Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee

21 Pages·2011·4.53 MB·English

Appetitive odor learning does not change olfactory coding
in a subpopulation of honeybee antennal lobe neurons
P. Peele . M. Ditzen . R. Menzel . C. G. Galizia
Abstract Odors elicit spatio-temporal patterns of modified by learning in this paradigm. The role that
activity in the olfactory bulb of vertebrates and the other projection neurons play in olfactory learning
antennal lobe of insects. There have been several re remains to be investigated.
ports of changes in these patterns following olfactory
learning. These studies pose a conundrum: how can an Keywords Learning and memory' Olfactory coding·
animal learn to efficiently respond to a particular od or Calcium imaging . Projection neurons . Coding
with an adequate response, if its primary representa invariance
tion already changes during this process? In this study,
we offer a possible solution for this problem. We Abbreviations
measured odor-evoked calcium responses in a sub AL Antennal lobe
population of uniglomerular AL output neurons in CR Conditioned response
honeybees. We show that their responses to odors are CS- Non-rewarded stimulus
remarkably resistant to plasticity following a variety of CS+ Rewarded (conditioned) stimulus
appetitive olfactory learning paradigms. There was no ITI Inter-trial interval
significant difference in the changes of odor-evoked I-ACT Lateral antenno-cerebralis tract
activity between single and multiple trial forward or LP Lateral protocerebrum
backward conditioning, differential conditioning, or m-ACT Medial antenno-cerebralis tract
unrewarded successive odor stimulation. In a behav MB Mushroom body
ioral learning experiment we show that these neurons ml-ACT Mediolateral antenno-cerebralis tract
are necessary for conditioned odor responses. We OB Olfactory bulb
conclude that these uniglomerular projection neurons OSN Olfactory sensory neuron
are necessary for reliable odor coding and are not PN Projection neuron
RMANOVA Repeated measures analysis of
variance
SEG Subesophageal ganglion
Tl-T4 Tracts 1-4 in the antennallobe
P. PeeIe . M. Ditzen . R. MenzelC. G. Galizia
Institute of Neurobiology, Freie Universitat Berlin, US Unconditioned stimulus
14195 Berlin, Germany PER Proboscis extension reflex
VUMmx1 Ventral unpaired neuron # 1 of the
C. G. Galizia
maxillary neuromere
Department of Entomology, University of California,
Riverside, CA 92521, USA
Introduction
Present Address:
C. G. Galizia ([2;])
The internal representation of the external world is
Universitat Konstanz, 78457 Konstanz, Germany
e-mail: [email protected] an inherent and fundamental function of all nervous
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systems that needs to be reliable to ensure the gener multiple conditioning trials induce a stable, long-term
ation of adequate behavioral responses. This is also memory that needs both translation and transcription
true for the chemical senses, which play a vital role for (MenzeI1999).
most animals. Primary olfactory information is pro The cellular changes leading to learning appear to be
cessed in the vertebrate olfactory bulb (OB) and its dispersed among different areas of the brain (Menzel
insect analogue, the antennal lobe (AL) (Hildebrand 1990; Gluck and Granger 1993; Tully et al. 1994; Ham
and Shepherd 1997). Both are subdivided into func mer and Menzel 1998; MenzeI1999). Amongst others,
tional processing units, the olfactory glomeruli. Odors neural plasticity due to olfactory learning has been
elicit specific spatio-temporal combinatorial patterns of found at the level of the first processing station in the
activated glomeruli in vertebrate OBs and invertebrate olfactory pathway, in the vertebrate OB (Woo et al.
ALs (Galizia and Menzel 2001; Friedrich 2002). A 1987; Sullivan and Wilson 1995; 10hnson et al. 1995;
particular glomerulus receives afferent input by a Brennan et al. 1998) and the insect AL (Faber et al.
specific olfactory sensory neuron type (OSN). Local 1999; Sandoz et al. 2003; Yu et al. 2004; Daly et al. 2004).
neurons within the OB or the AL modify the incoming Changes at the first odor processing level are likely to
activity (Mal un 1991; Yokoi et al. 1995; Mori et al. affect the neural representation of an odor. But how can
1999; Sachse and Galizia 2002), and the processed an animal learn to respond to a particular odor if its
information is relayed to higher order brain centers by neural representation is changing? Moreover, behav
vertebrate mitral/tufted cells or the insect projection ioral phenomena such as contextual, structural and
neurons (PNs). Higher order olfactory neuropiles in configural learning require a stable representation of
insects are the mushroom bodies (MB) and the lateral stimulus features (for example, that of an odor) in dif
pro to cerebrum (LP) (Mobbs 1982; Abel et al. 2001; ferent circuits that are selectively and specifically com
Muller et al. 2002; Marin et al. 2002; Wong et al. 2002; bined (Gerber and Menzel 2000; Sandoz and Menzel
Tanaka et al. 2004). In the honeybee, OSN afferents 2001; Menzel and Giurfa 2001; Deisig et al. 2001). We
travel in four different tracts, T1-T4, that innervate therefore investigated the effects of associative learning
about 160 glomeruli altogether. PNs run in three dif on the representation of olfactory information in the
ferent antenno-cerebral tracts (ACT). The medio-Iat AL. To this end, we combined a variety of appetitive
eral ACT (ml-ACT) contains pluriglomerular cells. olfactory conditioning paradigms with in vivo calcium
The lateral and median ACTs (1-and m-ACTs) contain imaging of one subpopulation of the olfactory PNs, the 1-
axons of uniglomerular PNs (Abel et al. 2001). It ACT PNs (Sachse and Galizia 2002). We measured odor
should be noted that the nomenclature for PN tracts responses before, during and after associative olfactory
differs for different insect species. For example, the learning, and found that od or representation in these
location of the honeybee I-ACT corresponds to the cells is reliable and stable throughout appetitive learn
Drosophila oACT, and whether the two are homolo ing experiments.
gous remains to be investigated.
Vertebrates as well as many invertebrates can be
conditioned to olfactory stimuli (Menzel 1990; Wilson Methods
and Stevenson 2003; Davis 2004). Sucrose stimulation
of antennal or proboscis chemoreceptors of a hungry Preparation
bee leads to the proboscis extension response (PER).
In an appetitive olfactory learning paradigm (PER Adult foraging bees, Apis melli/era carnica, were
conditioning), an odor, the conditioned stimulus (CS), caught at the hive in the afternoon 1 day before the
is paired with a subsequent sucrose reward as the experiments. Bees were expcrimentally naIve, but may
unconditioned stimulus (US). The animals form an have experienced odors during foraging flights. They
association between the two, so that an odor stimula were cooled for anesthesia, fixed in a recording stage
tion alone elicits the PER (conditioned response, CR) (Galizia et al. 1997), and fed with sucrose water (30%)
previously elicited only by the US. This effect is clearly until satiation. Bees were kept at 20°C in a dark
associative and involves classical conditioning (Bit humidified box over night. The next day PNs were
terman et al. 1983). The number of conditioning trials stained as follows: a window was cut in the head cap
applied to the honeybee influences which kind of sule and glands and trachea were carefully removed to
memory is induced. A single conditioning trial results allow visual access to the brain. A glass electrode was
in short-term memory which decays rapidly and is coated with crystals of FURA-dextran (potassium salt,
sensitive to amnesic treatments. This memory is inde 10.000 MW, Invitrogen, Germany), dissolved in 3%
pendent of translation and transcription. In contrast, bovine serum albumin solution (Albumin Fraction V,
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AppliChem GmbH, Germany), and injected into the using a computer controlled olfactometer (Galizia
right deutocerebrum dorsolateral to the alpha-lobe, et al. 1997).
aiming for the I-ACT. Using different activity indica For the US we delivered a drop of 2 fll 30% aqueous
tors including calcium dyes, other studies have suc sucrose solution to the antennae and the proboscis,
cessfully demonstrated plasticity in neural signa ling in using a 10 fll pipette with standard tips.
vertebrates and invertebrates (Faber et al. 1999; Faber
and Menzel 2001; Yu et al. 2004; Mutoh et al. 2005; Data analysis
Lohmann and Wong 2005). This indicates that calcium
sensitive dyes do not interfere with the intracellular All data were analyzed using custom software written
signaling cascades to a degree that would prevent any in IDL (Research systems, CO, USA). First, the raw
modulatory effects. The dye was left to travel along the fluorescent images were manually corrected for
tract for 4-8 h. Then, bees were prepared by fixing the movement within each measurement and for shifts
antennae with soft dental wax (Kerr, Sybron Dental between measurements. Scattered light correction was
Special ties, USA). Animals were immobilized by fixing applied to avoid that glomeruli with strong activity
the thorax and abdomen to the recording chamber with would cause fictive activity in neighboring silent
wax (Deiberit 502, Dr Boehme&Schoeps, Germany). glomeruli (Galizia and Vetter 2004). We calculated
The brain was rinsed with Ringer solution (130 mM F/ = Fi + n[Fi - sm(Fj)], where Fi is image number i,
NaCl,6 mM KCl, 4 mM MgCl2, 5 mM CaCl2, 160 mM and sm(F;) the same image after application of a spatial
sucrose, 25 mM glucose, 10 mM HEPES, pH 6.7, low-pass filter. We used a boxcar average filter with
500 mOsmol; all chemicals from Sigma-Aldrich, Ger kernel size of 20 flm, i.e. less than the radius of one
many). Strong movements of the brain needed to be glomerulus. The images F/ are the scattered-light
prevented to achieve stable imaging experiments. corrected images. The number n gives the strength of
Therefore, a second hole was cut ventrally to the the applied filter. In this study, we found n :::: 3 to give
antennae and the compact structure of muscles, the best results, as compared to n = 1 in previous work
esophagus, and supporting chitin was lifted and put (Galizia and Vetter 2004). Note that this procedure
under slight tension (Mauelshagen 1993). Thus stabil does not change the measured values in areas where
ization was accomplished without damage to the brain. there is no spatial contrast: in these areas, the low
However, this preparation impairs the PER. To allow passed filtered image has the same value as the original
for sucrose stimulation the proboscis was manually image, and consequently F;' = Fi•
extended and placed on a small glass capillary that was For each image i, we then calculated the percentage
fixed to the recording chamber. After surgery and be ratio Ri = (Fi 340nmlFi 380nm) X 100. Background ratio
fore recording the bees were left to recover for at least was determined by an average of five frames obtained
30 min. before stimulation and was subtracted from every ratio
frame of a measurement, thereby setting ratio values to
o
Imaging just before stimulus onset. The resulting values are
proportional to intracellular calcium concentration
Imaging was done using a CCD based imaging system changes, but are measured in arbitrary units, since we
(Polychrome IV with Imago-QE camera, T.I.L.L. Pho could not calibrate absolute calcium concentration.
tonics, Germany). Monochromatic excitation light The morphological structure allowed us to identify
alternated between 340 and 380 nm. Fluorescence was glomeruli on the basis of their borderlines using the
detected at a sampling rate of 5 Hz with a fluorescence digital atlas of the AL as a reference (Galizia et al.
microscope (Olympus BX-50WI, Japan) equipped with 1999a). This method has already been described in de
a 20x, NA = 0.95 dip objective (Olympus), 505 tail (Sachse et al. 1999; Galizia et al. 1999b). Honeybee
DRLPXR dichroic mirror and 515 nm LP filter (Omega glomeruli are labeled with a number and the name of the
Filters, VT, USA). Resolution was 172 x 130 pixels antennal nerve tract that innervates them, ranging from
binned on chip from 1,376 x 1,040 pixels, resulting in a Tl to T4, e.g. Tl-28 or T3-45 (Flanagan and Mercer
spatial sampling rate of 2.4 flm per pixel side. 1989). Since all glomeruli in this study were from the T1
Odors were diluted in mineral oil to 1 % (l-hexanol) tract, we have simplified their names by omitting T1
or 0.1 % (l-octanol and 1-nonanol, all odors from Sig from the name. We analyzed responses in glomeruli 17,
ma-Aldrich, Germany). Five microliter of the od or 23, 25, 28, 29, 33, 35, 36, 37, 38, 42, 47, 48, 49 and 60.
solution were applied onto a 1 cm2 piece of filter paper "Glomerular activity" in this study specifically refers to
and placed in a plastic syringe. Odors were injected activity of the uniglomerular PNs in each glomerulus,
into a continuous air stream directed to the antennae because only these neurons were stained, and therefore
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only this particular population of cells was measured in 3bw 3-trial backward conditioning with ITI of 2 min
each glomerulus. For each identified glomerulus time (n = 11)
courses were averaged taking an area of 9 x 9 pixels 3+ 3-trial forward conditioning with ITI of 2 min
(corresponding to 21.6 !lm x 21.6 !lm always within the (n = 11).
glomerulus chosen). Glomerular calcium response
magnitude (hereafter simply "response") was calcu This nomenclature indicates whether I-nonanol was
lated as the mean of the time course during odor stim unrewarded (-), forward paired with sucrose water (+)
ulation. Statistical analysis was performed using or backward paired with sucrose water (bw). The
Sigmastat (SPSS Inc, IL, USA). number (1, 3) corresponds to how often the condi
For false-color display, responses were calculated tioning trials were repeated.
for each pixel. The resulting image was median filtered Calcium responses for each glomerulus were ana
in space (3 x 3 pixels). Iyzed with a two-way repeated measures (RM) ANO
V A, with the different conditioning groups as one
Plasticity of odor responses factor and the "pre" -responses and "post" -responses
at different points in time as the repeated factor.
All experimental protocols are summarized in Fig. 1. 1- Multiple comparisons were performed using the
Holms-Sidak post-hoc test.
nonanol was always the reinforced odor (CS). In one
part of the experiments (set 1) we investigated changes
of the responses to 1-nonanol due to repeated stimula
Multiple od or experiments
tions, and due to pairing of this od or with a sucrose
reward in an absolute conditioning paradigm, with
We investigated the effect of sequential stimulation
special focus on progression in time after the condi
with three odors and differential conditioning (Fig. 1,
tioning phase. Thereafter, we included I-hexanol and 1-
set 2). Each experiment consisted of measuring od or
octanol into the stimulation protocols, allowing us to
responses before conditioning ("pre"), conditioning,
examine the effects of absolute conditioning on the
and measuring odor responses after conditioning
representation of non-conditioned odors and the effect
("post"), similar to experiments of set 1. "Pre" and
of differential conditioning on all odors involved (set 2).
"post" consisted of three blocks with three odor
stimulations each (l-hexanol, l-octanol and I-nona
Single odor experiments nol). Within "pre" and "post" blocks the odor se
quence was pseudorandomized to prevent odor
We investigated the effects of absolute conditioning on sequence effects (for example, an od or sequence could
the neural representation of the odor 1-nonanol be ABC, CAB, BCA, with each odor at a first, a second
(Fig. 1, set 1). The experiments consisted of three and a third position within the blocks). Different ani
parts: od or responses before conditioning ("pre"), mals were tested with different sequences. We aver
conditioning, and odor responses after conditioning aged the three "pre" responses and the three "post"
("post"). Odor stimulation was always 3 s long. We responses to yield one "pre" and one "post" response
determined the "naive" response to I-nonanol in the to every odor which we then compared. CS-US pairing
"pre"-phase during which all animals received two was done as for absolute conditioning (see above).
presentations of 1-nonanol with an inter-trial interval Stimulus sequence within the conditioning block was
(ITI) of 2 min. During conditioning a sucrose reward identical for all animals within each group.
was applied for 3 s to the ipsilateral antenna of the Seven groups were subject to the following condi
imaged AL and to the proboscis. For forward pairing tioning protocols:
the odor preceded the reinforcement, for backward 3N+ 3-trial absolute conditioning, ITI of 2 min
pairing that order was reversed, the overlap was 1 s in (no CS-) (n = 10)
both cases. During the "post" phase all animals were 3N+O- 3-trial differential conditioning, ITI of 1 min,
presented with I-nonanol after 1, 5 and 15 min fol CS+ 1-nonanol, CS- l-octanol (n = 9)
lowing conditioning. 5N+ 5-trial absolute conditioning, ITI of 2 min
Five groups were subject to the different condi (no CS-) (n = 15)
tioning protocols: 5N+O- 5-trial differential conditioning, ITI of 1 min,
r 1-trial unrewarded I-nonanol (n = 9) CS+ I-nonanol, CS- l-octanol (n = 9)
lbw 1-trial backward conditioning (n = 14) 5N+H- 5-trial differential conditioning, CS+ 1-
=
1+ I-trial forward conditioning (n 9) nonanol, CS- 1-hexanol (n = 10)
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Fig. 1 Stimulus protocols of pre conditioning post
the experiments. Stimulus
duration for odors and
sDuucrrionsge pwaaisri naglw, athyse 3tw so. 1- I• • I• . L ... 1min ... \ ~ . ~, \ I, .
stimuli overlapped for 1 s. bw
Grersopuecpt ilvaeb eclos nrdeiftlieocnt inthge - 1bw I. • I• • L ... 1min ... \ ~ • ~\ \ I. ,
,...
protocol. The number gives +
throiawl wofatse nre tpheea tceodn.d (it-i)o nstianngd s eCnl) 1+ I, . .I L ... 1min ... \ ~ • ~1 \ .I ,
fafoonrrd w u(anbrrwde )wp faaoirrrdi nebdga cowkdiwtohar ,rs du( +c)r ofsoer 3bw I. • I• , ,b"w I • bwI• •b wIi ,I ... 1min ... , ~ • ~1 \ I. .
pairing. 1-Nonanol was the + + +
othnely f iorsdt ofri vgei vgerno utphsr o(usgeth o1u) to f 3+ I. • I• • \ .I. I• • ~\ ... 1min ... \ ~ • ~\ \ I, .
-4 -3 -2 -1 5 15
the experiments. For the last
seven groups (set 2), odors
are indicated by capital .I+. I+ I+, 1 I 0 I 0 I 0 I I
letters: 1-hexanol (H), 3N+ ~~p~p~p~~\ \ • • 1\ ... 5m~ "',• i i I , i • i I i ,
l-octanol (0) and 1-nonanol
(N). Stimulations of the three 11 0 I 0 I 0 I I, + + + p p p .
odors were randomized in the 3WO' \~~~~~\ ... 5min ... \ ~ ~ ~ ~ ~ ~
i i i i i • i i i \
"pre" and "post" phase
+ + + + +
110101011, I I I I I 11 0 I 0 I 0 I I
Nd) 5N+ i • i i i i i i i , , •••••••••• I.. 5min ".\ ••••••• , ••
en
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ p~ p~ p~ ~ .
5N+O' ~~p~p~p~~\
\ 1. . 5min ... \
5N+H' ~~p~p~p~~\ \ ~ P ~ P ~ P~ ~ ~ ~ ~ ~ p~ p~ p~ ~ .
1. . 5min ... \
01 I I I 0 I 0 I, p p p .
5N'O- "~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
i i i I • i i i i I , \ .. 5min ... \
P~ ~ ~ ~ p~ p~ \ \~ P~ P ~ ~ ~ ~ ~ ~ \.. ~ ~ p~ p~ p~ ~ ,
5NH 5min ... \
-9 ·8 -7 -6 -5 -4 -3 -2 -1 5 6 7 8 9 1011 1213
time (min) time (min)
• 1-nonanol III!II 1-octanol 0 1-hexanol
+ =
bw= backward pairing forward pairing
5N-0- 5-trial stimulation as in 5N+0- but without ("post"), including all three odors I-hexanol, l-octanol
any reward (n = 10) and 1-nonanol.
5N-H- 5-trial stimulation as in 5N+H- but without Odor responses to the three odors in every glo
any reward (n = 9). merulus were analyzed with a two-way RM ANOVA,
with the different conditioning groups as one factor
In this nomenclature, the number (3, 5) indicates how and "pre"-responses and "post"-responses as the re
often conditioning trials were repeated. Conditioned peated factor time. Multiple comparisons were per
odors are indicated by capital letters: 1-hexanol (H), 1- formed using the Holms-Sidak post-hoc test.
octanol (0) and 1-nonanol (N), with a (+) for rein
forced odor or a (-) for unrewarded presentation. Behavior
Thus, 5N+0- represents a 5-trial differential condi
tioning, with 1-nonanol reinforced and l-octanol The animals were prepared and stained as in the
unrewarded. imaging experiments, with the difference that the
Five minutes after conditioning the animals were esophagus and the muscles around the proboscis were
subject to olfactory stimulation as described above not restrained in order to allow for the PER. First,
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animals were tested for PER to sucrose stimulation at on both antennae (data not shown). This indicates that
each antenna, i.e. both the ipsilateral and the contra no bilateral neurons contributed to our signal, notably
lateral antenna to the stained I-ACT PNs. Learning also not the VUMmxl neuron. Most likely, these neu
experiments started 30 min later to avoid any effects rons were not labeled with the calcium sensitive dye.
due to sensitization (Menzel et al. 1991).
Odor stimulation with I-nonanol was again 3 s, sim PN responses to sucrose are similar
ilar to the absolute conditioning experiments. Intertrial between animals
interval was 2 min throughout "pre" and conditioning.
All animals received two "pre" odor stimulations. In addition to odors, PNs also responded to sugar
Thereafter the animals were conditioned by a 3-trial water stimulus at the antenna. In Fig. 3 two typical
absolute conditioning as described above. Five minutes examples of an odor stimulation paired with a sucrose
after conditioning the animals were tested for PER to 1- US are shown, one forward (Fig. 3a) and one backward
nonanol stimulation at the ipsilateral antenna, at the (Fig. 3b). Sucrose solution evoked an activity pattern
contralateral antenna and at both antennae. The order which was consistent across animals, and identical to
of stimulation was randomized between animals. Stim stimulation with water alone. The response pattern
ulation of only one antenna was achieved by covering consisted of several activated glomeruli, suggesting a
the other antenna with a sealed plastic tube. All animals combinatorial representation as known for odors. The
were then examined for stained I-ACT PNs under a response pattern to water and sucrose solution in
fluorescent microscope. Only successfully stained ani cluded glomeruli that also respond to volatile odors
mals were included in the analysis. (such as glomerulus 17) and glomeruli for which no
volatile odor is yet known as a suitable stimulus (such
as glomerulus 42). The strongest activity was in glo
Results merulus 42 and the lowest was in 33 (Fig. 3c). PNs
responded only to sucrose solution when stimulated on
Odors elicit activity in PNs in the same glomeruli the ipsilateral antennae, but not when stimulated at the
across animals contralateral antenna or at the proboscis (data not
shown). Sucrose responses were clearly distinguishable
Insertion of FURA-dextran crystals into the I-ACT from mechanical stimulation (data not shown).
nerve tract led to a selective staining of PNs (Fig. 2a),
as previously reported (Sachse and Galizia 2002). We Repeated odor stimulation does not change
identified a set of 15 glomeruli in each of the 126 ani its representation
mals included in this study using the digital three
dimensional atlas of the honeybee antennal lobe All 54 animals of set 1 (see Fig. 1) received two "pre"
(Galizia et al. 1999a) (Fig. 2a, c). nonanol presentations. PN odor responses in glomer
When stimulating with I-hexanol, l-octanol or 1- ulus 17 and 33 across animals were indistinguishable
nonanol, PNs showed the same glomerular spatio for the two "pre" stimuli (Fig. 4a). The same held true
temporal actlVlty patterns published previously for all other glomeruli (RM two-way ANOVA , Holms
(Fig. 2b, d) (Sachse et al. 1999; Sachse and Galizia Sidak multiple comparisons, smallest P = 0.059 for
2002). Hexanol evoked a strong calcium response in glomerulus 17, all other P > 0.1).
glomerulus 28, and weaker responses in glomerulus 17, It is necessary to know the response variability to
36, 38 and 35, whereas calcium concentration in glo repeated odor stimulation alone before investigating
merulus 29 decreased (Fig. 2e). Octanol elicited strong learning effects. Six successive nonanol stimulations
responses in glomeruli 17 and 28, weak activity in (IN-group, Fig. 4b) over the 21 min of the experiment
glomerulus 33 and a Ca2+ decrease in glomeruli 23, 25 elicited responses that remained unchanged in both
and 29. Nonanol evoked a strong response in glomeruli magnitude (AN OVA) and shape (visual inspection) in
17 and 33 and a Ca2+ decrease in glomeruli 23 and 25. all glomeruli (see responses to glomeruli 17 and 33 in
All responses were consistent across animals, as can be Fig. 4c).
seen by the small error bars (Fig. 2e). The responses to
octanol were overlapping with those to nonanol, while Absolute conditioning does not change
the response to hexanol showed virtually no overlap of the representation of the learned odor
activity with nonanol in the 15 observed glomeruli.
No responses could be measured in the AL contra The glomerular odor responses were not statistically
lateral to the staining side when stimulating with odors different at any point in time (time) between the dif-
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Fig. 2 Odor responses in the B 1-nonanol l-octanol 1-hexanol
honeybee AL. a Raw A 11
fluorescence image of an AL
stained with FURA. b False
color coded spatial response
patterns to I-hexanol, 1-
octanol and I-nonanol.
-2
Glomerulus 17,28 and 33 are
circled with a dOlled white
line. c Schematic view of the 1-nonanol (n = 72) l-octanol (n = 72) 1-hexanol (n = 72)
AL with the 15 identified C D
glomeruli used in this study. 14 14 14
12 12 12 17
d Mean time traces of odor 10 10 10
responses in selected .r
28
glomeruli (n = 72 animals). N ro 29
Time trace colors correspond a~: 33
to glomeruli in c. Stimulus is 0 47
marked by the shaded area. ·2 ·2 ·2
e Odor responses to ·4 ·2·10123456 ·4 ·I2-·r1- -0f' -~1 -2' 3. -4 ~5 ~6 ·4 ·I2-·r1- -0f' -~1 2 -'3. -~4 ~5 6
I-nonanol, l-octanol and lime(s)
I-hexanol for 15 glomeruli
E 12
(± SEM, n = 72, average
during the 3 s odor
stimulation). The three most 10
• 1-nonanol
responsive glomeruli for these
odors are indicated by colored 8 1-octanol
squares (eJn) D 1-hexanol
c
0 6
0en. .
~
0 4
"0
0
2 n = 72
-2 [22]13311281 36 38 35 47 48 42 25 60 49 37 23 29
glomeruli
c
maximum response to sucrose
A forward pairing B backward pairing
(n = 20)
20 20
1-non8nol
15 15
10 10
5 5
o o
-5 -5
-10 +-.-,--.--.-,s_u,,-ga,...r-,--,--,-, -10 +-,--.-,s-,ug,,-a,...r-.--.--,,--r-.-
-2 -1 0 1 2 3 4 5 6 7 8 -2 -1 0 1 2 3 4 5 6 7 17 33 28 36 38 35 47 48 42 25 60 49 37 23 29
time (s) glomeruli
Fig. 3 PN responses to sucrose and nonanol. Time traces in five marked by a shaded area, sucrose is marked by the yellow bar.
glomeruli of an individual measurement for forward pairing (a) c Averages of maximum responses for 15 identified glomeruli
and backward pairing (b). The odor-stimulus (nonanol) is during sucrose stimulation (± SEM, n = 20)
1090
Fig. 4 Unrewarded odor A glomerulus 17 (n = 56) glomerulus 33 (n = 56)
responses are stable over
time. a Calcium responses in 12 3j10 12 3j10
c 1st 2nd c 1st 2nd
two identified glomeruli, 17 10 8.. B 10 8.. B
and 33, to the first and second C/l C/l
1ti-mnoe ntarancoel sotfi malul l5a4ti oann im(maelsa on f N. ror 86 "0@00 4 86 "o@8 64
set 1). The odor-stimulus is (e)r 4 4
marked by a shaded area. 2 2
IT! = 2 min. Inset shows
0 0
average odor responses
(± SEM, n = 54) during -2 -2
stimulation. b Group r , -4 -4
average responses ( ± SEM, -2 -1 0 1 2 3 4 5 6 7 8 9 10 -2 -1 0 1 2 3 4 5 6 7 8 9 10
n = 9) of 15 identified time (5)
glomeruli to six consecutive 1-
nonanol stimulations. C B group 1 (n=9)
Average time traces (group
10
r) for glomeruli 17 and 33
•
8 -4' •
-2'
0' 11
IcCD/ l 6 l' 0
0 5' 0
@CC/ll . 4 15' 0
0
"0
0 2
-2 17 33 28 36 38 35 47 48 42 25 60 49 37 23 29
glomeruli
C glomerulus 17, group 1-(n = 9) glomerulus 33, group 1-(n= 9)
12 12 -4' --
10 10 -2' ........
0' ___
8 8
l'
.r 6 6 5'
'"ro 4 4 15'
Q
0:: 2 2
0 0
-2 -2
-4 -4
-2 -1 0 1 2 3 4 5 6 7 8 9 10 -2 -1 0 1 2 3 4 5 6 7 8 9 10
time (5)
ferent stimulus protocols (group) in set 1 (Fig. 1), i.e. ing-independent way, i.e. without a statistical
successive odor stimulation, single and multiple trial significance for learning paradigm, but common across
conditioning, backward pairing and forward pairing all groups. Such changes are correlated with experi
(Fig. 5) (no significant interaction between time and mental time, and not with treatment. Indeed, pooling
group, RM two-way ANOV A, lowest P for glomerulus all animals, we found significant differences in some
17: df= 16, F = 1.614, P = 0.067, in all other cases odor responses. Responses to nonanol at 1 min after
P > 0.3). Therefore, absolute appetitive conditioning training were attenuated in glomerulus 17 (Holms-Si
did not change the neural representation of the learned dak, P = 0.001) and the inhibition in glomerulus 38 was
odor in I-ACT projection neurons. reduced (P = 0.024); at 15 min, odor responses in glo
Even in the absence of learning-related modula merulus 38 showed less inhibition (P = 0.001), whereas
=
tions, odor responses might be modulated in a learn- glomeruli 36 (P = 0.015) and 42 (P 0.03) were
1091
•
Fig. 5 Neural representation glomerulus 17 •
remains stable following glomerulus 17 glomerulus 33
glomerulus 33
sucrose reinforcement. PN
response traces of glomerulus 1- 12 12 10
c1o7 nadnidti o3n3i ntog 1(-"npornea"n) ol before (n = 9) ~ 8 8 <8cQn) . 86
compared with responses "Q'", 4 4 <e?n 4
("post") at 1,5 and 15 min er: 0 0 -(0; 2
following conditioning. The 0
third column shows the ·4 0
-2 0 2 4 6 8 10 0 2 4 6 8 10 pre l' 5' 15'
average odor responses of
time (s)
glomerulus 17 and 33
(± SEM, n is given in each 1+ 12 12 10
graph)
Q) 8
8 8 i!!
(n = 14) + 8. 6
'"Q' " 4 4 ~ <e?n 4
er: (;
0 0 -0 2
0
.'
-4 ·4 0
-2 0 2 4 6 8 10 -2 0 2 4 6 8 10 pre l' 5' 15'
time (s)
1b w 12 12 8
Q)
8 8 i!! 6
(n=9) r a0 .
"Ua'" ,: 4 4 ~ 4
(;
0 ..... 0 -0 2
0
-4 0
-2 0 2 4 6 8 10 0 2 4 6 8 10 pre l' 5' 15'
time (s)
3+ 12 12 10
8 8 c~ 8
(n=11) r 8. 6
"Q'", 4 4 <e?n 4
er: (;
0 0 -0 2
0
-4 -4 0
-2 0 2 4 6 8 10 -2 0 2 4 6 8 10 pre l' 5' 15'
time (s)
3bw 12 12 10
8 8 c~ 8
(n=11)r 8. 6
"ua'":, 4 4 \ <e?n 4
0 o ~ \ -.:...J".:' ". ~;j,',: .'{:f,-,v-. :' -(00; 2
·4 -4 0
-2 0 2 4 6 8 10 -2 0 2 4 6 8 10 pre l' 5' 15'
time(s)
slightly increased (data not shown). All other glomeruli ory induction (Muller 2000b). Three-trial backward
remained unchanged compared to the "pre" at all conditioning, however, does not lead to learning nor to
"post" time points (RM two-way ANOVA ). a PKA increase. Therefore, we investigated the re
sponses to the CS-US pairing during mUltiple trial
Both forward and backward pairing leave the conditioning. When comparing forward (group 3+)
learned odor unchanged with backward conditioning (group 3bW; Fig. 6), it was
not possible to compare responses to the CS only, be
While single trial conditioning leads to short-term cause CS and US overlap in different ways. For
memory only, 3-trial forward pairing leads to long-term example, during the first second of the CS stimulation
learning and to a prolonged elevation of PKA activity the backward group already includes the US (group
in the AL which is necessary for this long-term mem- 3hw), while the forward group includes the US only
1092
during the last second (group 3+) (Fig. 3). We there Appetitive conditioning does not change responses
fore analyzed the combined response to CS and US to multiple other odors (set 2)
during the conditioning trial (see Methods). Com
pound responses to the CS and US did not differ be Having established that olfactory conditioning does
tween the two paradigms (smallest P for glomerulus 48 not change the representation of the conditioned odor,
with df = 1, F = 1.972, P = 0.176 two-way RM ANO we wondered whether other odors might be modified
V A). There was no difference between successive trials by learning. To this end, we tested odor responses to
(smallest P for glomerulus 25 with df = 2, F = 1.399, three odors before and after training, and conditioned
P = 0.259) and no significant interaction between the nonanol alone or differentially against octanol or
paradigms and trials (smallest P for glomerulus 29 with hexanol (set 2, Fig. 1). Statistical analysis of the three
df = 2, F = 1.441, P = 0.295), with onc exception: "prc" odor responses and the three "post" odor re
Glomerulus 60 showed a difference in trial (df = 2, sponses revealed no significant differences within
F = 3.355, P = 0.045) and a Holms-Sidak multiple "pre" or within "post" (ANOVA for all odor re
comparison revealed that trial 2 was different from sponses in each glomerulus, df = 2, smallest P = 0.079).
both, trial 1 and trial 3 (P = 0.03, traces not shown). For statistical analysis, therefore, the average of all
There was no significant interaction between trial and three responses preceding the treatment was defined as
paradigm (P = 0.301) and in both groups the second the "pre" odor response for every odor and likewise
trial was slightly reduced. Glomerulus 60 neither for the "post" odor response.
showed a pronounced response to nonanol nor to su There was no significant effect of the conditioning
crose stimulation. We therefore regard this significant paradigms (group) on any of the od or responses (time)
value as a statistical effect typical for multiple com (i.e. the CS+, the CS-or the neutral odor; RM two-way
parison studies and as biologically not relevant. ANOVA , smallest P with df = 6, F = 2.152, P = 0.059,
all other P > 0.1). There was no significant interaction
between group and time. In more detail, the different
A backward, group 3bW (n=11) paradigms gave the following results. Odor response
8 D for the CS+ and the neutral odors were unchanged
trial 1
Cl after five trials of absolute conditioning (group 5N+;
trial 2
III trial 3 Fig. 7, compare with 3+, Fig. 5), as shown for the
responsive glomeruli 17,28 and 33 in Fig. 7.
Differential conditioning of nonanol against another
odor also did not induce any changes in the responses
of any of the odors involved. Responses to CS+, CS-or
the neutral od or (groups 5N+O-and 5N+H-, Fig. 8) did
not change, even for odors with highly overlapping
-2
17 33 28 36 38 35 47 48 42 25 60 49 37 23 29 glomerular patterns (group 5N+O-). Responses did
glomeruli
also not change in those groups which received the
B forward, group 3+ (n=11) same odor sequence but without rewarding nonanol
(groups 5WO- and 5N-H-, Fig. 8).
Even if od or responses are unchanged after condi
tioning as compared to before conditioning, odor re
sponses might change during the repeated learning
trials. In this analysis it is important to also include
CS+and CS-, because in behavioral studies of differ
ential conditioning the first reinforcement of the CS+
leads to an enhanced response to the subsequent CS-,
even though the CS- is not paired with the US, indi
17 33 28 36 38 35 47 48 42 25 60 49 37 23 29 cating that in this condition animals generalize be
glomeruli tween CS+ and CS-. As differential training
Fig. 6 Responses are stable during conditioning. The compound progresses, the response to the CS- returns to the
response to 1-nonanol and sucrose is not modulated during spontaneous response level, or falls below it (Bit
multiple trial conditioning. a Average compound responses
terman et aI. 1983). We therefore compared the CS+
(± SEM, n = 11) during trial 1,2 and 3 of backward conditioning
(group 3bw). b The same for forward conditioning (group 3+, and CS- responses during conditioning (where gener
n = 11) alization would be expected) with the respective