a pair of responses to 1 s of CS+
23
and CS– odorants was recorded twice, to ensure that its olfactory ability was preserved
throughout the experiment, but only the first presentation of each odour was kept for data
analysis, to avoid desensitization effects. Within each pair, the odorants were pseudorandomly ordered. Overall, 12 flies received CS+ first, and 14 flies received CS– first. During
an experimental session, special care was taken to sequentially image, with minimal delay (55
min on average), on the same hemisphere and with the same odour delivered first, one paired
and one unpaired fly grown in the same culture bottle, thus allowing treating paired and
unpaired flies as paired data for statistical analysis. For this experiment, we focused on the
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msmpr as GCAMP signal was stronger in this region. All measurements were performed
approximatively 3 hours after training.
Image analysis was performed offline with Matlab software using a custom-written program.
To obtain the time course of the relative variation of fluorescence, the light intensity was
averaged over a region of interest delimited by hand and surrounding the projections of the
MB-V2 neurons in the area of the msmpr and mushroom bodies. The resulting time trace was
normalized to a percent change of fluorescence 100*(F–F0)/F0, using a baseline value of the
fluorescence F0, that was estimated as the temporal mean over the 1 s period preceding the
switch of the valve. The peak response was calculated as the mean of this trace over a time
window shown in gray on Fig. 6g and Fig. 7e,f. The same procedure was applied on a pixelby-pixel basis, yielding a colour coded image of the spatial pattern of the peak response. The
brightness of each pixel was weighted by its mean fluorescence intensity over the duration of
the whole acquisition, resulting in a shaded colour-coded image which highlights responses in
regions that were actually labelled by the calcium reporter.
In the course of experiments on trained flies, comparison of responses to a given odorant was
clouded by significant variability among different animals. To overcome this, a normalized
value was calculated for each fly as the ratio of CS+ over CS– peak responses. In order to
24
accurately estimate the ratio of the mean responses, the natural logarithm of this value was
averaged over all flies that had been submitted to the same training13. For statistical
comparisons of two series of data, two-tailed t-tests were performed except when Lilliefors
test indicated significant (p < 0.05) shift from normal distribution for any of the two series, in
which case a Wilcoxon rank sum test was performed (Fig. 7j).
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Journal: Nature Neuroscience
Article Title:
Corresponding
Author:
Mushroom Body Efferent Neurons Responsible For Aversive Olfactory Memory
In Drosophila
Thomas Preat (thomas.preat@espci.fr)
Supplementary
Item & Number
Supplementary Figure 2
GAL4-expression patterns of NP2492 and MZ160 with Cha3.3kb-GAL80 and
NP2492-GAL80
Polarity of MB-V2 neurons visualised with various reporters
Supplementary Figure 3
Other behavioural analysis of NP2492/UAS-shits and MZ160/UAS-shits flies
Supplementary Figure 4
MB-V2 are not glutamatergic, dopaminergic or GABAergic
Supplementary Figure 5
Supplementary Figure 6
Blockade of MB-V2 neurons using R71D08 driver induces a 2-h memory
impairment
Colocalization of MB-V2 terminals with ChAT
Supplementary Figure 7
Colocalization of MB-V2 terminals with ChAT but not DVGLUT in the LH
Supplementary Figure 8
No colocalization of MB-V2 terminals and DVGLUT in the LH and msmpr
Supplementary Figure 9
Supplementary Video 1
No colocalization but proximity of MB-V2 terminals and GAD1 in the LH
and msmpr
Expression pattern of NP2492 in the brain
Supplementary Video 2
Expression pattern of MZ160 in the brain
Supplementary Video 3
Expression pattern of MZ160, Cha3.3kb-GAL80 in the brain
Supplementary Video 4
Expression pattern of MZ160, NP2492-GAL80 in the brain
Supplementary Video 5
Expression pattern of R71D08 in the brain
Supplementary Figure 1
peer-00653182, version 1 - 19 Dec 2011
Title or Caption
28
MB
msmpr
mCD8::GFP
'
LH
peer-00653182, version 1 - 19 Dec 2011
Syt::GFP
MB-V2
msmpr
msmpr
MB-V2’
'
LH
MB
MB
LH
'
LH
MB/msmpr
LH
LH
mCD8::GFP Syt::HA
mCD8::GFP
Syt::HA
Séjourné et al, Figure1
0.4
*
0.2
0.4
NS
0.6
0.6
0.6
0.5
0.5
0.5
NS
0.4
0.3
0.2
NS
0.3
0.2
Performance index
Time (h)
1
2
0.1
0.1
0.1
0.0
0.0
0.0
0.6
0.6
0.6
0.5
0.5
0.5
0.1
0.1
0.1
0.0
0.0
0.0
U
AS
-S
hi ts
/+
N
P2
N
P
49
24
2/
92
+
/+
;U
AS
-S
hi ts
/+
*
Temp (°C)
20
c
U
AS
-S
hi ts
/+
M
Z1
M
60
Z1
/+
60
/U
AS
-S
hi ts
0.2
33
w
ild
-ty
pe
0.3
0
Test
w
ild
-ty
pe
0.4
Train (1X)
Performance index
2
U
AS
-S
hi ts
/+
N
P2
N
P
49
24
2/
92
+
/+
;U
AS
-S
hi ts
/+
Time (h)
1
b
U
AS
-S
hi ts
/+
M
Z1
M
60
Z1
/+
60
/U
AS
-S
hi ts
Temp (°C)
20
Performance index
Temp (°C)
33
w
ild
-ty
pe
0
Test
Performance index
U
AS
-S
hi ts
/+
N
P2
N
P
49
24
2/
92
+
/+
;U
AS
-S
hi ts
/+
w
ild
-ty
pe
Performance index
Train (1X)
w
ild
-ty
pe
Performance index
0.3
U
AS
-S
hi ts
/+
M
Z1
M
60
Z1
/+
60
/U
AS
-S
hi ts
w
ild
-ty
pe
peer-00653182, version 1 - 19 Dec 2011
a
Train (1X)
0
Time (h)
Test
33
20
1
2
0.4
0.3
0.2
NS
0.4
0.3
0.2
a
b
MZ160
MZ160
NP2492-GAL80
e
peer-00653182, version 1 - 19 Dec 2011
d
c
MZ160
Cha3.3kb-GAL80
Séjourné et al, Figure3
0.4
0.2
***
0.0
0.2
1
Time (h)
24
0.3
0.2
**
0.3
**
0
0.4
0.4
0.3
0.1
0.1
0.1
0.0
0.0
0.0
0.1
0.1
0.0
0.0
U
AS
-S
hi ts
/+
N
P2
N
P
49
24
2/
92
+
/+
;U
AS
-S
hi ts
/+
*
0
Temp (°C)
Test
U
AS
-S
hi ts
/+
M
Z1
M
60
Z1
/+
60
/U
AS
-S
hi ts
0.2
20
Performance Index
0.4
33
w
ild
-ty
pe
0.3
Train (5 spaced cycles)
Performance Index
b
w
ild
-ty
pe
24
U
AS
-S
hi ts
/+
N
P2
N
P
49
24
2/
92
+
/+
;U
AS
-S
hi ts
/+
Time (h)
w
ild
-ty
pe
20
Temp (°C)
33
Performance Index
1
Test
U
AS
-S
hi ts
/+
M
Z1
M
60
Z1
/+
60
/U
AS
-S
hi ts
0.3
Performance Index
Temp (°C)
Train (5 massed cycles)
w
ild
-ty
pe
0.1
U
AS
-S
hi ts
/+
N
P2
N
P2
49
49
2/
2/
+
+
;U
AS
-S
hi ts
/+
w
ild
-ty
pe
Performance Index
0
U
AS
-S
hi ts
/+
M
Z1
M
60
Z1
/+
60
/U
AS
-S
hi ts
w
ild
-ty
pe
Performance Index
peer-00653182, version 1 - 19 Dec 2011
a
c
Train (5 spaced cycles)
1
2
Test
33
20
Time (h)
24
0.4
NS
0.2
0.4
0.3
NS
0.2
Séjourné et al., Figure 4
peer-00653182, version 1 - 19 Dec 2011
a
b
anterior
F/F0 (%)
c
140
i
g
120
ii
F/F0
100
iii
80
30 %
60
50 m
40
iv
0
20
posterior
d
Benzaldehyde
Paraffin oil (control)
e
f
1s
0
h
**
F/F0 (%)
100
NS
80
60
40
20
Methylcyclohexanol
Isoamylacetate
0
Oil
B
O
M
I
peer-00653182, version 1 - 19 Dec 2011
Octanol
Séjourné et al., Figure 6
a
b
Performance Index
0.8
NS
0.6
Paired training
CS +
odour
CS –
odour
CS +
or
CS –
CS –
or
CS +
“CS+”
odour
CS –
odour
“CS +”
or
CS –
CS –
or
“CS +”
0.4
0.2
Unpaired training
60 s
120 s
60 s
45 s
60 s
3h
1 s 180 s 1 s
U
w
ild
AS
-G
C
aM
N
P2
P3
49
/+
2/
N
+;
P2
U
49
AS
2/
-G
+
C
aM
P3
/+
-ty
pe
0.0
Octanol as CS+
c
OCT
Unpaired
e
OCT
MCH
140
150
ΔF/F0
(%)
ΔF/F0
(%)
OCT
Unpaired
0
f
MCH
MCH
0
140
70
ΔF/F0
(%)
ΔF/F0
(%)
OCT
0
MCH
0
h
Paired
Paired
OCT (CS +)
MCH
ΔF/F0
i
Paired
d
g
40 %
0
0
1s
j
Unpaired
ΔF/F0
OCT
MCH (CS +)
ΔF/F0
40 %
1s
Unpaired
ΔF/F0
OCT (“CS +”)
MCH
40 %
40 %
0
0
1s
OCT
MCH (“CS +”)
1s
k
l
**
Log(CS+ response/CS– response)
**
Log(CS+ response/CS– response)
peer-00653182, version 1 - 19 Dec 2011
Methylcyclohexanol as CS+
Paired
0.2
0
-0.2
-0.4
-0.6
Paired Unpaired
0.2
0
-0.2
-0.4
Paired Unpaired
Séjourné et al., Figure 7