Download Conditioning: Simple Neural Circuits in the Honeybee

This article was originally published in the Encyclopedia of Neuroscience
published by Elsevier, and the attached copy is provided by Elsevier for the
author's benefit and for the benefit of the author's institution, for noncommercial research and educational use including without limitation use in
instruction at your institution, sending it to specific colleagues who you know,
and providing a copy to your institution’s administrator.
All other uses, reproduction and distribution, including without limitation
commercial reprints, selling or licensing copies or access, or posting on open
internet sites, your personal or institution’s website or repository, are
prohibited. For exceptions, permission may be sought for such use through
Elsevier's permissions site at:
http://www.elsevier.com/locate/permissionusematerial
Menzel R (2009) Conditioning: Simple Neural Circuits in the Honeybee. In:
Squire LR (ed.) Encyclopedia of Neuroscience, volume 3, pp. 43-47.
Oxford: Academic Press.
Author's personal copy
Conditioning: Simple Neural Circuits in the Honeybee 43
Conditioning: Simple Neural Circuits in the Honeybee
R Menzel, Freie Universität Berlin, Berlin, Germany
ã 2009 Elsevier Ltd. All rights reserved.
The Antennal Lobe and the Mushroom
Body Are Sequentially Involved in the
Olfactory Memory Trace
In honeybee olfactory learning, the pathways for
the conditioned stimulus (CS, odor) and the unconditioned stimulus (US, sucrose) are well defined. The US
pathway is implemented in a single identified neuron,
the VUMmx1 neuron. The olfactory pathway (the CS
pathway) consists of the olfactory receptor neurons
projecting through the antennal nerve to the antennal
lobe (AL); the first-order integration neuropil; the
olfactory projection neurons (PNs), which connect
the AL with the mushroom body (MB); the lateral
horn (also called lateral protocerebrum); and other
higher-order brain regions. CS and US pathways converge anatomically at three sites: the AL, the lip region
of the MB, and the lateral horn. For two of these
regions, the AL and the MB, it has been shown with
local injections of the putative transmitter of the first
ventral unpaired median neuron of the maxillary segment (VUMmx1) as a substitute for the reward stimulus that olfactory memory induces learning by forward
pairing (but not by backward pairing). These findings
corroborate earlier observations using local cooling as
a retrograde amnestic treatment, which documented a
cooling-sensitive memory phase for the AL during the
first 1–2 min after a single learning trial and such a
phase for the MB for the first 5–6 min. The early and
late short-term-memory phases after single-trial olfactory conditioning were related to the AL and MB,
respectively, on the basis of such findings.
Single Neuron Activity Correlate with
Olfactory Learning
The reward neuron VUMmx1 responds to sucrose
with long-lasting spike activity and to various visual,
olfactory, and mechanosensory stimuli with low-frequency spike activity. Depolarizing VUMmx1 immediately after CS presentation (forward-pairing, CSþ)
leads to learning, but backward-pairing does not
(CS ). Thus VUMmx1 constitutes the neural correlate
of the US in associative olfactory learning. After differential conditioning in which an odor CSþ is forward
paired with sucrose reward (US) and another odor CS
is backward paired with US, presentation of the CSþ
alone activates and CS inhibits VUMmx1, indicating
that the reward pathway learns about the stimuli, a
property that may be the neural substrate of secondorder conditioning. In addition, VUMmx1’s response
to the US after presentation of the CSþ is greatly
reduced, but the response to CS remains normal,
indicating that the response of VUMmx1 to US after
conditioning depends on US expectation. This property of VUMmx1 is sufficient to explain the behavioral
phenomenon of blocking and may thus reflect its neural substrate. These results demonstrate that the single
identified neuron VUMmx1 is a sufficient neural substrate for the reinforcing function of the US sucrose in
olfactory conditioning and has properties that allow
explaining second-order conditioning and blocking.
Many other neurons belonging to different tracts
leading from the AL toward the MB, or which are
extrinsic to the MB, have been tested for associative
plasticity by means of the differential conditioning
paradigm. PNs connecting the AL with the MB
calyx were less clear in CSþ-specific plasticity when
intracellular recordings were applied, but recent
extracellular multiunit recordings provided compelling evidence for associative plasticity in the minute
range after conditioning. Most interesting, particular
PNs specifically enhance or reduce their responses to
CSþ in about equal proportions, and a CSþ-specific
shift in the power spectrum of local field potentials
indicates an increase of precise spike timing for the
CSþ but not for the CS . This property may explain
findings from optical imaging experiments in which
Ca2þ activity of the postsynaptic sites of lateral PN
neurons in the AL glomeruli was monitored during
differential olfactory conditioning. No specific CSþ
and CS effects were seen. It appears that the glomerulus as a whole reliably codes odors in a combinatorial pattern together with other glomeruli, whereas
single PNs undergo specific associative plasticity. In
the case of a balance between associative rise and fall
of activity in PNs originating in the same glomerulus,
no learning effect will be seen when the whole glomerulus is imaged. It will have to be elucidated in the
future whether the associative rise and fall in the rate
responses of particular PNs or the precise timing of
spikes in particular subsets of PNs represents the
learning-related signature in the AL-MB network.
Two other observations support the conclusion
that the AL network is indeed involved in an early
and short-lasting memory trace. First, Ca2þ imaging
of glomerular activity after bath application of the
Ca2þ indicator (Ca-green FM ester) gave consistently
higher responses to CSþ than to CS . The fluorescence
signal in such a situation may come predominantly
from the presynaptic terminals of olfactory receptor
Encyclopedia of Neuroscience (2009), vol. 3, pp. 43-47
Author's personal copy
44 Conditioning: Simple Neural Circuits in the Honeybee
neurons (and possibly also from local interneurons and
glia cells), indicating that synaptic processing inside
the glomerulus converts CSþ-specific plasticity into
both enhancement and reduction of odor responses.
Second, the postsynaptic sites of lateral PN neurons
within the glomeruli show spontaneous Ca2þ fluctuations. After stimulation with an odor, these spontaneous activity fluctuations are more strongly correlated
between the glomeruli that had been activated simultaneously by the odor, and the correlations between
the spontaneous activity fluctuations suffice to reconstruct the stimulus. It is concluded that these modifiable fluctuations could provide an ideal substrate for
Hebbian reverberations and sensory memory in a neural system. It is important to note that all these associative and nonassociative changes seen in the
AL glomeruli and PNs last only for a few minutes,
corroborating the conclusion that the AL may be
involved in the establishment of an olfactory memory
trace only shortly after the learning trial.
Two kinds of MB-extrinsic neurons have been studied with respect to associative plasticity, a single identified neuron, the pedunculus-extrinsic neuron 1
(PE1), and neurons in the protocerebral-calycal tract
(PCT). The PE1 neuron leaves the alpha lobe of the
MB and receives its input across the peduncle of
the MB at two bands of putative postsynaptic specializations. PE1 responds to a large range of odors.
Differential conditioning leads to a CSþ-specific reduction of odor responses, whereas US-only presentations
(sensitization) cause an increase of odor responses.
Since the CSþ-specific responses – as recorded intracellularly from isolated bee heads – were lost after
a few minutes, it was initially concluded that PE1
may be related to short-term memory. However,
recent extracellular recordings from the intact animal
lasting many hours have shown that the CSþ-specific
response reduction is stable over the lifetime of the
recording (several hours). Furthermore, the synapses
between the MB-intrinsic neurons (Kenyon cells)
and the PE1 were found to undergo long-term potentiation if PE1 was depolarized during the tetanic electric stimulation of the Kenyon cells. It is not yet clear
how learning-related response reduction and associative long-term potentiation in PE1 can be reconciled.
CSþ-specific effects – as compared with CS or
US-only effects – were seen in neurons of the PCT.
Many of these neurons are g-aminobutyric acid
immunoreactive and project back from the alpha
lobe of the MB to its input site, the calyx. They
terminate both at the presynaptic sites of the PNs
and the postsynaptic sites of the MB intrinsic neurons, the Kenyon cells. It is conceivable that PCT
neurons provide an adapted inhibitory feedback
from the output site of the MB to its input. Since
PCT neurons also cross between the sensory domains
of the MB, they could also be involved in contextspecific forms of learning.
Neural Circuits Underlying Olfactory
Conditioning: Signaling Cascades
Mediating CS and US Stimuli
The different contributions of AL and MB in associative learning are also evident at the level of the
molecular machinery implicated in learning and
memory formation. Processing CS and US stimuli –
even as early as during training – activates different
signaling cascades in vivo. Stimulation of the antenna
with sucrose, the US, induces a short transient activation of the cyclic adenosine monophosphate (cAMP)dependent protein kinase A (PKA) in the AL, whereas
odor stimulation (CS) or mechanical stimulation of
the antennae does not affect PKA activity in the ALs.
This US-induced PKA activation in the AL is
mediated by octopamine, the putative transmitter of
the reward neuron VUMmx1. The dense innervation
of the VUMmx1 in the AL, the main localization of
the PKA in AL interneurons, and the biochemical
measurements support a general modulatory function
of US-induced processes mediated via the cAMP/PKA
cascades in the AL. The specific reinforcing function
of octopamine during learning in the AL was supported by silencing the expression of the octopamine
receptor which impairs olfactory acquisition but not
odor discrimination.
Although the calyces, the olfactory input area of
the MB, are also densely innervated by the octopaminergic VUMmx1 neuron, stimulation with sucrose
(US) does not lead to PKA activation in the MB calyces
in vivo. This, and the fact that octopamine can, in
principle, stimulate PKA in cultured Kenyon cells of
the MBs, supports the idea that octopamine receptors
that receive input from VUMmx1 in the MB are most
likely coupled to Ca2þ-regulated pathways. The signaling cascade mediating the US function in olfactory
conditioning in the MB has not yet been identified.
Molecular Signaling Cascades in the ALs
Are Critical for the Induction of Olfactory
Memory Traces
Direct in vivo measurements demonstrate that the
temporal characteristics of the US-induced PKA activation in the AL is critically influenced by its pairing
with CS stimuli during olfactory conditioning.
A single CS/US forward pairing, which induces a
weak olfactory memory, leads to a transient increase
in PKA activity that returns to basal levels 60 s after
Encyclopedia of Neuroscience (2009), vol. 3, pp. 43-47
Author's personal copy
Conditioning: Simple Neural Circuits in the Honeybee 45
the conditioning trial. Three CS/US forward-pairings
in succession, which induce a long-term memory
(LTM), prolong PKA activation in the AL up to
more than 3 min. In contrast, US stimulation alone
and, independent of the number, US/CS backward
pairings induce a significantly shorter PKA activation. This close correlation between training parameters on the one hand and the temporal relation
between US and CS stimulation, the number of conditioning trials, and the dynamic properties of PKA
activation in the AL on the other hand leads to the
hypothesis that prolonged PKA activation in the AL is
involved in LTM formation. The hypothesis was
tested by photolytic release of caged cAMP in the
AL to artificially prolong PKA activation during
olfactory conditioning. A local replay of the prolonged PKA activation in the AL in vivo, combined
with a single conditioning trial, is sufficient to induce
long-lasting memory. This provides direct functional
evidence for a link between conditioning parameters,
PKA activation in the AL, and its contribution to
LTM formation in intact animals.
The use of the uncaging technique allowed further
identification of the molecular processes underlying
the prolonged PKA activation in the AL and thus the
mechanisms contributing to induction of LTM in
the AL. Nitric oxide, which is required for LTM
formation in the honeybee, mediates the prolongation
of the PKA activity by activation of the soluble
guanylate cyclase and increase of cyclic guanosine
monophosphate (cGMP). Inhibition of the soluble
guanylate cyclase impairs both the prolonged
PKA activation in the AL and LTM formation.
Similar to the uncaging of cAMP, photorelease of
caged cGMP in the AL – in combination with singletrial conditioning – induces long-lasting memory.
Although the target of cGMP has not yet been identified in vivo, the synergistic activation of honeybee
PKAII by cAMP and cGMP points to a direct function
of cGMP in the prolonged increase in PKA activity
(25%) in the AL.
These findings demonstrate that molecular processes localized in the neural circuit of the AL critically contribute to LTM formation. Prolongation of
PKA activation in the AL in conjunction with singletrial conditioning, however, does not reach the level
of conditioned responses after multiple-trial conditioning, and thus it seems feasible that additional
molecular processes or neural circuits may contribute
to the prolongation. The molecular and neural targets
of the early events in the AL that finally lead to LTM
are yet unknown.
The signaling cascade that involves the Ca2þphospholipid-dependent protein kinase C (PKC) is
implicated in a parallel-acting system contributing
to olfactory memory at the AL level. In contrast to
US-specific activation of the PKA in the AL, PKC is
activated by both the US and the CS. The temporal
pattern of PKC activation is independent of the
sequence of CS and US stimulation or the number
of conditioning trials. Since inhibition of PKC activation during the conditioning phase affects neither
acquisition nor memory formation, conditioninginduced PKC activation in the AL seems not to be
required for memory induction. The analysis of the
learning-induced changes in PKC activity in the
AL hours and days after training revealed a function
of PKC in memory maintenance. In contrast to a
single conditioning trial, repeated conditioning trials
that induce LTM cause an increase in PKC activity
beginning 1 h after conditioning and lasting up to
3 days. This long-lasting PKC activation can be dissected into two mechanistically independent phases.
In the early phase (1–16 h), a constitutively active
PKC, the PKM, contributes to the elevated activity.
PKM is formed by cleavage of the activated PKC by
the Ca2þ-dependent protease calpain. Blocking calpain activity during conditioning prevents PKM formation in the AL and impairs memory in a time
window between 1 and 16 h. This treatment does
not affect acquisition, the early memory phase up to
30 min, and memory after 1 day. The late phase of the
training-induced increase in PKC activity (1–3 days)
in the AL is unaffected by calpain blockers but
requires protein and RNA synthesis. Thus, PKM formation in the AL is an independent parallel process
required for maintaining a midterm memory phase.
The function of the late phase (1–3 days) is still
unclear but is probably one of several parallel
mechanisms occurring in different brain circuits
required for the formation of the late phase of LTM.
In summary, currently evidence exists for a function of the AL circuitry only in early processes of
learning and memory formation, like the nitric
oxide-mediated PKA activation involved in LTM
induction and the early PKM formation required
for maintaining a memory phase in the hours range.
Glutamate-Mediated Signaling Cascades
in the MBs Contribute to LTM Formation
Glutamate is the major excitatory transmitter in
mammals and plays a central role in neuronal plasticity in vertebrates. Its function in the insect brain,
however, is only poorly understood. Although glutamate receptors have been identified in honeybees
and manipulation of glutamate function using pharmacological tools supports a function in learning
processes, the results are a matter of controversial
discussion.
Encyclopedia of Neuroscience (2009), vol. 3, pp. 43-47
Author's personal copy
46 Conditioning: Simple Neural Circuits in the Honeybee
Photolytic uncaging of glutamate in the honeybee MB revealed direct evidence for a defined spatial and temporal contribution of glutamate in LTM
formation. Uncaging glutamate immediately after a
weak training protocol (single-trial training) in the
MB improves the formation of a late memory phase
(2 days) and thus mimics the effect of a strong training protocol. Uncaging shortly before the weak training has no effect, pointing to a defined function of
glutamate with respect to early memory processing
rather than learning. This function of glutamate is
restricted to the MB circuitry, since glutamate release
in the AL does not improve learning or retention.
Taken together, glutamate action is restricted to
the MBs, where it contributes to induction of a
late memory phase in a time window shortly after
training. Although it is still unclear whether this
late memory phase is mechanistically identical with
the LTM induced by a strong training (three-trial
conditioning), the findings provide an example for
parallel molecular processes underlying learning and
memory formation in vivo occurring in different neural circuits.
Parallel Molecular Processes Contribute
to LTM Memory Formation
As in other systems, LTM in honeybees can be divided
into an early phase (eLTM, 1–2 days), which requires
protein synthesis, and a transcription-dependent late
phase (lLTM, 3 days). In all model systems investigated so far, inhibition of PKA activity during the
training period results in a loss of both eLTM and
lLTM. Although this suggests a single PKA-triggered
process, experiments considering the impact of the
satiation level in appetitive learning and memory formation in the honeybee provided evidence of a more
complex function of the cAMP/PKA cascade in LTM
induction. Three-trial conditioning in hungry animals
leads to perfect acquisition and induction of midterm
memory and both LTM phases. However, feeding bees
4 h before olfactory conditioning impairs acquisition
and memory formation. This, and the different basal
PKA-activity in honeybee brains at different satiation
levels, points to a contribution of the cAMP/PKA
cascade. Elevating the low basal PKA-activity levels
in animals fed 4 h before conditioning specifically rescues the transcription-dependent lLTM. Acquisition,
midterm memory, and eLTM are still impaired. The
fact that both eLTM and lLTM require PKA for their
induction, but PKA rescue in fed animals improves
only lLTM but not eLTM, points to the existence of
two parallel cAMP/PKA pathways implicated in
memory formation: one process appears to trigger
events leading to translation-dependent eLTM; the
other is involved in inducing cascades required for
transcription-dependent lLTM. Localization of the
neural circuits related to these independent cAMP/
PKA pathways will provide important information
on the organization of the neural network involved
in LTM formation.
See also: Communication in the Honeybee; Conditioning:
Theories; Learning and Memory in Invertebrates: Honey
Bee; Olfaction in Invertebrates: Honeybee; Procedural
Learning: Classical Conditioning.
Further Reading
Abel R, Rybak J, and Menzel R (2001) Structure and response
patterns of olfactory interneurons in the honeybee, Apis mellifera. Journal of Comparative Neurology 437: 363–383.
Balfanz S, Strunker T, Frings S, and Baumann A (2005) A family of
octopamine receptors that specifically induce cyclic AMP production or Ca2þ release in Drosophila melanogaster. Journal of
Neurochemistry 93: 440–451.
Brandt R, Rohlfing T, Rybak J, et al. (2005) A three-dimensional
average-shape atlas of the honeybee brain and its applications.
Journal of Comparative Neurology 492: 1–19.
Faber T, Joerges J, and Menzel R (1999) Associative learning
modifies neural representations of odours in the insect brain.
Nature Neuroscience 2: 74–78.
Farooqui T, Robinson K, Vaessin H, and Smith BH (2003) Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee. Journal of Neuroscience
23: 5370–5380.
Fiala A, Müller U, and Menzel R (1999) Reversible downregulation
of protein kinase A during olfactory learning using antisense
technique impairs long-term memory formation in the honeybee, Apis mellifera. Journal of Neuroscience 19: 10125–10134.
Galán RF, Weidert M, Menzel R, Herz AV, and Galizia CG (2006)
Sensory memory for odors is encoded in spontaneous correlated
activity between olfactory glomeruli. Neural Computation 18:
10–25.
Ganeshina O and Menzel R (2001) GABA-immunoreactive neurons in the mushroom bodies of the honeybee: An electron
microscopic study. Journal of Comparative Neurology 437:
335–349.
Grohmann L, Blenau W, Erber J, Ebert PR, Strunker T, and
Baumann A (2003) Molecular and functional characterization
of an octopamine receptor from honeybee (Apis mellifera)
brain. Journal of Neurochemistry 86: 725–735.
Grünbaum L and Müller U (1998) Induction of a specific olfactory
memory leads to a long-lasting activation of protein kinase C in
the antennal lobe of the honeybee. Journal of Neuroscience 18:
4384–4392.
Grünewald B (1999) Physiological properties and response modulations of mushroom body feedback neurons during olfactory
learning in the honeybee Apis mellifera. Journal of Comparative
Physiology A 185: 565–576.
Hammer M (1993) An identified neuron mediates the unconditioned stimulus in associative olfactory learning in honeybees.
Nature 366: 59–63.
Hammer M (1997) The neural basis of associative reward learning
in honeybees. Trends in Neurosciences 20: 245–252.
Encyclopedia of Neuroscience (2009), vol. 3, pp. 43-47
Author's personal copy
Conditioning: Simple Neural Circuits in the Honeybee 47
Hammer M and Menzel R (1998) Multiple sites of associative
odour learning as revealed by local brain microinjections of
octopamine in honeybees. Learning & Memory 5: 146–156.
Hildebrandt H and Müller U (1995) Octopamine mediates rapid
stimulation of PKA in the antennal lobe of honeybees. Journal
of Neurobiology 27: 44–50.
Hildebrandt H and Müller U (1995) PKA activity in the antennal
lobe of honeybees is regulated by chemosensory stimulation
in vivo. Brain Research 679: 281–288.
Leboulle G and Müller U (2004) Synergistic activation of insect
cAMP-dependent protein kinase A (type II) by cyclicAMP and
cyclicGMP. FEBS Letters 576: 216–220.
Locatelli F, Bundrock G, and Müller U (2005) Focal and temporal
release of glutamate in the mushroom bodies improves olfactory memory in Apis mellifera. Journal of Neuroscience 25:
11614–11618.
Lopatina N, Ryzhova I, and Chesnokova E (2002) The role of nonNMDA-receptors in the process of associative learning in the
honeybee Apis mellifera. Journal of Evolutionary Biochemistry
and Physiology 38: 211–217.
Maleszka R, Helliwell P, and Kucharski R (2000) Pharmacological
interference with glutamate re-uptake impairs long-term memory in the honeybee Apis mellifera. Behavioural Brain Research
115: 49–53.
Mauelshagen J (1993) Neural correlates of olfactory learning in an
identified neuron in the honey bee brain. Journal of Neurophysiology 69: 609–625.
Menzel R (1999) Memory dynamics in the honeybee. Journal of
Comparative Physiology A 185: 323–340.
Menzel R, Erber J, and Masuhr T (1974) Learning and memory in
the honeybee. In: Barton-Browne L (ed.) Experimental Analysis
of Insect Behaviour, pp. 195–217. Berlin: Springer.
Menzel R and Manz G (2005) Neural plasticity of mushroom
body-extrinsic neurons in the honeybee brain. Journal of
Experimental Biology 208: 4317–4332.
Müller U (1996) Inhibition of nitric oxide synthase impairs
a distinct form of long-term memory in the honeybee, Apis
mellifera. Neuron 16: 541–549.
Müller U (1997) Neuronal cAMP-dependent protein kinase type II
is concentrated in mushroom bodies of Drosophila melanogaster and the honeybee Apis mellifera. Journal of Neurobiology 33: 33–44.
Müller U (2000) Prolonged activation of cAMP-dependent protein
kinase during conditioning induces long-term memory in honeybees. Neuron 27: 159–168.
Peele P, Ditzen M, Menzel R, and Galizia CG (2006) Appetitive
odour learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons. Journal of Comparative Physiology A 192(10): 1083–1103.
Riedel G, Platt B, and Micheau J (2003) Glutamate receptor
function in learning and memory. Behavioural Brain Research
140: 1–47.
Rybak J and Menzel R (1998) Integrative properties of the
Pe1-neuron, a unique mushroom body output neuron.
Learning & Memory 5: 133–145.
Si A, Helliwell P, and Maleszka R (2004) Effects of NMDA receptor antagonists on olfactory learning and memory in the
honeybee (Apis mellifera). Pharmacology, Biochemistry, and
Behavior 77: 191–197.
Zannat MT, Locatelli F, Rybak J, Menzel R, and Leboulle G (2006)
Identification and localisation of the NR1 sub-unit homologue
of the NMDA glutamate receptor in the honeybee brain. Neuroscience Letters 398: 274–279.
Encyclopedia of Neuroscience (2009), vol. 3, pp. 43-47