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J Comp Physiol A (1999) 185: 565±576
Ó Springer-Verlag 1999
ORIGINAL PAPER
B. GruÈnewald
Physiological properties and response modulations of mushroom body
feedback neurons during olfactory learning in the honeybee,
Apis mellifera
Accepted: 9 September 1999
Abstract Mushroom bodies are central brain structures
and essentially involved in insect olfactory learning.
Within the mushroom bodies c-aminobutyric acid
(GABA)-immunoreactive feedback neurons are the
most prominent neuron group. The plasticity of inhibitory neural activity within the mushroom body was
investigated by analyzing modulations of odor responses
of feedback neurons during olfactory learning in vivo.
In the honeybee, Apis mellifera, feedback neurons were
intracellularly recorded at their neurites. They produced
complex patterns of action potentials without experimental stimulation. Summating postsynaptic potentials
indicate that their synaptic input region lies within the
lobes. Odor and antennal sucrose stimuli evoked excitatory phasic-tonic responses. Individual neurons responded to various odors; responses of di€erent neurons
to the same odor were highly variable. Response modulations were determined by comparing odor responses
of feedback neurons before and after one-trial olfactory
conditioning or sensitisation. Shortly after pairing an
odor stimulus with a sucrose reward, odor-induced spike
activity of feedback neurons decreased. Repeated odor
stimulations alone, equally spaced as in the conditioning
experiment, did not a€ect the odor-induced excitation.
A single sensitisation trial also did not alter odor
responses. These ®ndings indicate that the level of odorinduced inhibition within the mushroom bodies is
speci®cally modulated by experience.
Key words Olfactory learning á Sensitisation á
Inhibitory feedback neurons á Insect á Intracellular
recording
B. GruÈnewald (&)
Institut fuÈr Neurobiologie, Freie UniversitaÈt Berlin,
KoÈnigin-Luise-Str. 28±30
D-14195 Berlin, Germany
e-mail: [email protected]
Tel.: +49-30-838-4298; Fax: +49-30-838-5455
Abbreviations CS conditioned stimulus á GABA
c-aminobutyric acid á MB mushroom body á PER
proboscis extension re¯ex á US unconditioned stimulus
Introduction
Studies on the neural correlates of learning behavior in
the honeybee, Apis mellifera, focused on the analysis of
the proboscis extension re¯ex, PER. This appetitive re¯ex can be conditioned by a single forward pairing of an
odor stimulus (conditioned stimulus, CS) with a sucrose
reward (unconditioned stimulus, US; Kuwabara 1957;
Vareschi 1971; Menzel et al. 1974; Bitterman et al.
1983). A single sucrose stimulation sensitises the response (Menzel et al. 1991; Hammer et al. 1994). Within
the insect brain, the mushroom bodies (MB) are essentially involved in olfactory learning and memory formation (Apis: Menzel et al. 1974; Erber et al. 1980,
1987; reviews: Hammer and Menzel 1995; Menzel and
MuÈller 1996; Drosophila: Heisenberg et al. 1985; De
Belle and Heisenberg 1994; reviews: Davis 1993; Dubnau and Tully 1998). The anatomical organization of the
MB, its connectivity within the insect brain and behavioural development have been described in detail (reviewed by Mobbs 1984, 1985; SchuÈrmann 1987; Menzel
et al. 1994; Fahrbach and Robinson 1995), and the
physiology of several of its neuronal elements have been
analyzed (Erber 1978; Homberg and Erber 1979;
Schildberger 1981, 1983, 1984; Homberg 1984;
Gronenberg 1986, 1987; Mauelshagen 1993; Laurent
and Naraghi 1994; SchaÈfer et al. 1994; Menzel et al.
1994; Li and Strausfeld 1997; Stopfer et al. 1997;
Goldberg et al. 1999).
The present study analyses the physiology of c-aminobutyric acid (GABA)-immunoreactive feedback neurons, which are major components of the insect MB
(ants: Goll 1967; crickets: SchuÈrmann 1973; ¯ies:
Strausfeld 1976; locusts: Weiss 1978; Schildberger 1983;
Leitch and Laurent 1996; moths: Homberg et al. 1987;
566
honeybees: Mobbs 1982; Bicker et al. 1985). In the
honeybee MB approximately 50 feedback neurons connect the dorsal and median a-lobe, the b-lobe, and the
pedunculus with all ipsilateral calycal subcompartments,
lip, collar, and basal ring (GruÈnewald 1999). Initial
physiological studies have shown that feedback neurons
respond to olfactory, visual, gustatory and mechanical
stimuli; often a single neuron responds to a variety of
stimuli of di€erent sensory modalities (Homberg and
Erber 1979; Schildberger 1981; Gronenberg 1987).
However, it was unknown whether feedback neurons
change their response behavior during olfactory learning.
In the honeybee MB, the individual, identi®ed MB
output neuron, PE1, undergoes modulations of its odor
responses, which are speci®c for associative and nonassociative stimulus paradigms (Mauelshagen 1993).
A single conditioning trial results in a decrease, a single
antennal sensitisation trial in a transient increase of odor
evoked spike frequency, indicative for a di€erential
neural representation of associative and non-associative
events at the MB level. The precise mechanisms that
underlie modulations of odor-evoked activity in MB
neurons are still unknown. Modulatory input from the
VUMmx1 neuron, which mediates the reinforcing
function of rewards during olfactory conditioning
(Hammer 1993, 1997) is probably involved. Since Mauelshagen (1993) suggested that response modulations of
the PE1-neuron may be due to altered input from presynaptic Kenyon cells, one may hypothesise that altering
the amount of inhibition within the MB regulates Kenyon cell excitation and thus a€ects odor responses of
MB output neurons like the PE1-neuron. Therefore, the
present study analyses responses of feedback neurons to
olfactory and gustatory stimuli that are relevant for olfactory learning. It further investigates whether a single
sensitisation or conditioning trial modulates the odor
responses of feedback neurons. The results indicate that
the level of odor-induced inhibition within the MB is
altered in an experience-dependent fashion.
Materials and methods
Animals and preparation
Worker honeybees (Apis mellifera carnica) were caught between
9:00 and 10:00 a.m. at the hive entrance. After immobilisation by
cooling, they were ®xed in small metal tubes with a strip of sticky
tape between head and thorax. After recovery animals were fed
with one drop of sucrose solution (25%). Only those bees which
readily showed proboscis extension upon feeding were selected for
experiments, which began at least 1 h after feeding. To achieve
stable intracellular recordings the head preparation developed by
Mauelshagen (1993) was used. For this the isolated head of the bee
was mounted on a perfusion chamber which was supplied continuously with aerated saline containing (in mmol á l)1): 135 NaCl, 5
KCl, 10 MgCl2, 1.6 CaCl2, and 80 tris(hydroxymethyl)aminomethane (TRIS), pH 7.25. After stabilising the brain by carefully
dissecting the oesophagus with its connecting muscles, the anterior
surface of the brain was exposed around the recording site. As a
consequence of the preparation the proboscis extension re¯ex was
irreversibly impaired.
Recording and labeling of neurons
Recording electrodes (borosilicate glass, 1.0 mm outer diameter,
0.58 mm inner diameter; Hilgenberg, Germany) were pulled with a
horizontal micropipette puller (P87, Sutter Instruments, Novato,
Calif.). Electrodes were back®lled with Neurobiotin (2% in
1 mol á l)1 KCl, Vector Laboratories, Burlingame, Calif.) or Lucifer
Yellow (5% in 0.1 mol á l)1 LiCl, Molecular Probes, Eugene, Ore.),
and had resistances in the tissue between 40±90 MW and between
100 and 180 MW, respectively. Electrodes were positioned in the
medio-lateral a-lobe, which was easy to recognise visually. They
were lowered vertically into the brain and their depth was monitored by using a potentiometer connected to the ®ne adjustment of
the micromanipulator (Leitz, Wetzlar, Germany).
After each experiment neurons were iontophoretically ®lled by
either injecting Neurobiotin (2±4 nA depolarising current) or Lucifer yellow (2±4 nA hyperpolarising current) for as long as the cell
could be held stabile (2±15 min). After dissection in saline, brains
were ®xed in paraformaldehyde (4% in phosphate-bu€ered saline
(PBS), for 12 h at 4 °C). Biotin-®lled brains were dehydrated in
graded ethanol, lipophilic substances solubilized in xylene, and
rehydrated in PBS. The ganglion sheath was permeabilized with
collagenase/hyaluronidase (1 mg ml)1, 1 mmol á l)1 CaCl2 added,
pH 7.4, 60 min, 37 °C; Sigma Chemicals, St. Louis, Mo.), endogenous peroxidases were blocked by H2O2 (0.03% in PBS). After
several bu€er rinses, specimens were incubated in biotinylised Avidin-HRP-complex (Vektor Laboratories, 5 h, 37 °C, 1% Triton
added). Following preincubation with 0.02% di-aminobenzidine
(Sigma, 45 min, 20 °C), di-azonium salt was precipitated by adding
0.001% H2O2. After dehydration in graded ethanol, specimens were
cleared with methyl salicylate. Lucifer Yellow-stained specimens
were dehydrated in graded ethanol, cleared up in methyl salicylate,
and examined using a ¯uorescence microscope (Wild-Polyvar, Leica,
Germany) under epi-illumination. During this study, intracellular
labeling of neurons was performed exclusively for morphological
identi®cation of a recorded cell. Due to the extended periods of up to
20 min needed for all test protocols, time for subsequent dye injection was limited and therefore most neurons were not completely
®lled. Only those recordings were analysed in which a single neuron
was stained and anatomically identi®ed as a feedback neuron. Thus,
52 recorded feedback neurons were included in this study.
Stimuli
Gustatory stimuli were manually applied and consisted of brie¯y
(<1 s) touching the distal tip of the antennae and/or the proboscis
with a wooden tooth stick soaked in sucrose solution (25%).
Olfactory stimuli (duration 2 s) were carnation and orange
blossom, citral and geraniol, which are components of ¯oral odors
and are frequently used as conditioned stimuli (CS) in olfactory
conditioning (e.g. Menzel et al. 1991). Pu€s of odors and plain air
were delivered to the antennae by a custom-made olfactometer.
Glass cylinders (1 ml volume), each ®lled with 0.4 ml of pure odor
substances, were placed into tightly closed brass containers (height
3 cm, diameter 2.5 cm), positioned ca. 50 cm from the head preparation. A continuous air¯ow (¯ow rate approximately 3 l min)1)
transported the odor streams via Te¯on tubes to the exhaust.
Switching a 3-way-magnetic valve directed the selected odor stream
through a nozzle (2 cm in front of the preparation) to the antennae.
The scented air was quickly exhausted through a large tube (diameter 5 cm, positioned 10 cm behind the recording chamber).
Stimulus onset and o€set (indicated by switching the magnetic
valves) were stored separately on tape. Stimulus latencies between
switching the valves and arrival at the antenna were estimated with
a thermistor and ranged between 80 ms and 120 ms.
Experimental design
To determine response modulations three experimental groups of
animals were formed. The conditioning group (n = 11) received a
single forward pairing of the CS (carnation) with a sucrose reward
567
(stimulation of both antennae and proboscis). Animals of the sensitisation group (n = 11) were stimulated with a sucrose solution
applied to both antennae without olfactory stimulation. Animals of
a control group (n = 8) were stimulated with an odor alone,
without sucrose application. In all groups the odor responses were
tested 2 min prior to the CS/US pairing or applying the sensitising
stimulus (reference response). After conditioning or sensitisation
responses during the presentation of the same odor were tested at
30 s, 1 min, and 2 min (test odor responses). Responses during the
tests were compared with their corresponding reference responses.
Data analysis
Intracellular signals were preampli®ed (d.c.-coupled, 10´;
Simmonds Ampli®er, Cambridge, UK), visualized on a storage
oscilloscope, and stored on a digital tape recorder (Biologic
DTR1800). The signal and stimulus traces were digitized (signal
sample rate 4000 Hz, stimulus sample rate 500 Hz) and stored on a
computer using a CED1401plus interface and Spike2 software
(version 2.24, Cambridge Electronic Design, Cambridge, UK).
To determine the spontaneous frequency the total number of
action potentials was counted during a 2 s interval immediately
before the ®rst odor response. Spike parameters (amplitude, duration, time-to-peak) were analyzed by measuring 4 individual spikes
per neuron and measures of several neurons were averaged. For
analyzes of odor responses (latency, peristimulus spike frequency,
maximum instantaneous frequency, burst depolarization, etc.), two
responses per neuron were analyzed and data from several neurons
averaged. The number of neurons analyzed is indicated in Table 1.
Response frequencies were calculated as the number of spikes
during the whole stimulus duration (2 s) and during peristimulus
time intervals of various durations (100±500 ms) after stimulus
onset. The spike frequency during a 2 s period prior to each
stimulus onset was subtracted from its corresponding response
frequency to correct for background spontaneous activity. Relative
response frequencies during the tests were expressed as the relative
deviation from the corresponding reference response frequencies,
Table 1 Physiological properties of feedback neurons
Parameter
General
Membrane potential
Spontaneous frequency
Action potential
Amplitude
Duration
Time-to-peak
Sucrose response
Duration
Membrane depolarization
Spike frequency
Maximum frequency
Odor responses
Latency
Membrane depolarization
Phasic response
Tonic response
Spike frequency
Phasic response
Tonic response
Maximum frequency
Phasic response
Tonic response
O€-responses
Latency
Duration
Spike frequency
ND ± Not determined
which was set to 1. Thus, a value of 1 indicates no change; 0 means
100% spike frequency reduction, and 2 indicates a 100% increase.
Statistics
All data are expressed as means with standard errors of means.
A 2-way ANOVA for repeated measures was employed for comparison of odor and air pu€ responses, and to evaluate di€erences
in relative frequencies and response latencies between the experimental groups. For subsequent post-hoc analyses, a NewmanKeuls test was chosen. Data were analysed using STATISTICA for
Windows (version 5.1; StatSoft, Tulsa, Okla.).
Results
Morphological identi®cation of feedback neurons
Intracellularly stained feedback neurons were identi®ed
according to several morphological features, that were
previously described in detail (GruÈnewald 1999). Their
somata are located ventrally at the anteriolateral
protocerebrum, close to the median border of the
lobula (Figs. 1, 2A). The primary neurite projects
dorsomedially and bifurcates at the dorsolateral margin
of the a-lobe. One branch projects dorsally and posteriorly within the protocerebral-calycal tract towards
the calyces. The other branch loops ventrally, penetrates the a-lobe at the a-exit point (Mobbs 1982), and
arborizes within the dorsal and median a-lobe layers. It
sends o€ branches posteriorly, which bifurcate at the
posterior border of the a-lobe, and arborize in the
pedunculus and the b-lobe. The protocerebral collateral
Range
Mean ‹ SEM
n
)35 to )65 mV
0±24 Hz
)41.3 ‹ 2.2 mV
5.2 ‹ 1.3 Hz
23
23
20±55 mV
2.55±4.67 ms
0.87±1.85 ms
33.3 ‹ 1.7 mV
3.38 ‹ 0.12 ms
1.16 ‹ 0.06 ms
23
23
23
105±1890 ms
2.5±10 mV
8±75 Hz
24±160 Hz
673 ‹ 102 ms
5.5 ‹ 0.58 mV
29.0 ‹ 3.9 Hz
86.7 ‹ 10.7 Hz
16
16
16
16
75±128 ms
96.6 ‹ 3.2 ms
19
2.4±11.7 mV
1.2±5.8 mV
3.3±48.0 Hz
1.5±35 Hz
16.1±219.3 Hz
ND
95±175 ms
160±750 ms
8±31 Hz
6.8 ‹ 0.69 mV
3.2 ‹ 0.35 mV
19
19
21.5 ‹ 2.8 Hz
12.3 ‹ 2.3 Hz
19
19
101.2 ‹ 13.5 Hz
ND
19
166 ‹ 14.1 ms
434 ‹ 97 ms
16.6 ‹ 3.6 Hz
6
6
6
568
Fig. 1A±F Morphology of
feedback neurons. A±C Lucifer
yellow-stained group of neurons, which connect the pedunculus with the lip of the calyces.
Neurons send several branches
(arrowheads in B) toward the
pedunculus. Three focal planes
(85, 100, 135 lm below the
frontal brain surface; wholemount). Arrow in A, primary
neurites; arrowhead points to aexit. Outlines of the a-lobe
indicated by broken lines in B, D.
Arrows in C point to inner-ring
tract, arrowhead to protocerebro-calycal tract. D, E
Staining of another, single neuron with arborizations in the
dorsal a-lobe (D) and in the
basal ring of the calyces (E;
broken lines indicate calycal
outlines; frontal view). F Fine
branches of yet another singlestained feedback neuron in the
collar region of the lateral calyx
(25 lm frontal section). No
stained pro®les in the lip and
basal ring. This neuron did not
respond to olfactory stimuli.
LC lateral calyx; MC median
calyx; Li lip; Co collar; Br basal
ring; d dorsal; l lateral. Scale
bars in A±E, 100 lm, in F,
50 lm
bifurcates ventrally of the calyces and innervates the
median and the lateral calyx. All calycal subcompartments, the lip, collar, and basal ring, are innervated by
feedback neurons. For this study only neurons were
selected that responded to olfactory stimuli. All these
neurons arborized either in the basal ring or the lip
region.
General physiological characteristics
Spontaneous activity
Recordings from neurites were performed at the a-exit
point. Impalement of neurons was indicated by a sudden
drop of the potential to )35 to )65 mV (see Table 1 for
569
Fig. 2A±C Wiring scheme and spike activity of feedback neurons.
A Schematic diagram of feedback neurons within the honeybee
mushroom body. Only the right brain hemisphere is drawn; arrows
represent the putative ¯ow of information within feedback neurons.
The calyces and the medio-dorsal a-lobe are shaded dark gray; star,
somata cluster. The recording site is indicated. a, a-lobe; b, b-lobe;
MC, median calyx; LC, lateral calyx; Pe, pedunculus; AL, antennal
lobe; LO, lobula; ME, medulla; CB, central body; OC, lateral ocellus.
Dorsal (d) and lateral (l) directions are indicated, scale bar = 100 lm.
B Spontaneous discharge pattern is a series of action potentials. In
most neurons relatively low frequency periods are interrupted by high
frequency, input activity (top three traces). Some neurons generate
continuous series of regularly spaced action potentials (lower
recording). Each trace represents a 10 s recording period from a
single neuron. C Typical shapes of action potentials generated by four
di€erent cells; asterisks in B, C summating excitatory postsynaptic
potentials (epsps); ®lled circles in B, ipsps
a list of physiological parameters). Most neurons (95%)
generated action potentials without experimental stimulation (Fig. 2B). The spontaneous background discharge frequency ranged between 0 and 24 Hz (Table 1)
and varied considerably among feedback neurons
(Fig. 2B). The spontaneous discharge activity of most
neurons was typically a complex temporal pattern,
which was intermitted by short periods of higher frequency episodic phasic activity rather than a continuous
series of equally spaced action potentials. This may indicate sensory input activity without experimental
stimulation (probably via antennal movements or airborne odors in the experimental environment). In most
neurons the action potentials (duration <5 ms) lacked
pronounced after-hyperpolarizations (Fig. 2C). Sum-
mating postsynaptic potentials (amplitude 2±10 mV)
occurred frequently at the recording site (asterisks in
Fig. 2B, C).
Responses to sucrose solution
Brie¯y touching the tip of the ipsilateral antenna with
sucrose solution evoked excitatory responses in 92%
of all recorded feedback neurons. Excitatory responses
were never observed after touching or closely approaching the antennal tip with a dry tooth pick. The
sucrose reaction tested by stimulating the ipsilateral
antenna lasted between 105 ms and 1890 ms (Fig. 3,
Table 1). Sucrose responses typically consisted of a
rapid membrane depolarisation that gradually declined
to baseline potential. During responses, spike frequency was increased to 29.0 ‹ 3.9 Hz above resting
values. Stimulation of the contralateral antenna
evoked responses in 78% of all neurons that responded to ipsilateral stimulation. Contralateral responses were weaker and shorter than during
ipsilateral stimulation (Fig. 3A), sucrose solution applied to the glossa rarely elicited any response. Repeated stimulation of the ipsilateral antenna at short
intervals (2±4 s, Fig. 3B) but not at long intervals
(30 s to 2 min, Fig. 3C) led to a gradual decline of
response strength as indicated by successively lower
response frequency (31.4±22 Hz) and shorter duration
(688±362 ms) after 5 stimulations.
570
Fig. 3A±C Responses of three di€erent feedback neurons to sucrose
stimulation. A Responses are excitatory and are stronger when
sucrose solution is applied to the ipsilateral (right) antenna than to the
contralateral (left) antenna. B Repeated ipsilateral antennal stimulation results in response decrement when the intervals between
stimulations are short (<5 s). Higher temporal resolution of the ®rst
and ®fth response are given above. C Spaced presentation (2 min
interval) of antennal sucrose solution did not result in response
decrement. The spike frequencies during the responses and the
response durations are indicated for each trace
Odor responses
Application of the test odors evoked either phasic or
phasic-tonic excitatory responses in feedback neurons
(Figs. 4±6). The phasic response component (duration
<500 ms) consisted of a rapid and steep rise of the
membrane potential with response latencies between
75 ms and 128 ms (Fig. 4, Table 1). The spike frequency
during the phasic response was increased to 3.3±48.0 Hz
above background activity, the instantaneous spike frequency may reach maximum values of up to 219 Hz.
The burst typically consisted of 1±40 spikes with reduced amplitudes (measured from the burst potential).
The end of the phasic response component is indicated
by an abrupt drop of the membrane potential and the
spike frequency to values still above resting potential
and spontaneous frequency (Fig. 4A, B). During the
tonic response component (Fig. 4A, C, D), which occurred in 74% of the recorded neurons, the membrane
remained slightly depolarized and the spike frequency
increased; summating postsynaptic potentials were frequently observed (Fig. 4A). Towards the end of the odor
stimulus both parameters declined to resting values; the
odor responses usually did not exceed stimulus duration.
However, some neurons (22%) generated excitatory o€responses after stimulus o€set (Fig. 4C, D, Table 1).
These o€-responses, which were not speci®c for a
particular odor, were short with a latency of
Fig. 4A±D Odor responses of feedback neurons. A Burst responses
consist of almost instantaneous membrane depolarisation and high
spike frequency (often exceeding 100 Hz). Marked area of A enlarged
below. The membrane remains depolarized after the burst and during
the tonic response component. Summating epsps (asterisks); and ipsps
(®lled circle) occur during odor responses. B Pure phasic response.
C, D O€-responses (C shows same neuron as in A). Stimulus
durations (orange in A; carnation in B, D; geraniol in C) are indicated
as a bar below each signal trace; response frequencies and durations of
the response component are given for the traces in C, D
166 ‹ 14.1 ms (measured from stimulus o€set), which
was substantially longer than during the on-responses of
the same neurons during the same responses (99.8 ‹
2.3 ms, n = 6).
Stimulations with pu€s of plain air (duration 2 s)
evoked purely phasic, excitatory responses (Figs. 5B, 6)
with spike frequencies between 0.5 Hz and 14.5 Hz
above spontaneous activity. Carnation stimuli induced
stronger excitatory responses in the very same neurons.
Comparing spike activity during odor and during air
pu€ responses (Fig. 5) revealed signi®cant di€erences
between the tests (df = 1; F = 6.83; P < 0.02; 2-way
MANOVA): Post-hoc tests showed weaker responses
during air pu€s then during odor stimuli (P < 0.02,
Newman-Keuls post-hoc test), but no signi®cant changes in background activity (P = 0.65, n.s.). Obviously, a
portion of the phasic odor response component was
571
Fig. 6A±C Variability of odor responses among di€erent feedback
neurons. Each column (A±C) represents responses of one individual
neuron to the various odors and to a plain air pu€ (top to bottom
rows, stimulus indicated at the right)
third neuron, that are almost indistinguishable from airpu€ stimulation (column C).
Modulations of odor responses
Fig. 5A, B Comparison of responses to odor (A) and air-pu€
stimulation (B) of neurons that responded to both stimuli. Peristimulus time histograms starting 1 s prior to stimulus onset (error bars,
SEM; bin width 100 ms). The odor (carnation) induced a spike
frequency of 13.4 ‹ 2.8 Hz above spontaneous activity (n = 16,
measured for the whole stimulus duration). The very same neurons
responded to stimulation with plain air pu€s with an increased
frequency of 3.3 ‹ 1.14 Hz. C Subtraction of A-B yielded odor
component of the whole response. Stimulus duration is indicated by
the horizontal bar
caused by activation of mechanosensory a€erences at
stimulus onset.
Various test odors evoked excitatory responses in a
given feedback neuron (Fig. 6, columns). The responses
of a given neuron to the di€erent test odors di€er
slightly, but di€erences in odor responses were more
pronounced between di€erent neurons (Fig. 6, rows).
One given odor (e.g., geraniol, row 4 of Fig. 6) may elicit
weak phasic responses, hardly above the spontaneous
activity in one neuron (e.g., column A of Fig. 6); in
another neuron the same odor may evoke high-frequency, phasic-tonic responses with an o€-component
(column B), and almost purely phasic responses in a
During these experiments spike activity of feedback
neurons was recorded during olfactory learning and
sensitisation. The results indicate that one-trial conditioning induces changes in odor responses of MB feedback neurons. Examples of original recording traces
show similar temporal patterns of spike activity during
odor responses of feedback neurons before and after
conditioning or sensitisation (Fig. 7A, B). After a single
pairing of an odor with a sucrose reward most feedback
neurons generated weaker CS responses during the 30 s
test and the 1 min test as compared to the reference
test (91% and 82% of neurons, respectively; n = 11;
Table 2). The mean relative frequencies indicated a
response decrement during the tests. A single antennal
sensitising stimulus led to stronger odor responses in
55% of feedback neurons during the 30 s test (n = 11)
and in 78% during the 1 min test (n = 9). The mean
relative frequencies indicated a response enhancement
after sensitisation. Repeated presentations of a given
odor (control group) did not systematically change response frequencies of feedback neurons, since almost the
same number of neurons responded stronger or weaker
during the tests as compared to the reference test.
Statistical analyses revealed di€erences in relative
response frequencies (calculated for the whole stimulus
duration) between the experimental groups (Fig. 8A,
Table 2). Thus, the relative frequency of the CS response
30 s test after conditioning was decreased as compared
to the odor control group (P < 0.002; n = 8; Newman-
572
Fig. 7A±C Recording traces of feedback neurons during conditioning
(A), and sensitisation (B), and stimulus con®guration (C). Responses
of feedback neurons to the odor before ()2 min car, carnation) either
the pairing with compound sucrose stimulation (A), or a single
sensitisation trial (B), and during subsequent tests (0.5 min, 1 min).
Response decrement during the tests in A was not due to an unspeci®c
weakening of the sensory input, since subsequent response to orange
stimulation (or, 3 min after conditioning) illustrates that the neurons
is still capable of generating stronger excitation. Also given for
comparison is the response during presentation of geraniol in B (ger,
5.5 min after sensitisation). These other odors were not tested prior to
sensitisation or conditioning and responses may, therefore, not be
compared quantitatively. C Time course and sequence of olfactory
(open rectangles) and gustatory stimuli ( ®lled rectangles) used in the
three experimental groups. Each experiment consisted of the three
phases, reference test (ref.), training (train., conditioning, sensitisation,
odor), and tests. Occasionally, after the last test additional odors were
presented at various intervals. The time scale is given below
Keuls post-hoc test), but not during the 1 min test (see
Table 2 for statistical details). Relative frequencies
during odor responses after sensitisation di€er from
those after conditioning, but not from those of the
control group. Similar results were obtained by comparing relative frequencies during the phasic response
component, determined during the 1st 500 ms interval
after stimulus onset (Table 2, Fig. 8B). Relative frequencies during the 30 s test are reduced after conditioning as compared to the odor control group
(P < 0.05; n = 8; Newman-Keuls post-hoc test). Sensitisation did not induce modulations of the phasic odor
response as compared to repeated odor presentations.
Thus, conditioning produces a transient odor response
decrement, whereas antennal sensitisation does not induce any signi®cant response modulations.
Response latencies did not change signi®cantly in any
experimental group (Table 2). No di€erences were observed between groups (df = 2; F = 1.24; P = 0.31;
n.s.; two-way ANOVA for repeated measures), nor
within groups between references and tests (df = 2;
F = 0.06; P = 0.94; n.s.).
Discussion
The present study showed that mushroom body feedback neurons in the honeybee receive excitatory olfactory input. Individual feedback neurons respond to
various odors. The odor-induced inhibitory activity in
the mushroom body can be modulated by a single
classical conditioning trial, but not by a single sensitisation trial.
Physiological properties of feedback neurons
Previous studies presented morphological evidences that
the information ¯ow within feedback neurons is directed
from the lobes toward the calyces, i.e. from the MB main
output toward its main input regions (Mobbs 1982;
Rybak and Menzel 1993; GruÈnewald 1999). This view
can now be con®rmed by the physiological ®ndings
presented here. First, the occurrence of summating
postsynaptic potentials at the recording site indicated
that feedback neurons possess synaptic input regions
close to the a-exit, probably within the lobes and pedunculus. This is generally consistent with the appearance
of spiny processes at the feedback neuron terminals
(honeybees: Mobbs 1982; Gronenberg 1987; Rybak and
Menzel 1993) and ultrastructural evidence for output
synapses from Kenyon cells onto feedback neurons in
these areas (locust: Leitch and Laurent 1996). Second,
the amplitudes of action potentials imply that spikes are
passively propagated to the recording site. The spike
generating zone should be close to the a-exit, however,
573
Fig. 8A±C Quanti®cation of learning experiments. A Relative frequencies were calculated for a 2 s peristimulus interval during the 30 s
(left) and the 1 min (right) test. B Relative frequencies during the 1st
500 ms interval after stimulus onset, representing the phasic response
component. Response frequencies during the reference test were set to
1; thus, downward bars indicate response decrements, upward bars
indicate response enhancements. Stars indicate statistically signi®cant
(P > 0.05, Newman-Keuls post-hoc test) di€erences between experimental groups
since afterhyperpolarisations were recorded in some
neurons. Thus, feedback neurons probably form dendrites within the output regions of the MB, where they
receive input from Kenyon cells and recurrently transmit
this information to the MB input sites. There, in the
calyces, they probably synapse onto dendrites of Kenyon cells, which express GABA receptor-mediated Cl)
currents in vitro (honeybees: Rosenboom et al. 1994;
crickets: Cayre et al. 1999). In locusts Kenyon cells
generate ipsps, which are evoked by spike activity in
feedback neurons (Laurent and Naraghi 1994) and
Kenyon cells of ¯ies express GABA receptors (Drosophila: Harrison et al. 1996; Calliphora: Brotz et al. 1997).
Odour stimuli evoked excitatory responses in most
feedback neurons. A given odor evokes a wide variety of
responses in di€erent feedback neurons ranging from
weak phasic to high frequency phasic-tonic activity.
Assuming that odors activate a large number of feedback neurons, olfactory stimuli induce a complex spatiotemporal pattern of inhibitory activity that
counteracts the odor-induced excitation within the MB.
This massive odor-induced activity of feedback neurons
is striking, because antennal information is processed
mainly within the ventral a-lobe (Mobbs 1982), whereas
the dendritic ®elds of feedback neurons within the a-lobe
are restricted to its median and dorsal portions (GruÈnewald 1999). The question of where the feedback neurons receive olfactory information is yet unsolved
(cf. discussion in GruÈnewald 1999).
Individual feedback neurons do not show a pronounced odor speci®city, since they respond to various
odors, which was also previously observed (Gronenberg
1987) and they respond similarly to olfactory, mechanical, and gustatory stimuli. This may be due to their
extended dendritic ®elds in the lobes and pedunculus
(GruÈnewald 1999), where, like many MB output neurons
Table 2 Response modulations of feedback neurons
Parameter
Test
Conditioning
Sensitization
Odor
Statistics1
Number of ¯/­ neurons2
30 s
1 min
10/1 (11)3
9/2 (11)
4/6 (11)
2/7 (9)
3/3 (8)
2/3 (8)
n.t.
30
1
30
1
30
1
30
1
0.58
0.67
0.66
0.77
0.58
0.61
85.2
89.6
1.58
1.94
1.23
1.36
1.06
1.52
90.0
89.1
Relative frequencies
Whole stimulus
1st 500 ms
2nd 500 ms
Latencies
s
min
s
min
s
min
s
min
Post-hoc analyses5
Cond. vs Odor
Sens. vs Odor
Cond. vs Sens.
30
1
30
1
30
1
s
min
s
min
s
min
‹
‹
‹
‹
‹
‹
‹
‹
0.08
0.16
0.08
0.08
0.16
0.02
3.2 ms
4.4 ms
‹
‹
‹
‹
‹
‹
‹
‹
0.35
0.44
0.21
0.27
0.4
0.4
5.9 ms
5.4 ms
1.28
1.38
1.23
1.00
0.62
0.75
95.8
95.4
‹
‹
‹
‹
‹
‹
‹
‹
0.28
0.33
0.14
0.21
0.13
0.13
5.4 ms
4.2 ms
Whole stimulus duration
1st 500 ms interval
P
P
P
P
P
P
P
P
P
P
P
P
<
=
=
=
<
<
0.002*, n = 8
0.051, n = 8
0.18, n = 8
0.14, n = 8
0.05*, n = 11
0.002*, n = 9
1
Two-way ANOVA for repeated measures; n.t., not tested; n.s.,
not signi®cant
2
Number of neurons showing weaker (¯) or stronger (­) odor
responses during the 30 s and 1 min test as compared to the
reference test. Responses were de®ned as weaker (stronger) as
reference test, if they showed a more than 10% reduction (increase)
<
=
=
=
<
=
df = 2; F = 6.64;
P < 0.005*
df = 2; F = 4.83;
P < 0.02*
n.t.4
df = 2; F = 1.24;
P = 0.31; n.s.
0.05*, n = 8
0.25, n = 8
0.54, n = 8
0.27, n = 8
0.015*, n = 11
0.09, n = 9
of response strength
3
Total number of observations in brackets
4
Number of observations to low, because ca. 30% of the neurons
do not show tonic responses
5
Newman-Keuls post-hoc test on relative frequencies
574
(Rybak and Menzel 1993, 1998), the feedback neuron
may integrate information from di€erent Kenyon cell
populations. Such multimodal sensitivity of MB extrinsic neurons has been reported in a variety of systems
(honeybees: Erber 1978; Homberg and Erber 1979;
Gronenberg 1987; Mauelshagen 1993; Rybak and
Menzel 1998; crickets: Schildberger 1981, 1984; cockroaches: Li and Strausfeld 1997).
Experience-dependent response modulations
Feedback neurons show experience-dependent modulations of their odor responses. These ®ndings can be
directly compared with intracellular studies by Mauelshagen (1993) and with behavioral analyses (e.g. Bitterman et al. 1983; Hammer et al. 1994), because the
experimental design and stimulus con®guration were
designed according to those previous experiments.
The head preparation allowed frequent impalements
and stable intracellular recordings of central neurons in
the honeybee brain, while sensory a€erences and sensory processing are left intact. Thus, intracellular
activity of MB neurons can be recorded during simple
learning tasks, such as one-trial conditioning and sensitisation. The response frequency of feedback neurons
to an odor stimulus (CS) decreased after a single
olfactory conditioning trial, which was similarly observed for the PE1-neuron (Mauelshagen 1993). The
initial hypothesis, that inhibitory activity of feedback
neurons may mediate the learning-dependent decrement
of response frequency of the MB output neuron PE1,
must therefore be modi®ed. Although the feedback
neurons showed learning-dependent response modulations after one-trial conditioning, these are not antagonistic to those of the PE1-neuron as was expected, but
rather similar, and cannot explain in a simple model
the response decrement in the PE1-neuron. How then
may the response modulations of MB output neurons
be explained? First, activity of inhibitory feedback
neurons may not a€ect odor processing of MB output
neurons. The experience-dependent frequency modulations of output neurons and feedback neurons may
then be accomplished further upstream of both neuron
groups at the level of olfactory relay neurons within the
antennal lobes or the MB calyces. Second, feedback
neurons might presynaptically control transmitter
release of the VUMmx1 neuron in the calyces. This
modulatory neuron mediates the US reinforcing stimulus in appetitive olfactory conditioning (Hammer
1993). A reduced odor-induced presynaptic inhibition
via feedback neurons in the lip region of the calyces
may enable increased release of modulatory transmitter
from the VUMmx1 neuron onto Kenyon cells.
Output neurons like the PE1 and feedback neurons
receive convergent synaptic input from thousands of
Kenyon cells. Therefore, activity in these MB extrinsic
neurons probably re¯ects the overall level of odorevoked excitation within the MB, which is transiently
reduced after a single olfactory conditioning trial. Behavioural studies on the proboscis extension re¯ex (PER)
have shown that a single pairing of an odor with a sucrose
reward immediately enhances the probability of odorinduced PER for hours (Bitterman et al. 1983; Menzel
1990). This increased responsiveness is not paralleled by
an increased odor-evoked activity within the MB.
Rather, odor-induced MB neural activity is transiently
reduced after single-trial conditioning. Interestingly,
Hammer and Menzel (1998) showed recently that pairing
an odor with octopamine injection into the calyces produces long-term enhancement of PER. However, during
acquisition, the probability of odor-evoked PER was
signi®cantly lower than during consecutive memory tests,
which indicates inhibitory phenomena within the MB
during acquisition. This would be consistent with a
transiently decreased neural activity within MB neurons,
like the PE1-neuron and the feedback neurons.
Functional roles of inhibitory feedback neurons
Inhibitory feedback connections are essential components in most neuronal systems, where they often prevent overshooting excitation by controlling the stimulusinduced excitatory level during information processing.
If this also holds true for the insect MB, the balance
between excitatory and inhibitory neural activity can be
shifted by experience. It may, therefore, be one role of
feedback neurons during olfactory learning in insects to
regulate odor-induced MB excitation.
Alternatively, feedback neurons may be involved in
olfactory information processing. Odor stimuli induce
coherent oscillatory activity in olfactory projection
neurons (locusts: Laurent and Naraghi 1994; honeybees:
Stopfer et al. 1997). This odor-evoked oscillatory activity is supposed to be necessary for ®ne odor discrimination and is maintained within the MB (MacLeod et al.
1998). Experience-dependent modulations of activity in
inhibitory feedback neurons may modulate these odorinduced oscillations within the MB.
Inhibitory feedback loops are involved in learningdependent plasticity in a variety of systems. During
induction of hippocampal long-term potentiation, for
example, dendritic GABAa inhibition is reduced, resulting in enhanced postsynaptic depolarisation by facilitation of the NMDA current component (Bliss and
Collingridge 1993; Tomasulo et al. 1993). During eyeblink conditioning recurrent inhibition via GABAergic
cerebello-olivary projections regulates processing of the
US (Thompson and Krupa 1994; Hesslow and Ivarsson
1996) and mediates the behavioural phenomenon of
blocking (Kim et al. 1998). In the somatosensory cortex
the balance between GABAergic and cholinergic input
may control neural plasticity of cortical neurons by
regulating their receptive ®eld size (Dykes 1997).
Whether inhibitory feedback neurons in the insect
mushroom body play similar roles during memory formation awaits further exploration.
575
Acknowledgements This paper is dedicated to Dr. Juliane
Mauelshagen. The author was grateful for her expert comments
and continuous support. I thank Drs. Randolf Menzel and Richard
B. Levine for helpful comments and critically reading the manuscript and Mary Wurm for correcting the English manuscript. This
work was supported by the Deutsche Forschungsgemeinschaft (Pf
128/6-4 and SFB 515/C5).
References
Bicker G, SchaÈfer S, Kingan TG (1985) Mushroom body feedback
interneurons in the honeybee show GABA-like immunoreactivity. Brain Res 360: 394±397
Bitterman ME, Menzel R, Fietz A, SchaÈfer S (1983) Classical
conditioning of proboscis extension in honeybees (Apis mellifera). J Comp Psyc 97: 107±119
Bliss TVP, Collingridge GL (1993) A synaptic model of memory:
long-term potentiation in the hippocampus. Nature 361: 31±39
Brotz TM, Bochenek B, Aronstein K, Ffrench-Constant RH, Borst
A (1997) gamma-Aminobutyric acid receptor distribution in the
mushroom bodies of a ¯y (Calliphora erythrocephala): a functional subdivision of Kenyon cells? J Comp Neurol 383: 42±48
Cayre M, Buckingham SD, Yagodin S, Sattelle DB (1999) Cultured
insect mushroom body neurons express functional receptors for
acetylcholine, GABA, glutamate, octopamine, and dopamine.
J Neurophysiol 81: 1±14
Davis RL (1993) Mushroom bodies and Drosophila learning.
Neuron 11: 1±14
De Belle JS, Heisenberg M (1994) Associative learning in Drosophila abolished by chemical ablation of mushroom bodies.
Science 263: 692±695
Dubnau J, Tully T (1998) Gene discovery in Drosophila. Annu Rev
Neurosci 21: 407±444
Dykes RW (1997) Mechanisms controlling neuronal plasticity in
somatosensory cortex. Can J Physiol Pharmacol 75: 535±545
Erber J (1978) Response characteristics and after e€ects of multimodal neurons in the mushroom body area of the honey bee.
Physiol Entomol 3: 77±89
Erber J, Homberg U, Gronenberg W (1987) Functional roles of the
mushroom bodies in insects. In: Gupta AP (ed) Arthropod
brain: its evolution, development, structure, and functions.
John Wiley and Sons, New York, pp 485±511
Erber J, Masuhr T, Menzel R (1980) Localization of short-term
memory in the brain of the bee, Apis mellifera. Physiol Entomol
5: 343±358
Fahrbach SE, Robinson GE (1995) Behavioural development in
the honey bee: toward the study of learning under natural
conditions. Learn & Mem 2: 199±224
Goldberg F, GruÈnewald B, Rosenboom H, Menzel R (1999) Nicotinic acetylcholine currents of cultured Kenyon cells from the
mushroom bodies of the honey bee Apis mellifera. J Physiol
514: 759±768
Goll W (1967) Strukturuntersuchungen am Gehirn von Formica.
Z Morphol Oekol Tiere 59: 145±210
Gronenberg W (1986) Physiological and anatomical properties of
optical input-®bres to the mushroom body in the bee brain.
J Insect Physiol 32: 695±704
Gronenberg W (1987) Anatomical and physiological properties of
feedback neurons of the mushroom bodies in the bee brain. Exp
Biol 46: 115±125
GruÈnewald B (1999) Morphology of feedback neurons in the
mushroom body of the honeybee, Apis mellifera. J Comp
Neurol 404: 114±126
Hammer M (1993) An identi®ed 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 Neurosci 20: 245±252
Hammer M, Braun G, Mauelshagen J (1994) Food-induced
arousal and nonassociative learning in honeybees: dependence
of sensitisation on the application site and duration of food
stimulation. Behav Neural Biol 62: 210±223
Hammer M, Menzel R (1995) Learning and memory in the honeybee. J Neurosci 15: 1617±1630
Hammer M, Menzel R (1998) Multiple sites of associative odor
learning as revealed by local brain microinjections of octopamine in honeybees. Learn & Mem 5: 146±156
Harrison JB, Chen HH, Sattelle E, Barker PJ, Huskisson NS, Rauh
JJ, Sattelle DB (1999) Immunocytochemical mapping of a Cterminus anti-peptide antibody to the GABA receptor subunit,
RDL in the nervous system of Drosophila melanogaster. Cell
Tissue Res 284: 269±278
Heisenberg M, Borst A, Wagner S, Byers D (1985) Drosophila
mushroom body mutants are de®cient in olfactory learning.
J Neurogenet 2: 1±30
Hesslow G, Ivarsson M (1996) Inhibition of the inferior olive
during conditioned responses in the decerebrate ferret. Exp
Brain Res 110: 36±46
Homberg U (1984) Processing of antennal information in extrinsic
mushroom body neurons of the bee brain. J Comp Physiol A
154: 825±836
Homberg U, Erber J (1979) Response characteristics and identi®cation of extrinsic mushroom body neurons of the brain.
Z Naturf 34c: 612±615
Homberg U, Kingan TG, Hildebrand JG (1987) Immunocytochemistry of GABA in the brain and suboesophageal ganglion
of Manduca sexta. Cell Tissue Res 248: 1±24
Kim JJ, Krupa DJ, Thompson RF (1998) Inhibitory cerebelloolivary projections and blocking e€ect in classical conditioning.
Science 279: 570±573
Kuwabara M (1957) Bildung des bedingten Re¯exes von Pavlovs
Typus bei der Honigbiene, Apis melli®ca. J Fac Sci Hokkaido
Univ Ser VI Zool 13: 458±464
Laurent G, Naraghi M (1994) Odorant-induced oscillations in the
mushroom bodies of the locust. J Neurosci 14: 2993±3004
Leitch B, Laurent G (1996) GABAergic synapses in the antennal
lobe and mushroom body of the locust olfactory system.
J Comp Neurol 372: 478±514
Li Y, Strausfeld NJ (1997) Morphology and sensory modality
of mushroom body extrinsic neurons in the brain of the
cockroach, Periplaneta americana. J Comp Neurol 387: 631±
650
MacLeod K, BaÈcker A, Laurent G (1998) Who reads temporal
information contained across synchronized and oscillatory
spike trains? Nature 395: 693±698
Mauelshagen J (1993) Neural correlates of olfactory learning
paradigms in an identi®ed neuron in the honeybee brain.
J Neurophysiol 69: 609±625
Menzel R (1990) Learning, memory, and ``cognition'' in honey
bees. In: Kesner RP, Olton DS (eds) Neurobiology of comparative cognition. Lawrence Erlbaum, Hillsdale, New Jersey,
pp 237±292
Menzel R, Durst C, Erber J, EichmuÈller S, Hammer M, Hildebrandt H, Mauelshagen J, MuÈller U, Rosenboom H, Rybak J,
SchaÈfer S, Scheidler A (1994) The mushroom bodies in the
honeybee: from molecules to behaviour. In: Schildberger K,
Elsner N (eds) Fortschritte der Zoologie, vol 39. Neural basis of
behavioural adaptations. Fischer, Stuttgart, pp 81±102
Menzel R, Erber J, Masuhr T (1974) Learning and memory in the
honeybee. In: Barton Browne L (ed) Experimental analysis of
insect behaviour. Springer, Berlin Heidelberg New York, pp
195±217
Menzel R, Hammer M, Braun G, Mauelshagen J (1991) Neurobiology of learning and memory in honeybees. In: Goodman
LJ, Fisher RC (eds) The behaviour and physiology of bees.
C.A.B. International, Wallingford, pp 323±353
Menzel R, MuÈller U (1996) Learning and memory in honeybees:
from behaviour to neural substrates. Annu Rev Neurosci 19:
379±404
Mobbs PG (1982) The brain of the honeybee Apis mellifera. I. The
connections and spatial organization of the mushroom bodies.
Phil Trans R Soc Lond B 298: 309±354
576
Mobbs PG (1984) Neural networks in the mushroom bodies of the
honeybee. J Insect Physiol 30: 43±58
Mobbs PG (1985) Brain structure. In: Kerkut GA, Gilbert LI (eds)
Comprehensive insect physiology, biochemistry, and pharmacology. Pergamon Press, Oxford, pp 299±370
Rosenboom H, Goldberg F, SchaÈfer S, Menzel R (1994) Ionic
currents of Kenyon cells possibly involved in olfactory learning
in insects. Soc Neurosci Abstr 20: 333.19
Rybak J, Menzel R (1993) Anatomy of the mushroom bodies in the
honey bee brain: the neuronal connections of the alpha-lobe.
J Comp Neurol 334: 444±465
Rybak, J Menzel R (1998) Integrative properties of the Pe1 neuron,
a unique mushroom body output neuron. Learn & Mem 5: 133±
145
SchaÈfer S, Rosenboom H, Menzel R (1994) Ionic currents of
Kenyon cells from the mushroom body of the honeybee.
J Neurosci 14: 4600±4612
SchuÈrmann F-W (1973) UÈber die Struktur der PilzkoÈrper des
Insektengehirns III. Die Anatomie der Nervenfasern in den
Corpora pedunculata bei Acheta domesticus L (Orthoptera):
Eine Golgi-Studie. Z Zellforsch 145: 247±285
SchuÈrmann F-W (1987) The architecture of the mushroom bodies
and related neuropils in the insect brain. In: Gupta AP (ed)
Arthropod brain. Its evolution, development, structure, and
functions. John Wiley and Sons, New York, pp 231±256
Schildberger K (1981) Some physiological features of mushroom-body linked ®bers in the house cricket brain. Naturwissenschaften 67: 623±624
Schildberger K (1983) Local interneurons associated with the
mushroom bodies and the central body in the brain of Acheta
domesticus. Cell Tissue Res 230: 573±587
Schildberger K (1984) Multimodal interneurons in the cricket
brain: properties of identi®ed extrinsic mushroom body cells.
J Comp Physiol A 154: 71±79
Stopfer M, Bhagavan S, Smith BH, Laurent G (1997) Impaired
odour discrimination on desynchronization of odour-encoding
neural assemblies. Nature (Lond) 390: 70±74
Strausfeld NJ (1976) Atlas of an insect brain. Springer, Berlin
Heidelberg New York
Thompson RF, Krupa DJ (1994) Organization of memory traces in
the mammalian brain. Annu Rev Neurosci 17: 519±549
Tomasulo RA, Ramirez JJ, Steward O (1993) Synaptic inhibition
regulates associative interactions between a€erents during the
induction of long-term potentiation and depression. Proc Natl
Acad Sci USA 90: 11578±11582
Vareschi E (1971) Odor discrimination in the honey bee ± single cell
and behavioural response. Z Vergl Physiol 75: 143±173
Weiss MJ (1978) Giant ®ber associated with mushroom body
neuropiles in an insect brain: a possible one-cell feedback loop.
Soc Neurosci Abstr 4: 210