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The Journal of Neuroscience, June 1, 1998, 18(11):4384-4392
Induction of a Specific Olfactory Memory Leads to a
Long-Lasting Activation of Protein Kinase C in the Antennal Lobe of
the Honeybee
Lore
Grünbaum and
Uli
Müller
Institut für Neurobiologie der Freien Universität
Berlin, D-14195 Berlin, Germany
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ABSTRACT |
In this study we investigated the role of protein kinase C (PKC) in
associative learning of Apis mellifera. Changes in PKC activity induced by olfactory conditioning were measured in the antennal lobes, a brain structure involved in associative learning. Multiple conditioning trials inducing a memory different from that
induced by a single conditioning trial specifically cause an increase
in PKC activity. This increase begins 1 hr after conditioning, lasts up
to 3 d, and is attributable to an increased level of constitutive
PKC. The increased level of constitutive PKC consists of an early
proteolysis-dependent phase and a late phase that requires RNA and
protein synthesis. Inhibition of the pathways resulting in constitutive
PKC selectively impairs distinct phases of multiple-trial induced
memory. The inhibition of the proteolytic mechanism has an instant
effect on an early phase of multiple-trial induced memory but does not
affect acquisition and the late phase of memory. Blocking of the
transient PKC activation during conditioning does not affect the
induction of memory formation. Thus, the constitutive PKC in the
antennal lobe seems to contribute to the early phase of memory that is
induced by multiple-trial conditioning.
Key words:
antennal lobe; memory phases; olfactory learning; protein
kinase C; thiol proteases; translation; transcription
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INTRODUCTION |
The molecular analysis of learning
and memory has confirmed the concept that memory consists of different
phases, which are induced and supported by different mechanisms. The
neural substrate of both short- and long-term memory is believed to
reside in the synaptic connections between neurons. Recent work has
documented convincingly the central role of second messenger-induced
phosphorylation cascades in the modulation of neuronal activity and
their involvement in learning and memory. One of these signaling
cascades is the protein kinase C (PKC) pathway, which has been
implicated in later stages of memory in vertebrate studies. A
long-lasting activation and relocation of PKC consecutive to learning
suggests a function of the PKC pathway in the expression of long-term
memory (LTM) (Olds et al., 1989 ; Scharenberg et al., 1991; Van der Zee
et al., 1992; Golski et al., 1995). Similar findings have been
demonstrated for long-term potentiation (LTP) in the mammalian
hippocampus, which is discussed as a cellular correlate of associative
learning (Colley et al., 1989; Roberson et al., 1996). Mechanisms
underlying the long-lasting PKC activation in LTP have been
investigated, but the results are still controversial (Akers et al.,
1986; Klann et al., 1993; Sacktor et al., 1993; Powell et al., 1994),
and no conclusion can be drawn as to whether these mechanisms are also
responsible for PKC modulation in associative learning.
The molecular mechanisms of learning and memory have been investigated
extensively in Aplysia, Drosophila, and the
honeybee, but very little is known about the role of PKC in associative learning in these invertebrates. However, PKC has been implicated in
synaptic plasticity in Aplysia, Hermissenda, and
Drosophila (Braha et al., 1990; Farley and Schuman, 1991;
Sugita et al., 1992; McPhie et al., 1993; Sossin et al., 1994; Kane et
al., 1997).
Associative learning in honeybees has several features similar to
higher forms of learning observed in vertebrates (Menzel, 1990; Menzel
and Müller, 1996). One learning paradigm is olfactory conditioning of the proboscis extension response (PER). This paradigm for olfactory learning consists of the pairing of an odor stimulus (CS)
with a sucrose reward (US) (Menzel, 1985). Depending on the number of
conditioning trials, different memories are induced. Whereas
single-trial conditioning induces a memory (sLTM) that is insensitive
to the inhibition of nitric oxide synthase (NOS) during training,
multiple-trial conditioning additionally induces a distinct
NO-dependent memory (mLTM) (Müller, 1996). Interestingly, in the
honeybee brain, PKC is expressed strongly in two regions that are both
involved in associative olfactory learning: the antennal lobe (AL) and
the mushroom body (MB) (Hammer and Menzel, 1995; Menzel and
Müller, 1996). In this study we investigated the role of PKC in
the AL. We report here that multiple-trial conditioning selectively
induces a long-lasting PKC activation after conditioning. This
activation involves the formation of constitutively active PKC and is
separable into two mechanistically different phases that contribute to
distinct memory phases.
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MATERIALS AND METHODS |
Materials. [ -32P]ATP (5000 Ci/mmol)
was purchased from NEN Life Science Products (Brussels, Belgium), and E
64 was obtained from Biomol (Hamburg, Germany). Gö 7874 hydrochloride (Kleinschroth et al., 1995) and the protein kinase C
inhibitor peptide (19-31) were obtained from Calbiochem-Novabiochem
(Bad Soden, Germany). The ELISA plates were purchased from Falcon,
Becton Dickinson (Heidelberg, Germany). All other chemicals were
obtained from Sigma (Deisenhofen, Germany). All of the chemicals used
were of analytical grade. Protein kinase C (PKC) was purified from bee brains, as has been described previously (Altfelder et al., 1991).
Animal treatment and injection procedure. Honeybees
(Apis mellifera carnica) were caught at the hive entrance,
immobilized by cooling, and mounted in metal harnesses by a strip of
tape between the head and the thorax. Bees were fed with a sucrose solution (1.4 M) until satiation and kept in darkness in a
container at 70% relative humidity and at 20-25°C until the
training started the next day. Bees that were used in the long-term
experiments also were kept in the containers between sessions and fed
every evening with sucrose solution. Drugs were injected into the
hemolymph of the thorax by using a microcapillary, as has been
described previously (Müller and Hildebrandt, 1995).
Classical olfactory conditioning. Behavioral experiments
were performed on different days and in different treatment
combinations (drug injections). Each combination always included one
PBS-injected control group. Olfactory conditioning of the proboscis
extension response (PER) consisted of the temporal pairing of an odor
(carnation) as the conditioned stimulus (CS) with sucrose as the
unconditioned stimulus (US) (Müller, 1996). Three conditioning
trials were presented, with an intertrial interval of 2 min. After
successful conditioning, the presentation of the CS alone elicited PER.
Animals that did not respond to the US before conditioning were
excluded from the experiments (<5%). The PER was calculated for each
treatment by dividing the sum of responding animals by the total number of animals tested after the respective treatment. Differences between
the PBS-injected control and the drug-injected animals were evaluated
by the  2 test.
Preparation of tissues. Preparation was performed at
different times after stimulation or conditioning. In experiments in which bees were killed at times longer than 1 hr after conditioning, the conditioned PER was tested 30 min before dissection. Only bees that
showed conditioned PER in this test were dissected. The bees were
cooled quickly in ice water, their heads were cut off, and the AL was
isolated. Each AL was transferred into a separate capillary and
homogenized on the surface of 10 µl of frozen extraction buffer
[containing (in mM) 50 Tris-HCl, pH 7.5, 10 MgCl2, 1 EDTA, 1 EGTA, and 10 2-mercaptoethanol with
0.1 M NaCl). The whole procedure, from the cooling of the
bee to the freezing of the AL, took ~1 min. AL were kept in liquid
nitrogen until they were used in the in vitro
phosphorylation assay.
In vitro phosphorylation assay. PKC activity was
measured by the in vitro phosphorylation of exogenously
added myristoylated alanine-rich C kinase substrate (MARCKS) protein
from bovine brain, which is a specific substrate for PKC from honeybee
brain (Müller, 1997a). Homogenates from single AL were
phosphorylated in a final volume of 20 µl containing (in
mM) 50 Tris-HCl, pH 7.5, 10 MgCl2, 1 EDTA, 1 EGTA, and 10 2-mercaptoethanol plus 0.1 M NaCl, 10 µM ATP, 1 µg of MARCKS protein, and 0.1 µCi
[-32P]ATP (3000 Ci/mmol). PKC activators (1.5 mM CaCl2, 0.02 µg of diolein, and 0.8 µg of phosphatidylserine) were included in the mixture, unless
indicated otherwise. To confirm the specific phosphorylation of MARCKS
by PKC, we included the PKC inhibitor peptide (19-31) in the
phosphorylation assay where indicated. The samples were phosphorylated
for 20 sec at 15°C. The reaction was terminated by the addition of 3 µl of sample buffer (0.25 M Tris-HCl, pH 6.7, containing
20% glycerol, 5% SDS, and 5% 2-mercaptoethanol). SDS-PAGE and
autoradiography were performed as described (Hildebrandt and
Müller, 1995). Conditions of the film exposure were adjusted to
keep the signals of labeled proteins in a linear range. For the
calibration of the film exposure, a scintillation counter was used to
determine 32P incorporation into MARCKS. Autoradiograms
were scanned by a UMAX UC840 Scanner, and the 32P
incorporation into the MARCKS protein was quantified by National Institutes of Health Image software. The statistical analysis was
performed with Student's t test.
PKC immunohistochemistry. Immunohistochemistry was performed
as described in Müller (1997b). PKC immunoreactivity was
visualized with polyclonal antiserum generated against
calcium-dependent PKC purified from the honeybee brain and alkaline
phosphatase coupled to the immunocomplex via the biotin streptavidin
system.
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RESULTS |
Localization of PKC in the honeybee brain
The calcium-dependent PKC purified from honeybee brain shows
remarkable similarities to the corresponding vertebrate PKC (Altfelder et al., 1991). Interestingly, the staining of honeybee brain with a
polyclonal PKC antiserum reveals a strong, distinct labeling of the
antennal lobes (AL) and the mushroom bodies (MB) (Fig. 1). AL and MB are both involved in
olfactory learning in the honeybee. Local cooling experiments and local
injections of octopamine into the AL indicate that the AL may establish
a memory trace independently of the MB (Erber et al., 1980; Hammer and
Menzel, 1995). However, the AL primarily process chemosensory
information, and PKC immunoreactivity is located predominantly in the
AL interneurons. Sensory neurons projecting via the antennal nerve and
innervating the rind areas of the glomeruli are stained very weakly. In
contrast to the AL, the MB are involved in higher integrative functions
and receive input from different sensory modalities (Menzel et al.,
1994). The MB show high levels of PKC expression. Thus, because the AL predominantly process olfactory information, we concentrated on this
structure of the honeybee brain to study the role of PKC in associative
olfactory learning.
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Figure 1.
Comparison of the distribution of PKC in the
honeybee brain with the neuropils critical to olfactory learning. The
distribution of PKC in the adult honeybee brain, as analyzed by
immunohistochemistry (A), is compared with the
neuropils that are involved in olfactory learning (schematic).
B, The antennal lobes (al), which
receive mainly chemosensory input, show strong staining in the central
core (asterisk), in the central regions of the glomeruli
(g), and in the somata of the interneurons
(arrow). Strong labeling is found throughout the
mushroom bodies (mb), which process multimodal input and
are innervated by AL interneurons. The lateral protocerebral lobes
(lp) are stained also. In contrast, the optical lobes
(ol) are stained very weakly. Scale bar, 100 µm.
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Stimuli used in olfactory learning induce a transient PKC
activation in the AL
To detect changes in PKC activity induced by in vivo
stimulation, we applied a fast phosphorylation assay similar to that described for the PKA (Hildebrandt and Müller, 1995), using
MARCKS as a PKC-specific substrate. We recently demonstrated that
MARCKS is phosphorylated specifically by PKC, but not by PKA or CaMII kinase from honeybee brain (Müller, 1997a). MARCKS also is
phosphorylated by the calcium-independent PKC activity of PKM (see
Table 2). In brain homogenate that was depleted of PKC, MARCKS
phosphorylation was below 10% of the phosphorylation in the original
homogenate containing PKC (see Table 2). To confirm further the
specific phosphorylation of MARCKS by PKC, we included the PKC
inhibitor peptide (19-31) in the phosphorylation assay. This PKC
inhibitor peptide selectively inhibits MARCKS phosphorylation by PKC
purified from honeybee brain (IC50  10 µM)
without affecting PKA and CaMII kinase activity. In brain homogenates
it reduces MARCKS phosphorylation to below 10% of the phosphorylation
in the absence of the inhibitor peptide (see Table 2). These data
strongly confirm that MARCKS phosphorylation of honeybee brain
homogenates is mediated exclusively by PKC.
To discriminate between two different forms of PKC activation, (1)
transient calcium-dependent activation and (2) constitutive calcium-independent activation, we measured PKC activity in either the
presence or the absence of activators for classical PKC [calcium and
diacylglycerol (DAG)]. All of the stimuli used for olfactory conditioning induce comparable transient PKC activation in the AL
regardless of the stimuli and the sequence of application (Fig. 2). A similar, although prolonged,
transient activation also is induced by three forward or backward
pairings with intertrial intervals (ITI) of 2 min (Fig. 2). In the
absence of PKC activators (calcium and DAG) in the in vitro
assay, the PKC activity is reduced to <5% of the activity measured in
presence of the activators. This suggests that in vivo
stimulation during conditioning causes a transient calcium-dependent
activation of PKC in the AL.
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Figure 2.
PKC activation induced by stimuli used in
olfactory conditioning. Animals received either a single
CS or US, a single forward
(CS-US) or backward pairing (US-CS), or
three forward or backward pairings delivered with an intertrial
interval (ITI) of 2 min. Animals were dissected for the determination
of PKC activity in the antennal lobes (AL) either immediately
(0), 1.5, or 10 min after stimulation. In each
experiment, values were normalized to PKC activity in unstimulated
animals ( ). Each column represents the mean ± SEM of n measurements as indicated by the
numbers on the bars. For all stimulations
PKC activity at 0 is significantly different from PKC
activity in unstimulated animals (p < 0.05;
t test).
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To confirm that PKC activation after stimulation takes place in
vivo and is not produced during handling in vitro
(i.e., during homogenization, phosphorylation, etc.), we analyzed the
in vivo effect of PKC inhibitors on PKC activity. Of various
available PKC inhibitors (chelerythrin, hypericin, calphostin C, GF
109203x, and sphingosin) only Gö 7874 (Kleinschroth et al., 1995)
led to a specific and dose-dependent reduction of calcium-dependent activation of PKC when it was used in vitro
(IC50 0.5 µM). Interestingly, calcium-independent PKC activity of PKM was not blocked significantly by Gö 7874 (10 µM) in vitro (<10%
reduction in MARCKS phosphorylation; p > 0.05;
t test). Using appropriate substrates (Altfelder et al.,
1991; Müller, 1997b), we showed that Gö 7874 at a
concentration of 10 µM did not affect the activity of PKA
and CaMII kinase purified from honeybee brain in
vitro. In vivo application of Gö 7874 (10 µM final concentration) did not affect PKC activity
measured in the AL of unstimulated animals in the presence of calcium
and DAG. However, stimulus-induced PKC activation (Fig. 2) was fully suppressed in Gö 7874-treated animals (Table
1). This,
together with the finding that Gö 7874 does not inhibit
calcium-independent PKC activity in vitro, suggests that
Gö 7874 selectively interferes with calcium-dependent activation
of PKC. Independent of the stimulation the animals received, the
addition of PKC peptide inhibitor (100 µM) to the
phosphorylation mixture strongly reduced MARCKS phosphorylation (Tables
2, 3). This confirms that changes in
MARCKS phosphorylation induced by in vivo stimulation
reflect PKC activity.
Inhibition of transient PKC activation during conditioning does not
interfere with associative olfactory learning
Forward and backward pairings both result in a comparable
activation of PKC in the AL. This suggests that PKC might not be essential for the induction of memory during acquisition. To test this
hypothesis, we investigated the effect of the PKC inhibitor Gö
7874 on the acquisition and retention of memory induced by single- or
multiple-trial conditioning. In neither case was the conditioned PER of
Gö 7874-injected animals significantly different from that of
PBS-injected control animals (Table 4).
Thus, inhibition of the transient calcium-dependent activation of PKC
during conditioning (Table 1) seems not to interfere with the
mechanisms of associative learning.
Learning-induced long-lasting changes in PKC activation
Training-induced long-lasting changes in PKC activation as well as
in PKC distribution have been observed in various species (Bank et al.,
1988; Olds et al., 1989; Burchuladze et al., 1990; Van der Zee et al.,
1992; Golski et al., 1995). In these studies it has been shown that
learning induces changes of membrane-bound PKC activity or of PKC
activity in subcellular fractions in the range of 10-40% of the
corresponding values from control animals. Therefore, we have analyzed
changes in total PKC activity in the AL of honeybees at different times
after single- and three-trial conditioning. Only bees that showed
conditioned PER after conditioning were used. This enabled us to
compensate for the distinct levels in conditioned PER, depending on the
number of conditioning trials, and to detect potential differences in
the mechanisms underlying single- and multiple-trial conditioning.
Untreated animals and animals that had received the corresponding
backward pairings (US-CS), which cause no excitatory learning, were
used as controls.
No change in PKC activity was detectable in animals of the control
groups at different times after stimulation (data not shown). The same
holds for animals that showed conditioned PER after single-trial conditioning, which induces sLTM (Fig.
3). In contrast, after three-trial
conditioning, which induces mLTM, a characteristic change of PKC
activity was detected. This learning-induced MARCKS phosphorylation is
PKC-specific, because it is strongly inhibited by the addition of the
PKC inhibitor peptide to the phosphorylation assay (Table 3). The
decrease in PKC activity observed ~30 min after training was highly
variable and not significantly different from controls. Increased PKC
activity was detected first at 1 hr and reached its maximum ~3 hr
after training. It remained at this elevated level until 3 d after
conditioning (Fig. 3). However, at 4 and 5 d after conditioning,
PKC activity was not distinguishable from the activity measured in
untrained animals. The observed 20-30% increase of total PKC activity
is in agreement with changes within the same range observed for
membrane-associated PKC in other systems (Bank et al., 1988; Olds et
al., 1989; Burchuladze et al., 1990; Golski et al., 1995). The specific
change in PKC activity, only detected in animals that show a
conditioned PER after three-trial conditioning, indicates a role of the
long-lasting elevation of PKC activity in the formation of this
distinct memory.
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Figure 3.
Time course of PKC activity after one and three
conditioning trials. PKC activity in the AL was determined at different
times after one or three conditioning trials (ITI, 2 min). In each
experiment, values were normalized to PKC activity in unstimulated
naive animals (dashed line). Each point
represents the mean ± SEM of n measurements.
Values that are significantly different from PKC activity in
unstimulated naive animals are marked by an asterisk
(p < 0.05; t test).
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Long-lasting PKC activation is attributable to constitutively
active PKC
Evidence from other studies suggests that PKC activation after
learning involves the formation of a constitutively active form of PKC
that is independent of calcium and DAG (Klann et al., 1991, 1993). To
distinguish between transient and constitutive activity, we measured
PKC activity in the presence and absence of calcium and DAG. Again, the
MARCKS phosphorylation measured either in the presence or in the
absence of calcium and DAG is PKC-specific, because it is strongly
reduced by the addition of the PKC inhibitor peptide to the
phosphorylation assay (Table 3).
Whereas constitutive activity is marginal in untrained animals (<5%
of basal calcium-dependent PKC activity), three-trial conditioning
produces an at least 600% increase in constitutive activity, as
compared with that in untrained animals (Fig.
4A). This increase is
similar at 3 and 18 hr after conditioning and corresponds to the
learning-induced elevation of PKC activity measured in the presence of
calcium and DAG. Hence, the constitutive PKC activity may be fully
responsible for the long-lasting PKC activation induced by
conditioning. Whereas the transient activation of PKC induced by single
or paired stimuli is blocked in the presence of the PKC inhibitor
Gö 7874 (Table 1), long-lasting activation 3 and 18 hr after
conditioning is not susceptible to this inhibition (Fig.
4B). Thus, the calcium independence and the lack of
inhibition by Gö 7874 support the hypothesis that long-lasting
and transient PKC activation are mediated by different mechanisms.
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Figure 4.
Characterization of the long-lasting PKC
activation induced by three-trial conditioning. A, PKC
activity in the AL was measured at 3 and 18 hr after three-trial
conditioning (ITI, 2 min) in the presence or absence of calcium and
diacylglycerol (DAG). In a control group of untrained
animals the PKC activity was measured by following the same procedure
as in trained animals. In each experiment, values were normalized with
respect to PKC activity in untrained animals, measured in the presence
of calcium and DAG. B, Animals were dissected at 3 and
18 hr after three-trial conditioning. Conditioned animals were each
injected with 1 µl of either Gö 7874 (1 mM) or PBS
20 min before dissection. Untrained animals were handled in parallel in
the same way as trained animals. In all experimental groups the PKC
activity was measured in the presence of calcium and DAG. In each
experiment, values were normalized with respect to PKC activity in
PBS-injected untrained animals. Each column represents
the mean ± SEM of n measurements as indicated by
the numbers on the bars
(*p < 0.05; t test).
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PKC activation after three-trial conditioning is separable into two
mechanistically different phases
Because PKC activation is induced as early as 1 hr after
three-trial conditioning but remains stable until 3 d later,
different mechanisms may be responsible for the elevation at different
times after conditioning. Permanent membrane translocation and
proteolytic formation of the catalytic fragment PKM have been proposed
in this regard (Akers et al., 1986; Klann et al., 1993; Sacktor et al.,
1993). However, the observed long-lasting increase in PKC activity (see
Fig. 3) should, finally, require a change of gene expression. We
investigated the contribution of these different mechanisms to
long-lasting PKC activation for two time intervals: 2-4 hr (3 hr) and
14-24 hr (18 hr) after conditioning.
To test whether long-lasting PKC activation involves membrane
translocation, we quantified PKC with antibodies in soluble and
particulate fractions of AL homogenate at different times after
conditioning. Using the ELISA technique (Müller, 1997b), we
detected no change in total PKC expression or PKC distribution (data
not shown). Nevertheless, subtle changes that can cause drastic
alterations of PKC activity, which are not detected by this assay, may
occur. It is also possible that the learning-induced changes of PKC
activity described above are attributable to PKC isoforms that are not
detected with the antibodies that were used. To detect a possible
formation of the constitutively active fragment PKM directly, we
performed immunoblot analysis. Unfortunately, none of the presently
available antibodies against honeybee PKC did detect PKM.
Because the direct detection of PKM was not possible, the in
vivo effect of protease inhibitors on conditioning-induced PKC activation was investigated. The formation of PKM probably is mediated
by the thiol protease calpain (Suzuki et al., 1992). Calpain homologs
have been purified from honeybee brains and can be inhibited by the
thiol protease inhibitor E 64 (Müller and Altfelder, 1991) (our
unpublished results). E 64 was injected before conditioning, because
the induction of a proteolytic mechanism in LTP was described as taking
place during or shortly after training (Sacktor et al., 1993).
Elevation of PKC activity 3 hr after conditioning was blocked in E
64-treated bees, as compared with that in PBS-injected control animals
(Fig. 5A). However, 18 hr
after conditioning, PKC activation in E 64-treated bees was not
distinguishable from that in PBS-injected controls. PKC activity in
untrained animals was not changed by E 64. Hence, a proteolytic
mechanism is required for the induction of PKC activation during the
first hour after conditioning.
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Figure 5.
Effect of E 64, actinomycin D, and
anisomycin/cycloheximide on conditioning-induced PKC activation.
A, Animals were injected with 1 µl of the thiol
protease inhibitor E 64 (1 mM) or PBS 20 min before
three-trial conditioning (ITI, 2 min). PKC activity was measured at 3 and 18 hr after conditioning. B, Animals were injected
with 1 µl of actinomycin D (2 mg/ml), anisomycin/cycloheximide
(CHX; 2.5 respectively, 5 mg/ml), or PBS 1 hr after
three-trial conditioning. PKC activity was measured at 3 and 18 hr
after conditioning. PKC activity was determined in parallel in groups
of untrained animals, injected according to the protocol for trained
animals. In each experiment, values were normalized to PKC activity in
PBS-injected untrained animals. Each column represents
the mean ± SEM of n measurements as indicated by
the numbers on the bars
(*p < 0.05; t test).
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To investigate the contribution of gene expression to PKC activation
after conditioning, we tested transcription and translation inhibitors
for their ability to interfere with PKC activation in vivo.
We assumed that RNA and protein synthesis are not required during, but
after, training. Therefore, inhibitors of RNA (actinomycin D) and
protein synthesis (a mixture of cycloheximide and anisomycin) were
injected 1 hr after training. Actinomycin D did not interfere with PKC
activation 3 hr after conditioning. However, 18 hr after conditioning,
no activation of PKC was measured in bees treated with transcription or
translation inhibitors. Taken together, these data suggest that the
early phase of conditioning-induced PKC activation is dependent on
proteolysis and is not required for the induction of the late phase of
conditioning-induced PKC activation, which requires the synthesis of
RNA and proteins.
Memory formation after three-trial conditioning also can be
dissected into different phases
To test whether the long-lasting PKC activation is related
functionally to olfactory learning, we analyzed E 64 and actinomycin D,
which suppress PKC activation at different times after multiple-trial conditioning, with regard to their effect on memory formation.
Whereas E 64 does not affect the conditioned PER at any time after
single-trial conditioning, it significantly reduces the conditioned PER
during the first hour after three-trial conditioning (Fig.
6). The acquisition, however, does not
differ from that of PBS-injected animals. Interestingly, the effect of
E 64 on the conditioned PER, similar to the suppression of PKC
activation by E 64, is transient, and the conditioned PER tested at
times longer than 1 d after three-trial conditioning is not
compromised by E 64. The coincidence of these time windows suggests
that the proteolysis-dependent PKC activation might contribute
selectively to an early phase of multiple-trial induced memory.
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Figure 6.
Dissection of memory phases by inhibition of thiol
proteases or transcription. Animals were injected with 1 µl of PBS or
1 µl of the thiol protease inhibitor E 64 (1 mM) 20 min
before conditioning, or they were injected with 1 µl of PBS or
actinomycin D (2 mg/ml) 1 hr after conditioning. Animals received
either one conditioning trial (A) or three
conditioning trials (B). The probability of the
CS-elicited proboscis extension response (PER) was
tested during acquisition (acq) and at different times
after conditioning (retr). The PER of animals that
received PBS injection before or after conditioning did not differ
between the groups. Thus the values were combined in the PBS control
group. The data show the PER of n animals, as indicated.
Sequence from top to bottom: PBS, actino,
and E 64 (*p < 0.001; 2
test).
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Actinomycin D, like E 64, does not change the conditioned PER after
single-trial conditioning (Fig. 6). Although actinomycin D suppresses
the increase in PKC activity already at 1 d after conditioning,
the conditioned PER of actinomycin-treated animals is not affected
until 3 d after conditioning. Thus, the late phase of PKC
activation in the AL, requiring RNA and protein synthesis, does not
coincide with a defect in memory formation.
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DISCUSSION |
In this study we have investigated the role of PKC in associative
learning and memory in the honeybee. The method of shock freezing the
bees in liquid nitrogen allowed us to preserve the in vivo
state of activation of PKC and to measure directly the PKC activity in
homogenates of the frozen AL by specific substrate phosphorylation. The
MARCKS protein serves as a highly specific substrate for PKC from
honeybee brain (Müller, 1997a). Using the PKC inhibitor peptide
(19-31), we demonstrated that MARCKS is phosphorylated by
calcium-dependent as well as calcium-independent PKC (Table 2). Thus,
we were able to monitor changes in both forms of PKC activity.
Interestingly, the PKC inhibitor Gö 7874 inhibits only
calcium-dependent PKC activity and is not effective on
calcium-independent PKC in vitro. Thus, Gö 7874 was
used to distinguish between these different forms of PKC activation.
However, a detailed analysis of the calcium-dependent and
calcium-independent PKC isoforms in the honeybee is still missing.
Although this method allowed us to determine the time course of PKC
activation with a resolution of ~1 min, we did not detect a
learning-specific modulation of PKC activity at the stage of acquisition. Single and combined stimuli, used in olfactory
conditioning, evoked a comparable, transient, and second
messenger-dependent activation of PKC, irrespective of whether the
stimulations resulted in the induction of memory or not. Interestingly,
in vivo inhibition of PKC during conditioning did not affect
acquisition and memory at different times after training (Table 4).
These findings suggest that the activation of PKC immediately after
conditioning is not essential for the induction of memory but, rather,
may be attributed to a role for PKC in the processing of chemosensory
information in the AL in general. In contrast, three-trial conditioning
that promotes the formation of mLTM specifically leads to a
long-lasting but reversible activation of PKC from 1 hr up to 3 d
after conditioning.
This is in agreement with behavioral studies in vertebrates, in which
PKC inhibitors interfered with memory formation, whereas the
acquisition of memory was not compromised (Burchuladze et al., 1990;
Zhao et al., 1994). A membrane translocation and regional redistribution of PKC in the hippocampus were detected within 1 hr, but
also 1 d or more after learning, suggesting that learning might
lead to a long-lasting activation of PKC (Bank et al., 1988; Olds et
al., 1989, 1990; Scharenberg et al., 1991; Van der Zee et al., 1992;
Golski et al., 1995). However, it was not shown whether these
PKC-related changes are reversible.
The role and the time course of PKC activity were investigated in more
detail in the vertebrate model system for synaptic plasticity, LTP.
Although PKC is not involved in the initial induction, it contributes
to the expression of LTP at later times (Colley et al., 1989; Roberson
et al., 1996). A long-lasting activation of PKC in LTP was demonstrated
by using assays for kinase activity, and PKC inhibitor studies
confirmed the requirement for ongoing PKC activity during the first
hour after LTP induction (Lovinger et al., 1987; Colley et al., 1990;
Klann et al., 1991, 1993; Wang and Feng, 1992). Interestingly,
injection of the PKC inhibitor polymyxin B (PMXB) before the induction
of LTP did not affect initial potentiation but led to a decay of LTP
within 2 hr after induction. This decay also was observed when PMXB was
injected 15 or 30 min after LTP induction. Another inhibitor, H7, which interacts with the catalytic domain of PKC, was effective even when it
was injected up to 4 hr after LTP induction (Colley et al., 1990).
These data suggest the existence of two postinitiation components of
PKC activation in LTP.
Permanent membrane translocation, proteolytic removal of the regulatory
region leading to generation of constitutively active PKM, and
phosphorylation of PKC may be responsible for persistent activation of
PKC (Akers et al., 1986; Suzuki et al., 1992; Klann et al., 1993;
Sacktor et al., 1993; Powell et al., 1994; Osten et al., 1996).
However, it remains unclear how these mechanisms are related to the
function of PKC in learning in vivo.
In Drosophila, the role of PKC was investigated only for the
associative conditioning of courtship: transgenic flies with reduced
PKC activity do not express initial learning in their behavior,
although the expression of memory after conditioning is not compromised
(Kane et al., 1997). These data suggest that the immediate performance
of the task during acquisition and the induction of memory formation
may be mediated by different mechanisms acting in parallel. However, in
these transgenic flies PKC is inhibited permanently, and the effect of
a precisely timed inhibition of PKC during or at different times after
conditioning cannot be determined.
In the AL of the honeybee, we have dissected different mechanisms
contributing to long-lasting PKC activation at distinct times after
conditioning. A proteolytic mechanism, probably involving the thiol
protease calpain, is needed during training for the induction of PKC
activation in the range of just a few hours after conditioning, whereas
PKC activation ~18 hr after conditioning is dependent on RNA and
protein synthesis. Both mechanisms are independent of each other (see
Fig. 5).
The dissection of memory phases in Drosophila has led to the
concept that memory, at a distinct time after learning, may consist of
mechanistically different components (Tully et al., 1990, 1994). In the
honeybee it has been demonstrated that, depending on the numbers of
conditioning trials, at least two distinguishable memories are induced:
mLTM induced by multiple-trial conditioning is strongly reduced by NOS
inhibition, whereas sLTM, induced by single-trial conditioning, is not
affected by this treatment (Müller, 1996). Our data now present
additional evidence that PKC also contributes specifically to mLTM: (1)
only multiple-trial conditioning induces a long-lasting, but
reversible, activation of PKC, which depends on an early proteolytic
mechanism and requires RNA and protein synthesis in the late phase; (2)
inhibition of these mechanisms selectively interferes with distinct
phases of mLTM.
However, only the protease inhibitor E 64 has a coinciding effect on
memory and on suppression of PKC activation, whereas the effect of
actinomycin D affects memory and PKC activation in different time
windows (compare Figs. 5 and 6). Because PKC activity was determined
exclusively for the AL whereas drug injection was performed
systemically, these results may reflect directly the contribution of
the AL to distinct phases of associative learning.
Because E 64 exerts its action on PKC and PER within coinciding time
windows, the activation of the PKC in the AL may contribute to an early
phase of mLTM. Interestingly, application of E 64 selectively
suppresses the conditioned PER in the range of hours, but neither
interferes with acquisition nor with the late phase of mLTM (Fig. 6).
Thus, the rapidly induced proteolysis-dependent activation of PKC in
the AL might serve to bridge the time gap until other RNA- and protein
synthesis-dependent mechanisms take effect.
The RNA and protein synthesis-dependent PKC activation in the AL,
however, seems to have no immediate effect on memory formation. This
suggests that, in the range of days after conditioning, other brain
areas than the AL are involved predominantly in memory formation. This
is in agreement with findings that the AL and the MB seem to contribute
to different features of memory formation (Erber et al., 1980; Hammer
and Menzel, 1995). Therefore, the late phase of PKC activation in the
AL may be only one of several parallel mechanisms that contribute to
the formation of mLTM with different time courses and occur in
different brain compartments. Although we have evidence that the
proteolytic activation of PKC in the AL contributes to an early phase
of mLTM, future analysis will reveal the role of PKC in brain areas
like the MB and its interaction with the RNA and protein
synthesis-dependent PKC activation in the AL.
An interesting speculation is that long-time activation of PKC may
depend on a preceding activation of PKA. PKA and its nuclear substrate,
cAMP response element-binding proteins (CREB), have been shown to
mediate changes in gene expression in long-term facilitation, late LTP,
and LTM (Frey et al., 1993; Kaang et al., 1993; Bourtchouladze et al.,
1994; Yin et al., 1994; Abel et al., 1997). Future analysis will have
to show whether PKC is a direct target of conditioning-induced gene
expression or whether the expression of another protein could be
responsible for constitutive PKC activation. Moreover, it is of special
interest to learn how these changes in PKC activity affect the
phosphorylation and function of substrates and thus finally contribute
to structural changes in LTM.
| |
FOOTNOTES |
Received Dec. 2, 1997; revised March 2, 1998; accepted March 18, 1998.
This investigation was supported by the Deutsche Forschungsgemeinschaft
(SFB 515/C3). We would like to thank R. Menzel for helpful suggestions
on an earlier version of this manuscript and D. Alexander and S. Meuser
for their help with the preparation of this manuscript.
Correspondence should be addressed to Dr. Uli Müller, Institut
für Neurobiologie der Freien Universität Berlin,
Königin-Luise-Strasse 28/30, D-14195 Berlin, Germany.
| |
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Top
Abstract
Introduction
