Protein kinase Cs (PKCs) are important effectors of synaptic plasticity. In Aplysia, there are two major phorbol ester-activated PKCs,
by translocating from the cytoplasm to the membrane, we used fluorescently tagged PKCs to determine the isoform and cell-type
specificity of translocation in living Aplysia neurons. In Sf9 cells, low levels of diacylglycerol translocate Apl II, but not Apl I, which
the absence of neuronal firing translocates Apl II, but not Apl I, consistent with the role of Apl II in short-term facilitation. This
neuron; firing alone is ineffective. Because combined 5-HT and firing are required for the induction of one type of intermediate-term
negative Apl I, but not Apl II, blocks intermediate-term facilitation. Thus, different isoforms of PKC translocate under different condi-
Protein kinase C (PKC) plays an important role in regulating
synaptic strength by phosphorylating and regulating proteins
in neurotransmitter release that, in turn, control synaptic
strength (Keenan and Kelleher, 1998; Majewski and Iannazzo,
1998). Persistent changes in synaptic strength can depend on
persistent activation of PKC, suggesting that regulation of PKC
itself may be central in some memory systems (Malinow et al.,
1988; Grunbaum and Muller, 1998; Sutton and Carew, 2000;
Ling et al., 2002; Linden, 2003; Sutton et al., 2004). Thus, under-
standing how and when PKCs are activated is important for un-
derstanding diverse types of synaptic plasticity.
Behavioral sensitization in Aplysia is mediated in part by an
increase in the strength of the connections between mechanore-
ceptor sensory neurons (SNs) and motor neurons (Kandel,
2001). Synaptic facilitation is mediated by the neurotransmitter
serotonin (5-HT), which can induce facilitation in isolated gan-
glia and in cocultures of sensory neurons and motor neurons
(Byrne et al., 1993; Byrne and Kandel, 1996). Interestingly, the
mechanisms determining short-term facilitation (STF) depend
activation of protein kinase A (PKA), whereas facilitation at syn-
apses that have been depressed by previous activity depends on
activation of PKC (Braha et al., 1990; Ghirardi et al., 1992; Klein,
1993). There is also a distinction between PKA and PKC in more
itation (ITF)] caused by 5-HT. Although ITF induced by spaced
application of 5-HT depends on persistent activation of PKA,
pairing 5-HT with firing in the sensory neuron leads to a form of
ITF that depends on persistent activation of PKC (Sutton and
Aplysia PKC to neuronal membranes (Sacktor and Schwartz,
1990). Biochemical experiments indicate that in the Aplysia ner-
vous system, there are only two major phorbol ester-activated
(Kruger et al., 1991; Sossin et al., 1993). Biochemical analysis of
PKC activation in pleural-pedal ganglia suggested that 5-HT ac-
tivates Apl I but not Apl II (Sossin and Schwartz, 1992). More-
Research (CIHR) Grant MOP 12046 (W.S.S.), and a grant from the W. M. Keck Foundation (K.C.M.). C.A.-F. is the
recipientofapostdoctoralfellowshipfromtheFondsdelaRechercheenSante ´duQue ´bec,andW.S.S.isaKillamand
TheJournalofNeuroscience,August23,2006 • 26(34):8847–8856 • 8847
I was easier to activate, even in the absence of calcium, than was
Apl II (Pepio et al., 1998). However, 5-HT-mediated reversal of
synaptic depression was blocked by dominant-negative forms of
(Manseau et al., 2001).
cells using fluorescently tagged PKCs to examine the isoform
specificity of enzyme translocation. These results are consistent
with distinct physiological roles for the two different isoforms of
PKC in sensory neurons, with Apl II responsible for one type of
STF and Apl I necessary for a form of ITF.
Plasmid construction. The pNEX3 enhanced green fluorescent protein
have been described previously (Manseau et al., 2001). To construct
monocistronic red fluorescent protein (mRFP) PKC Apl II, mRFP was
amplified from an mRFP plasmid (Campbell et al., 2002) using overlap
PCR such that the PCR piece had KpnI and PvuI sites at the ends and
the resulting mRFP-Apl II had the identical linker as eGFP-Apl II
(Manseau et al., 2001). This piece was then inserted into pNEX3-eGFP-
Apl II that had eGFP cut with KpnI (Partial) and PvuI.
Sf9 cell culture. The Sf9 cells were purchased from Sigma-Aldrich
(Sigma-Aldrich, Oakville, Ontario, Canada). Sf9 cells were grown in
Grace’s medium (Invitrogen, Burlington, Ontario, Canada) supple-
ada) as a monolayer at 27°C. For transfection, cells were plated on Mat-
Tek glass bottom culture dishes (MatTek Corporation, Ashland, MA)
with a glass surface of 14 mm and a coverslip thickness of 1.5 mm at a
density of 0.11 ? 106cells/35 mm dish. Cells were transfected using the
Cellfectin reagent (Invitrogen, Burlington, Ontario, Canada) following
microscope was performed 48–72 h posttransfection. Cells were serum
starved for 6 h before imaging sessions. 1,2-Dioctanoyl-sn-glycerol
(DOG) (Avanti Polar Lipids, Alabaster, AL) and Ionomycin (Sigma-
Aldrich) were dissolved in dimethyl sulfoxide and diluted to the final
ing the experiment, the cells were not exposed to dimethyl sulfoxide
concentrations ?1%. All of the experiments were performed in a
temperature-controlled chamber at 27°C and on at least three different
Confocal microscopy in SF9 cells. Cells expressing eGFP-Apl I and
mRFP-Apl II were examined using a Zeiss laser scanning microscope
(Zeiss, Oberkochen, Germany) with an Axiovert 200 and a 63? oil im-
to the dish at 30 s, and a series of 20 confocal images were recorded for
each experiment at time intervals of 30 s.
Image analysis of SF9 cells. The time series was analyzed using NIH
plasma membrane and three rectangles at random locations in the cy-
tosol. The relative increase in the amount of enzyme localized in the
plasma membrane for each time point was calculated by using the ratio
r ? Average (Im)/Average (Ic) where Average (Im) is the average fluores-
cence intensity at the plasma membrane and Average (Ic) is the average
lished procedures (Schacher and Proshansky, 1983; Klein, 1993). Adult
Alacrity Marine Biological Services, Redondo Beach, CA) were anesthe-
(ASW; see below for composition) containing 1% protease type IX
(Sigma). Tail SNs and siphon (LFS) motor neurons were isolated and
plated in L15 containing 10% or 50% Aplysia hemolymph on dishes
(Glass Bottom Microwell Dish; MatTek Corporation) pretreated with
poly-L-lysine (molecular weight, ?300,000; Sigma). Dishes were pre-
pared with isolated sensory neurons, isolated motor neurons, or cocul-
tures of sensory neurons and motor neurons for synapse formation.
Microinjection of plasmid vectors. On day 1 after isolation, solutions of
plasmids in distilled water containing 0.25% fast green were microin-
jected from back-filled glass micropipettes using a General Valve Driver
into the cell nucleus, and short pressure pulses (10–50 ms duration; 20
psi) were delivered until the nucleus became uniformly green. The cells
were incubated overnight at room temperature for experiments on the
following day. For experiments at later times, cultures were kept at 4°C
cell imaging: neurons expressing eGFP-PKC were imaged on a Zeiss
Pascal scanning laser microscope using a 63? (1.2 numerical aperture)
water immersion objective and 10% of a 25 mW argon laser. Optical
sections (Z-stack interval of 0.8 ?m) were acquired before and after
incubation with 5-HT (10 ?M) or phorbol dibutyrate (PDBu; 1 nM).
Immunocytochemistry was as follows: cultures were incubated with
ASW (control) or 5-HT (10 ?M) for 1 or 5 min, fixed in 4% paraformal-
dehyde/30% sucrose/PBS for 10 min and then permeabilized with 0.3%
Triton X-100 for 10 min. After quenching free aldehyde groups with 50
temperature, cultures were incubated with Apl II antibody in 10% goat se-
Fluorescence microscopy. Cultures were viewed at 20 or 40? with a
fluorescence excitation. A combination of optical filters and dichroic
emission measurements were centered at 535 nm. Images were acquired
with a cooled CCD camera (Retiga EXi) controlled by IPLab software
(version 3.65; Scanalytics, Fairfax, VA). Sequences of 7–30 images of
neuronal cell bodies taken at planes of focus separated by 1–3 ?m were
deconvolved using one of the haze-removing functions of IPLab
to five sections were analyzed for translocation.
Analysis of fluorescent images of neurons. Because the distribution of
fluorescence on neuronal membranes was less uniform than that in Sf9
cells, a different quantitative technique was used that allowed unbiased
measurement of the entire membrane (see Fig. 5A). For images of cell
bodies acquired with either the confocal microscope or the Nikon Dia-
phot, background fluorescence was first subtracted, and then the fluo-
rescence of concentric rings one pixel in width from the periphery to the
center of the cell body was measured. Translocation resulted in an in-
crease in the perimembrane fluorescence and a concomitant decrease in
translocation of Apl II by 5-HT is shown in Figure 5A. As seen in the
figure, application of 5-HT results in an intersection of the fluorescence
plot in the presence of 5-HT with the plots before 5-HT and after wash-
out. We took the total fluorescence between the edge of the cell and the
point of intersection as representing membrane-associated Apl II, and
the plots coincided, as representing cytoplasmic Apl II (see Fig. 5A). We
then took the ratio between the membrane fluorescence and the cyto-
plasmic fluorescence as a measure of the distribution of the fluorescent
PKC between the membrane and the cytoplasm. Translocation is ex-
pressed as the change in this ratio with the various treatments (see Figs.
5C, 6B). All measurements on the same neuron were done in exactly the
brane and the cytoplasm under different experimental conditions. Be-
cause it is impossible to delineate exactly the boundary between the
membrane and the cytoplasm, all of the measured changes are
Alto, CA) and glass micropipettes (tip resistance, 10–20 M?) filled with
0.5 M potassium chloride and 2 M potassium acetate were used for intra-
8848 • J.Neurosci.,August23,2006 • 26(34):8847–8856Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticity
cellular recordings. All recordings were done in ASW (in mM: 460 NaCl,
10 KCl, 11 CaCl2, 55 MgCl2, and 10 HEPES, pH 7.5). The membrane
potential of sensory neurons or of isolated motor neurons was held at
?55 mV. A train (10 Hz, 1–10 s) of short depolarizing pulses (5 ms) was
delivered through the intracellular electrode to trigger action potentials
where indicated. In cocultures, the motor neuron was impaled first, and
its membrane potential was maintained at ?80 mV. A supramaximal
concentration of 5-HT (creatinine sulfate; Sigma) was delivered as a
bolus of 100 ?l (100 ?M) or 20 ?l (1 mM) in
ASW by a hand-held pipette directly to the
bath, ?5 mm from the cells.
In the experiments on intermediate-term fa-
(10 ?M) was added to the bath for a total of 5
four trains of action potentials (10 Hz for 2 s)
were triggered in the sensory neuron at 1 min
intervals. The 5-HT was then washed out, and
the EPSP was tested after 30 and 60 min. EPSP
amplitude is expressed relative to the test EPSP.
All experiments were performed at room
SF9 cells were cotransfected with pNEX3-
eGFP-Apl I and pNEX3-mRFP-Apl II
a permeable analog of diacylglycerol
diate concentrations of DOG (2.5–25 ?g/
ml), mRFP-Apl II translocated, but eGFP-
Apl I did not (Fig. 1). This was not a result
of the different fluorescent tags, because
eGFP-Apl II translocated at similar con-
centrations of DOG (data not shown).
There was also no competitive effect, be-
cause eGFP-Apl I did not translocate at
these concentrations of DOG when trans-
fected alone (data not shown). This was
surprising, because kinase activity mea-
that Apl I was more easily activated than
Apl II by low concentrations of DOG
(Pepio et al., 1998; Manseau et al., 2001).
At high concentrations of DOG (50 ?g/
in the presence of calcium (Pepio et al.,
1998), we asked whether calcium could
enhance translocation of Apl I. High con-
centrations of ionomycin (1 ?M), a cal-
cium ionophore, were sufficient to trans-
locate eGFP-Apl I to membranes but did
not translocate mRFP-Apl II (Fig. 2A).
The failure of Apl II to translocate in re-
sponse to increases in calcium concentra-
tion is expected, because the C2 domain of Apl II does not bind
lipids in a calcium-dependent manner (Pepio et al., 1998). At
lower concentrations of ionomycin (0.5 ?M), translocation of
eGFP-Apl I was small and transient (Fig. 2B). However, if this
of DOG (5 ?g/ml), eGFP-Apl I translocated robustly (Fig. 2C).
cence images of Sf9 cells coexpressing eGFP-PKC Apl I (squares) and mRFP-PKC Apl II (diamonds) at different points of the
Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticityJ.Neurosci.,August23,2006 • 26(34):8847–8856 • 8849
Thus, Apl I translocated only when DOG
was paired with calcium influx, whereas
DOG alone was sufficient to translocate
PKC Apl II.
strate translocation of Apl II from the sol-
uble to the membrane fraction of homog-
enates of Aplysia ganglia after exposure to
the facilitatory transmitter 5-HT (Sossin
and Schwartz, 1992; Sossin et al., 1994).
However, in subsequent physiological ex-
periments, overexpression of a dominant-
negative version of Apl II in Aplysia sen-
sory neurons blocked the facilitation of
tic depression (Manseau et al., 2001), sug-
gesting that Apl II is activated by 5-HT in
sensory neurons. Biochemical experi-
ments lack the sensitivity to detect re-
stricted translocation in specific cells or
specific compartments. We therefore ex-
amined the translocation of Apl II using
immunocytochemistry of sensorimotor
cocultures either in the presence or in the
absence of 5-HT. Our results indicated
that 5-HT induced translocation of Apl II
to the plasma membrane in sensory neu-
rons but not in motor neurons (Fig. 3).
Immunocytochemical experiments do
not allow before and after comparison of
individual cells, and there was consider-
able variability in the amount of Apl II
membrane staining. Therefore, to more
quantitatively characterize the transloca-
tion of Apl II by 5-HT in sensory neurons,
we used live imaging of sensory neurons
expressing eGFP-Apl II. Consistent with
the immunocytochemistry results, Apl II visibly translocated to
membranes in sensory neurons (Fig. 4). Translocation of Apl II
by 5-HT did not require the presence of a motor neuron in the
of isolated sensory neurons (Fig. 4A). Moreover, we could also
detect translocation in sensory neuron varicosities adjoining
neurites of motor neurons in cocultures where synaptic connec-
tions were verified with electrophysiological recording (Fig. 4B).
Thus, Apl II appears to undergo translocation by 5-HT at puta-
reversal of synaptic depression (Manseau et al., 2001).
Although detectable translocation was observed in 20 of 24
isolated sensory neurons and in 37 of 42 sensory neurons in co-
cultures, no translocation was seen in experiments with seven
motor neurons in isolation or five motor neurons in cocultures
(Fig. 4C). Application of PDBu, a nonspecific activator of PKCs,
caused translocation of Apl II in seven of seven motor neurons
ing that the differential translocation of Apl II by 5-HT was not
compared with controls (left), but this shift in immunofluorescence is not apparent in motor
8850 • J.Neurosci.,August23,2006 • 26(34):8847–8856Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticity
the result of a deficiency in the ability of eGFP-Apl II to translo-
cate in the motor neuron or of insufficient sensitivity of our
solely to the plasma membrane but also to what appear to be
cytoskeletal compartments (Fig. 4C). This is consistent with
previous biochemical experiments, demonstrating that PDBu
translocates PKC to both actin filaments and microtubules
(Nakhost et al., 1998, 2002; Kabir et al., 2001). In contrast, in
sensory neurons, 5-HT mainly translocated Apl II to the
in the ratio between the fluorescence associated with the mem-
brane and the fluorescence in the cytoplasm (see Materials and
Methods). When the fluorescence intensity is plotted as a func-
tion of distance from the edge of the neuron, translocation ap-
by a decrease in the fluorescence further into the cell (Fig. 5A).
Consistent with the absence of PKC in the nucleus, the fluores-
cence of the nucleus does not change.
Using this method of quantifying translocation, we examined
the time course of translocation of Apl II in response to 5-HT
(Fig. 5B). As seen in the summary plot (Fig. 5C), translocation
could be detected by 30 s, the earliest time
the presence of 5-HT for up to 5 min, the
latest time we examined in this set of ex-
induced again by subsequent application
of 5-HT (data not shown).
Translocation of eGFP-Apl I in Sf9 cells re-
quires both DOG and a rise in intracellular
calcium concentration (Fig. 2). In contrast,
previous biochemical results indicated that
5-HT alone could increase Apl I activity on
membranes in pleural-pedal ganglia (Sossin
and Schwartz, 1992; Sossin et al., 1994).
However, in these dissected ganglia, there
may have been considerable calcium entry
To determine the requirements for Apl
I translocation in sensory neurons, we ex-
amined translocation of eGFP-Apl I (Fig.
6). Although high concentrations of cal-
cium could translocate eGFP-Apl I in Sf9
ron with a microelectrode (data not
shown), which would increase intracellu-
stimulation of the neuron, which also re-
sults in a significant increase in the intra-
cellular calcium concentration (Boyle et
al., 1984), had little effect on the subcellu-
lar localization of Apl I (Fig. 6A,B), indi-
cating that these levels of calcium entry
were not sufficient to translocate the en-
zyme. Likewise, application of 5-HT alone
did not result in translocation. However,
electrical stimulation of the sensory neuron in the presence of
membrane (Fig. 6). In nine experiments, firing of the sensory
neuron in the presence of 5-HT resulted in an increase of 30 ?
8.6% (SEM) in the translocation index (calculated as in Fig. 5A)
compared with 5-HT alone ( p ? 0.01) (Fig. 6B). When stimula-
subsequent train of action potentials in the presence of 5-HT, and
this too reversed when the stimulation ended (data not shown).
Thus, eGFP-Apl I can be translocated by physiological levels of cal-
cium when 5-HT is present, suggesting that activation of Apl I in
sensory neurons could act as a detector of coincident 5-HT and
The same protocol of 5-HT application and electrical stimu-
theless significant, translocation of fluorescent eGFP-Apl I (Fig.
6B). In 10 experiments on motor neurons, the translocation in-
dex increased by 4.2 ? 1.7% ( p ? 0.05) with firing in the pres-
rons ( p ? 0.02). In contrast, application of PDBu to motor
neurons in two experiments resulted in increases of 27 and 31%
Bright-field image of a coculture of a sensory neuron expressing eGFP-Apl II and a motor neuron (MN) from which a synaptic
potential was recorded. Right, Detail of fluorescence images before (PRE) and after application of 5-HT. Fluorescence in the
Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticityJ.Neurosci.,August23,2006 • 26(34):8847–8856 • 8851
in the translocation index ( p ? 0.02 com-
pared with 5-HT), which is comparable to
the translocation induced by concurrent
5-HT and firing in sensory neurons.
Sensorimotor synapses of Aplysia display
several different forms of facilitation,
which can be distinguished by the condi-
tions required for their induction, by their
requirement for transcription or transla-
tion and by their time course (Montarolo
et al., 1986; Ghirardi et al., 1995; Sutton
and Carew, 2000; Sharma et al., 2003). In
contrast to the short-term synaptic facili-
tation that is induced by 5-HT alone, re-
peated stimulation of the sensory neuron
in the presence of 5-HT leads to an ITF
that can last several hours (Bailey et al.,
2000; Sutton and Carew, 2000; Sutton et
al., 2004). The induction of ITF can be
blocked by inhibitors of PKC (Sutton and
Carew, 2000; Sutton et al., 2004), indicat-
ing that PKC activity contributes to this
experiments did not reveal which isoform
presynaptic or the postsynaptic neuron.
Given the requirement for both 5-HT and
II into the sensory neuron was shown to be
synapses by 5-HT alone (Manseau et al.,
be blocked by overexpressing a dominant-
in intact ganglia (Sutton and Carew,
2000). In cultures, a form of ITF depen-
dent on 5-HT and activity has also been
described (Bailey et al., 2000), but using a
different protocol. First, we determined
that we could induce ITF in sensorimotor
cocultures with a protocol similar to the
ganglia. Four trains of high-frequency fir-
ing in the sensory neuron in the presence
of 5-HT resulted in an increase in the
postsynaptic potential that lasted for at
acteristic of ITF (Fig. 7). In contrast, the
STF induced by application of 5-HT alone
decayed by 30 min, and firing the sensory
lead to ITF (Fig. 7).
shells is plotted against distance from the edge of the neuron before (Pre) and during application of 5-HT and after washout
box at the bottom. B, Projections of a sensory neuron expressing eGFP-Apl II before, during, and after application of 5-HT. C,
Summary plot showing ratio of fluorescence intensity of the membrane to that of the cytoplasm in three to seven separate
body (top) and neurite (bottom) of a sensory neuron expressing eGFP-Apl I in control condition (Pre), with stimulation of the
sensory neuron at 10 Hz (Stim), subsequent application of 5-HT, stimulation in the presence of 5-HT (5-HT?Stim), and after
8852 • J.Neurosci.,August23,2006 • 26(34):8847–8856Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticity
Overexpression in the sensory neuron of an altered form of
eGFP-Apl I in which the catalytic lysine is converted to arginine
and the autophosphorylation sites required for correct folding
are converted to glutamic acid (Manseau et al., 2001) completely
blocked the facilitation of the postsynaptic potential at 30 min
and 1 h (Fig. 8) but had no effect on STF caused by 5-HT alone
( p ? 0.4; t test). Overexpression of wild-type Apl I-eGFP had no
effect on the ITF induced by pairing of firing with 5-HT at any
time point (Fig. 8B). Similarly, overexpression of dominant-
negative Apl II in the sensory neurons also did not reduce ITF
(Fig. 9) in contrast to the inhibition by this construct of short-
term reversal of depression caused by 5-HT alone. These experi-
ments thus demonstrate that a functional calcium-dependent
pairing of 5-HT with sensory neuron firing.
The induction of different forms of synaptic plasticity and their
behavioral consequences varies widely depending on the stimu-
lation protocol, neuronal activity, and the time elapsed since the
presentation of the stimulus (Neveu and Zucker, 1996; Nguyen
and Kandel, 1996; Wang and Kelly, 1996; Huang, 1998; Zucker,
2000; Yasuda et al., 2003). Different kinds of plasticity are distin-
persistent enzyme activity, or the synthesis of new RNA or pro-
tein. The sensorimotor synapses that mediate defensive with-
drawal in Aplysia display diverse types of short-term,
intermediate-term, and long-term facilitation, depending on
whether the facilitatory neurotransmitter is presented once or
repeatedly, and whether or not the presence of the transmitter is
plasticity involve the activity of protein kinases, as well as the
actions of other enzymes such as phosphatases and proteases.
At sensorimotor synapses of Aplysia, 5-HT acting through
PKC is involved in two types of synaptic facilitation, STF of de-
pressed synapses, and ITF resulting from concurrent 5-HT and
sensory neuron firing. We now show how each of these forms of
facilitation relies on the respective properties of two isoforms of
PKC. Unlike biochemical assays, these imaging experiments
demonstrate activation of PKCs under physiological conditions
in individual living neurons and in real time.
STF of depressed synapses is mediated by the calcium-
independent PKC Apl II in the sensory neurons (Manseau et al.,
2001). We show by direct visualization of Apl II in living sensory
neurons that its activation requires only the presence of 5-HT,
firing. A, Postsynaptic potentials in motor neurons elicited with single action potentials in
the sensory neuron. Only combined 5-HT and firing resulted in significant facilitation of the
the combination of 5-HT and firing in the sensory neurons gives rise to intermediate-term
Intermediate-term facilitation induced by combined 5-HT and sensory neuron
expressed dominant-negative Apl I ( p ? 0.001 at 30 min, p ? 0.01 at 60 min; two-way
Overexpression of a dominant-negative version of Apl I in the sensory neuron
Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticityJ.Neurosci.,August23,2006 • 26(34):8847–8856 • 8853
and that this activation can be detected in several cellular com-
partments, including presynaptic terminals (Fig. 4). Consistent
with the physiological experiments of Manseau et al. (2001), Apl
II translocates from cytoplasm to membrane in sensory neurons
but not in motor neurons (Fig. 4C).
ITF induced by pairing 5-HT with sensory neuron firing re-
quires PKC activity for its induction and depends on persistent
PKC activity for its maintenance (Sutton and Carew, 2000; Sut-
ton et al., 2004). Our finding that translocation of calcium-
dependent isoform Apl I to the membrane also requires conjoint
5-HT and firing (Fig. 6), suggested that Apl I could be the PKC
that is responsible for this form of facilitation. This hypothesis is
supported by the total block of ITF by dominant-negative Apl I
We find that 5-HT alone translocates only Apl II in sensory neu-
rons, using both immunocytochemical detection of endogenous
Apl II (Fig. 3) and imaging of eGFP-Apl II (Fig. 4). The fluores-
vitro (Manseau et al., 2001). Also, the level of overexpression is
not extremely high, because expressing cells show less than two-
Although contributions from background staining and/or non-
linearities in immunofluorescence are not known, we are likely
looking at translocation under close to physiological conditions.
Moreover, these results are consistent with the finding that
dominant-negative Apl II, but not Apl I, blocked reversal of syn-
aptic depression by 5-HT (Manseau et al., 2001).
In Sf9 cells, low concentrations of DOG translocated Apl II,
but not Apl I (Fig. 1). Thus, a likely explanation for the selective
phospholipase C, producing levels of DAG sufficient to translo-
cate Apl II. However, phospholipase C activity should also pro-
duce IP-3 that releases calcium from internal stores, and 5-HT
does not increase resting calcium levels in sensory neurons,
it is possible that 5-HT activates phospholipase D, producing
phosphatidic acid, which is then converted to DAG (Becker and
Hannun, 2005); in this case, IP3is not produced. The failure of
Apl II to translocate in motor neurons is most simply explained
by the lack of a 5-HT GPCR coupled to DAG production.
iments that failed to detect Apl II activation after short applica-
tions of 5-HT (Sossin and Schwartz, 1992; Sossin et al., 1994).
The most likely explanation for this is the limited sensitivity of
subset of neurons.
The better translocation of Apl II by DOG alone compared with
Apl II for DOG, as has been shown for Ca2?-independent PKCs
in vertebrates (Giorgione et al., 2006). Although the C1 domains
of Apl I and Apl II bind phorbol esters to a similar extent (Pepio
et al., 1998), this does not necessarily predict affinities for DOG
(Slater et al., 1996). Alternatively, because the availability of the
C1 domain to DAG is restricted by the C2 domain (Pepio and
Sossin, 1998; Medkova and Cho, 1999; Slater et al., 2002), cal-
I, whereas cofactors that allow access are either not required for
Apl II, or are already present in cells.
of purified Aplysia PKCs (Pepio et al., 1998) or PKCs from Aply-
sia nervous system extracts (Sossin and Schwartz, 1992; Sossin et
al., 1993). In these assays, Apl I did not require calcium for acti-
vation and indeed, its kinase activity was increased at similar or
lower amounts of DOG than for Apl II, even in the absence of
calcium (Pepio et al., 1998). This discrepancy could reflect non-
physiological aspects of the kinase assay, where the lipids are
vivo translocation is required for activity. In support of this, the
physiological results using dominant-negative kinases match the
in vivo translocation results, not the in vitro kinase assays.
uli for their induction. Long-term potentiation and long-term
glutamate to NMDA receptors at CA3-to-CA1 synapses in the
ment for coincidence of signals limits the induction of the plas-
ticity to a subset of neurons or synapses, thus providing for re-
sponse specificity. In most cases, induction requires an influx of
calcium, either from the outside through plasma membrane
channels, or by the release of calcium from intracellular sources
B, Summary data for cultures expressing dominant-negative Apl II (DN) and uninjected con-
Overexpression of a dominant-negative version of Apl II in the sensory neuron
8854 • J.Neurosci.,August23,2006 • 26(34):8847–8856Zhaoetal.•RolesofPKCIsoformsinSynapticPlasticity
(Zucker, 1999). Increased calcium then activates enzymatic pro-
cesses that bring about changes in synaptic transmission.
The form of ITF that we examine here requires that the pres-
ence of the modulatory transmitter 5-HT coincide with firing in
the sensory neuron. This facilitation depends on the generation
of a constitutively active protein kinase, termed PKM, that is
derived from proteolysis of PKC by the calcium-activated pro-
tease calpain (Sutton et al., 2004). Firing in the sensory neuron
provides the increase in calcium that activates calpain.
We now show that increased calcium is needed for an addi-
tional step in induction: activation of calcium-dependent Apl I.
both are required. The complete block of ITF by dominant-
negative Apl I (Fig. 8) demonstrates that Apl I is necessary for
induction. Calcium thus plays a dual role: it is necessary for the
initial activation of Apl I, and it is also needed for the generation
of the persistently active PKM by calpain. The coincidence re-
quirement is probably a result of the synergy between DAG and
calcium required for translocation of Apl I, as seen in Sf9 cells
(Fig. 2). It should be noted, however, that coupling of 5-HT and
levels of calcium may also be required for activation of Apl I.
Our experiments indicate that Apl I is apparently the PKC
isoform that is cleaved by calpain to yield the persistently active
PKM. Sutton et al. (2004) showed that both Apl I and Apl II are
cleaved by calpain in vitro but were unable to detect proteolytic
the sensitivity of their assay. The failure of dominant-negative
Apl II to reduce ITF (Fig. 9) rules out the possibility that Apl II
plays a critical role. The fact that functional Apl II and Apl I are
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