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Neuroscience 268 (2014) 75–86


C. XU, * Q.-Y. LIU AND D. L. ALKON to be induced by bryostatin. Ó 2014 IBRO. Published by
Blanchette Rockefeller Neurosciences Institute, Morgantown, Elsevier Ltd. All rights reserved.
WV 26506, United States of America

Abstract—Bryostatin-1, a potent agonist of protein kinase C Key words: bryostatin, GABA, PKCa, PKCe, synaptic plasi-
(PKC), has recently been found to enhance spatial learning ticity.
and long-term memory in rats, mice, rabbits and the nudi-
branch Hermissenda, and to exert profound neuroprotective
effects on Alzheimer’s disease (AD) in transgenic mice.
However, details of the mechanistic effects of bryostatin INTRODUCTION
on learning and memory remain unclear. To address this Bryostatin, a potent agonist of protein kinase C (PKC),
issue, whole-cell recording, a dual-recording approach and
particularly a & e isozymes, has recently been found to
extracellular recording techniques were performed on
enhance spatial learning and long-term memory in rats,
young (2–4 months) Brown-Norway rats. We found that
bath-applied bryostatin-1 significantly increased the fre- mice, rabbits and the nudibranch Hermissenda
quency and amplitude of spontaneous inhibitory postsynap- (Kazanietz et al., 1994; Sun and Alkon, 2005; Kuzirian
tic currents (sIPSCs). The firing rate of GABAergic et al., 2006; Sun et al., 2008; Wang et al., 2008;
interneurons significantly was also increased as recorded Hongpaisan et al., 2013). Bryostatin is also found to
with a loosely-attached extracellular recording configura- increase the levels of synaptic proteins spinophilin and
tion. Simultaneous recordings from communicating cell synaptophysin and cause structural changes in
pairs of interneuron and pyramidal neuron revealed unique synapses (Hongpaisan and Alkon, 2007). Furthermore,
activity-dependent properties of GABAergic synapses. Fur- bryostatin exerts profound neruoprotective effects on AD
thermore, the bryostatin-induced increase of the frequency
transgenic mice (Etcheberrigaray et al., 2004). It has
and amplitude of IPSCs was blocked by methionine enkeph-
been known that an inhibition or impairment of PKC
alin which selectively suppressed the excitability of inter-
neurons. Pretreatment with RO-32-0432, a relatively activity leads to learning and memory disorders
specific PKCa antagonist, blocked the effect of bryostatin (Takashima et al., 1991), therefore, an appropriate
on sIPSCs. Finally, bryostatin increased paired-pulse ratio activation of PKC isozymes such as PKCa or PKCe
of GABAergic synapses that lasted for at least 20 min while results in the restoration of learning and memory (Sun
pretreatment with RO-32-0432 significantly reduced the and Alkon, 2010).
ratio. In addition, 8-[2-(2-pentyl-cyclopropylmethl)-cyclopro- Bryostatin-1, enhances spatial learning and memory
pyl]-octanoic acid (DCP-LA), a selective PKCe activator, also by synaptic or structural remodeling and synaptogenesis
increased the frequency and amplitude of sIPSCs. Taken in the hippocampus and related cortical areas
together, these results suggest that bryostatin enhances
(Hongpaisan and Alkon, 2007). DHA-CP6, a novel
GABAergic neurotransmission in pyramidal neurons by
PKCe activator, reduced b-amyloid level by increasing
activating the PKCa & e-dependent pathway and by a pre-
synaptic mechanism with excitation of GABAergic interneu- b-amyloid degradation through endothelin-converting
rons. These effects of bryostatin on GABAergic enzyme (ECE) (Nelson et al., 2009). PKC activation has
transmissions and modifiability may contribute to the been shown to reduce apoptotic neuronal cell death
improvement of learning and memory previously observed secondary to oxidative stress (Maher, 2001).
Additionally, PKC activation enhances Ca2+ currents
*Corresponding author. Tel:+1-240-477-3662. and elevates cytosolic-free Ca2+, resulting in
E-mail address: (C. Xu). neurotransmitter release (Swartz et al., 1993; Hussain
Abbreviations: Ach, acetylcholine; aCSF, artificial cerebrospinal fluid; and Carpenter, 2003), and potassium channel (Hoffman
AD, Alzheimer’s disease; AP5, DL-2-amino-5-phosphonovaleric acid; and Johnston, 1998) and sodium channel inhibition
DCP-LA, 8-[2-(2-pentyl-cyclopropylmethl)-cyclopropyl]-octanoic acid;
DNQX, 6,7-dinitroquinoxaline-2,3-dione; ECE, endothelin-converting
(Chen et al., 2005). 8-[2-(2-pentyl-cyclopropylmethl)-
enzyme; EGTA, ethylene glycol tetraacetic acid; HEPES, 2-[4-(2- cyclopropyl]-octanoic acid (DCP-LA), another compound
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; K-S test, Kolmogorov– that selectively and directly activates PKCe, enhances
Smirnov test; LTP, long-term potentiation; PKC, protein kinase C; PPD, the response of presynaptic a7 acetylcholine (Ach)
paired-pulse depression; PPF, paired-pulse facilitation; sIPSCs,
spontaneous inhibitory postsynaptic currents; uIPSCs, unitary receptors that are involved in glutamate and GABA
inhibitory postsynaptic currents. release (Yamamoto et al., 2005; Kanno et al., 2005).
0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

76 C. Xu et al. / Neuroscience 268 (2014) 75–86

DCP-LA or bryostatin-1 also enhances a transient (Bethesda, MD). After sedation with isoflurane, the rat
potentiation and/or long-term potentiation (LTP) in CA1 was decapitated and its brain bisected sagittally and
region of rat hippocampal slices (Yamamoto et al., removed, placed into ice-cold sucrose buffer containing
2005; Kim et al., 2012). In this study, we explored the the following (in mM): 254 sucrose,10 D-glucose, 26
effect of bryostatin on GABAergic neurotransmission in NaHCO3, 2 CaCl2, 2 MgSO4, 3 KCl, and 1.25 NaH2PO4,
rat hippocampal CA1 pyramidal neurons and saturated with 95% O2/5% CO2, at pH 7.4. Transverse
interneurons by using electrophysiological recordings hippocampal slices (250–300 lM thick) were cut with a
from hippocampal slices. These experiments were VT 1000S microtome (Leica, Deerfield, IL). Slices were
designed to address the following questions: (1) Does transferred immediately into a holding chamber and
bryostatin directly enhance or reduce GABAergic incubated at 32–33 °C for a 30-min recovery period in a
neurotransmission in rat hippocampal CA1 pyramidal mixture of 50% sucrose saline and 50% artificial
neurons? (2) Is the effect of bryostatin on GABAergic cerebrospinal fluid (aCSF) containing the following (in
neurotransmission through a presynaptic or postsynaptic mM): 128 NaCl, 10 D-glucose, 26 NaHCO3, 2 CaCl2, 2
mechanism? (3) Do interneurons have direct contact on MgSO4, 3 KCl and 1.25 NaH2PO4, slices were then
pyramidal neurons in the hippocampal CA1 sector? (4) placed on a nylon mesh, submerged in normal aCSF
Which PKC isoforms are involved in PKC activation in bubbled with 95% O2/5% CO2 continuously, and
GABAergic neurotransmission? (5) How does bryostatin maintained at room temperature (21–24 °C) until
affect short-term and long-term plasticity of GABAergic whole cell patch clamp recording (30 min to 5 h).
Electrophysiological recordings
Slices were transferred to a submersion-type recording
Spatial water maze tasks chamber (Warner Instruments, Hamden, CT) on a
Effects of bryostatin-1 on spatial memory were evaluated Burleigh Gibraltar fixed stage system (Burleigh
in rats in vivo with the Morris water maze task. Male adult Instruments, Fisher, NY), secured beneath a nylon harp,
Brown Norway rats (2–3 months old; 260–300 g; from and perfused with aCSF heated to 30–33 °C at a rate of
National Institute on Aging, Bethesda) were housed in 2–3 ml per min. CA1 pyramidal cells and interneurons
temperature-controlled (20–24 °C) room for a week, (oriens-alveus inteneurons of the hippocampus; see
allowed free access to food and water, and kept on a Fig. 9) were identified visually by using an Axioskop
12-h light/dark cycle. All rats were randomly assigned to 2FS microscope (Zeiss) equipped with a 40X water-
different groups (10 each except for bryostatin + immersion objective coupled with an infrared differential
RO-32-0432 group 7 rats) and swam for 2 min in a interference contrast camera system. Whole-cell patch-
1.5-m (diameter)  0.6-m (depth) pool, filled with water clamp recordings were established using a dual-
to a depth of 40 cm (24 ± 1 °C). On the following day, headstage MultiClamp 700A amplifier (Axon
rats were trained in a three-trails-per-day task for Instruments, Union City, CA). Membrane current and
consecutive days. Each training trial lasted for up to potential signals were digitized and analyzed with
2 min, during which rats learned to escape from the Digidata 1322A and pClamp 8.2 systems (Molecular
water by finding a hidden platform that was placed at a Devices). Patch pipettes of 5 MO were pulled with a
fixed location and submerged 1–2 cm below the water Narishige PP-830 puller (Narishige, Greencale, NY).
surface. The navigation of the rats was viewed on-line The pipette solution had the following composition (in
by the investigators (Video Monitor BWM9, Javelin mM) unless otherwise stated: 140 KCl, 0.1 CaCl2, 5
Electronics), who were obscured from the rats’ view and EGTA, 10 HEPES, 4 ATP-Mg2+, 0.4 GTP-2Na+, 1
tracked by a video-camera. The escape latency and the QX314 (Lidocaine N-ethyl bromide), pH 7.2 and
route of rats’ swimming across the pool to the platform 290 mOsm. The diffusion potential (liquid junction
were recorded with a video-tracking system (poly-Track potential) was 4 mV calculated by Clampex software.
Video Tracking System, San Diego Instruments) for a Under these conditions, synaptic currents were acquired
quantitative analysis. A probe test was used to evaluate at a holding potential of 70 mV, the high
retention of the learned navigation experience. The concentrations of chloride in the pipette caused the
probe test (1 min) was performed after removing the inhibitory postsynaptic current (IPSC) to appear as an
platform, 24 h after the last training trial, by monitoring inward current. QX314 was added to the pipette solution
the distance swum by each rat in the quadrants with the to block the GABAB-mediated currents and to prevent
same video-tracking system. At 60 min before water the generation of Na+-dependent action potential.
maze training on days 1, 3, and 5, tail-vein injection was Spontaneous excitatory amino acid currents (sEPSCs)
used to administer RO-32-0432 (500 lg/kg body weight) were excluded from recordings by adding glutamate
or vehicle. After 30 min, bryostatin-1 or vehicle was receptor antagonists DNQX (6,7-dinitroquinoxaline-2,3-
administered i.p. at 5 lg/kg body weight (60 lg/m2 dione, 20 lM) and AP5 (DL-2-amino-5-
body surface). phosphonovaleric acid, 20 lM); therefore all of the
recorded inward currents were spontaneous IPSCs
(sIPSCs). The method of recording inhibitory synaptic
Hippocampal slices
currents was set up according to Rodriguez-Moreno’s
Young (2–3 months of age) male Brown Norway (BN) rats method and with some adjustment (Rodriguez-Moreno
were obtained from the National Institute on Aging et al., 2000).
C. Xu et al. / Neuroscience 268 (2014) 75–86 77

Paired-pulse stimulation and dual whole cell patch- by changes in noise level or by membrane fluctuations.
clamp recordings If the background noise increased during the recording,
the data from that cell were discarded. The data
A paired-pulse stimulation protocol was used to evaluate
generated from these measurements were used to plot
short-term GABAergic synaptic plasticity in hippocampal
cumulative probability amplitude and interevent interval
slices (more than five animals each group). The pipette
graphs, with each distribution normalized to a maximal
solution contained the following (in mM): 90 K+
value of 1. Cumulative probability plots obtained under
gluconate, 45 KCl, 1.7 NaCl, 0.1 CaCl2, 2.7 MgCl2, 10
different experimental conditions were compared using
HEPES, 1.1 EGTA, 5 phosphocreatine-Na+, 3.5 ATP-
the nonparametric Kolmogorov–Smirnov (K-S test),
K+, 0.3 GTP-Na+, was titrated at pH 7.2 and
which estimates the probability that two cumulative
290 mOsm. GABAergic, inhibitory postsynaptic currents
distributions differ from each other by chance alone (Xu
(IPSCs) are evoked using a concentric bipolar
et al., 2009). The significance level for the K-S test was
stimulation electrode placed in the stratum pyramidale
set at a value of p < 0.05. All numerical values are
about 80 lm from the recording cell, which was kept at
expressed as a mean ± standard error of the mean
a holding potential of 70 mV. DNQX and AP-5 were
(SEM) and statistic analyses were performed by using a
included in the perfusion solution to block glutamatergic
paired Student’s t-test or the analysis of variance (one-
currents. Only one cell was recorded from each brain
way analysis of variance [ANOVA]), whenever
slice. Dual whole-cell patch-clamp recordings were
appropriate. The differences are considered significant
obtained simultaneously from two hippocampal neurons
at p < 0.05.
(most pairs of a pyramidal cell and an interneuron) in
the hippocampal CA1-isolated slices under visual
AP5, DNQX and Bicuculline were purchased from Tocris
Extracellular cell-attached recording
Cookson Inc, Ellisville, MO. TTX, QX314, Staurosporine
Cell-attached recordings were made on interneurons aglycone and RO-32-0432 (Bisindolylmaleimide XI
using a dual-headstage MultiClamp 700A amplifier and hydrochloride) were purchased from Sigma. Bryostatin-1
pClamp 8.2 software. The recording solution included was purchased from BioMol international, LP.
DNQX and AP-5. Cell-attached electrodes were pulled Methionine enkephalin acetate salt was purchased from
from the walled borosilicate glass capillaries, were filled Bachem Bioscience Inc., PA. Staurosporine aglycone
with 150 mM NaCl, and had resistance of 3–7 MX. and DNQX were dissolved in DMSO. All stock solutions
While approaching the cell, positive pressure was were stored as frozen aliquots and diluted to test
applied to the patch electrode. The seal (100–300 MX) concentration in aCSF by a factor of 1000-fold. Drugs
between the recording pipette and the cell membrane were administered by bath application.
was obtained by applying suction to the electrode.
Action potential currents were recorded in voltage-clamp
mode (Perkins, 2006). RESULTS
Bryostatin enhanced Brown Norway rat water maze
Acquisition and analysis of synaptic currents spatial learning and memory
GABAA-mediated inward currents were recorded with Effects of bryostatin-1 on spatial learning and memory
whole-cell electrodes containing high concentrations were previously evaluated in Male adult Wistar rats,
of chloride and 1 mM QX314 using the continuous using a hidden-platform water maze and it was found
single-electrode voltage-clamp mode. Access that bryostatin enhanced rat water maze spatial learning
resistance (<25 MO) was regularly monitored during and memory (Sun and Alkon, 2005). Here we want to
recordings, and cells were rejected if resistance explore the effect of bryostatin-1 on Brown Norway rats.
changed >15% during the experiment. If the access As shown in Fig. 1A, the latency of escape to the
resistance increased during the course of the hidden platform in the three groups of rats decreased
experiment and caused significant reductions in the gradually during the training sessions, indicating that all
synaptic current amplitudes, efforts were made to rats were able to learn the task through training and the
improve access (such as applying additional suction or learning was progressive. However, there was a
slight positive pressure); if this failed, the experiment significant group difference in escape latency
was discontinued. (F(2,14) = 12.86, p < 0.001), indicating difference in
Spontaneously occurring synaptic currents were learning between the treatments. The difference
filtered at 2 kHz, and digitized at 10 kHz using Digidata between rats that received bryostatin-1 or vehicle was
1322A. Synaptic currents were collected at 60 s for significant (F(1,9) = 16.16, p < 0.05), indicating that
each experimental condition. Off-line analysis of spatial learning in rats that were injected with bryostatin-
synaptic currents was performed using the Minianalysis 1 was faster than that of the rats injected with vehicle.
software (Version 6.0; Synaptosoft, Decatur, GA). The difference between rats that received bryostatin-1
Synaptic currents were screened automatically using an or co-administration of bryostatin-1 and RO-32-0432
amplitude threshold of 3 pA. Events were then visually was significant (F(1,9) = 9.15, p < 0.05), indicating
screened to ensure that the analysis was not distorted RO-32-0432, a more selective PKCa inhibitor, blocked
78 C. Xu et al. / Neuroscience 268 (2014) 75–86

A 100
Bryostatin significantly increased the frequency and
amplitude of sIPSCs but not mIPSCs in CA1
pyramidal neurons
80 Bryostatin-1
Escape latency (s)

To explore the effect of bryostatin on spontaneous

Bryostatin-1 GABAA receptor-mediated inhibitory postsynaptic
+ RO-32-0432 currents (sIPSCs) and miniature GABAA receptor-
mediated currents (mIPSCs), we performed patch-clamp
recordings of CA1 pyramidal neurons in the presence of
DNQX (20 mM) and AP5 (20 mM). sIPSCs and mIPSCs
20 were confirmed by the application of GABAA receptor
antagonist bicuculline. As shown in Fig. 2A, C, the
mean frequency and amplitude of sIPSCs were
1 2 3 4 5 1.77 ± 0.29 Hz and 39.11 ± 2.46 pA at the control
Training days condition. The mean frequency and amplitude of
sIPSCs were 2.76 ± 0.41 Hz and 48.43 ± 4.05 pA at
2 min following the bath application of 10 nM bryostatin.
B 60
# Bryostatin significantly increased the mean frequency
** and amplitude of sIPSCs (⁄⁄p < 0.01 or ⁄p < 0.05;
50 Bryostatin-1 Paired Student’s t-test, n = 12). The representative
Swimming distance in

traces of sIPSCs are shown in Fig. 2A. The cumulative

quadrant (% of total)

40 + RO-32-0432 # frequency and amplitude histograms for representative
cells (Fig. 2B) demonstrated a significant increase in
frequency and amplitude of sIPSCs with the bath
application of bryostatin (KS-T, p < 0.0001). However,
as shown in Fig. 2D, E, the mean mIPSC frequency and
amplitude in the control condition were 1.67 ± 0.38 Hz
and 26.65 ± 3.94 pA (n = 7); the mean mIPSC
frequency and amplitude in the presence of bryostatin
(10 nM) were 1.58 ± 0.37 Hz and 24.92 ± 5.06 pA
0 Opp (n = 7). There was no significant difference in the
Aj-R Aj-R Target
frequency and amplitude of mIPSCs between the control
Quadrant group and bath application bryostatin group (P > 0.05;
Fig. 1. Effects of bryostatin-1 on rat performance in the hidden
Paired Student’s t-test, n = 7). Therefore, bryostatin
platform water maze task. (A) Escape latency (means ± SEM) in significantly increased the frequency and amplitude of
water maze training for 5 days in control. Bryostatin (n = 10) and sIPSCs but not mIPSCs in CA1 pyramidal neurons.
bryostatin+RO-32-0432 group (n = 7). (B) Results of the quadrant
preference test, conducted at the next day after the end of training.
(⁄⁄p < 0.01 compared with the control group (n = 10); #p < 0.05 Simultaneous recordings from communicating cell
compared with other quadrants). Opp, opposite to target quadrant;
Aj-r, adjacent right quadrant; Aj-L, adjacent left quadrant.
pairs of interneuron and pyramidal neuron revealed
unique activity-dependent properties of GABAergic
Bryostatin significantly increased the inhibitory
the enhancement of learning induced by bryostatin-1. As postsynaptic currents, so we tested the effect of
shown in Fig. 1B, quadrant tests 24 h after the last training bryostatin on interneurons in hippocampal CA1 sector
trial showed that all three groups showed a target using a loosely-attached extracellular recording
quadrant preference (Control group: F(3,39) = 3.93, configuration. Under these recording conditions,
P < 0.05; Bryostatin-1 group: F(3,39) = 47.85, bryostatin significantly increased the firing rate of
P < 0.001; Bryostatin plus RO-32-0432 group: interneurons. As shown in Fig. 3A, B, the mean
F(3,27) = 11.45, P < 0.001). The difference in the frequency of firing rate of interneurons was 2.1 ± 0.5 Hz
target quadrant preference between groups was in control. When 10 nM bryostatin was bath-applied at
significant (F(3,11) = 9.91, P < 0.01), indicating that 2 min, the mean frequency of firing rate of interneurons
the bryostatin-1 rats spent significantly more time than increased to 3.75 ± 0.84 Hz (⁄p < 0.05 comparing with
the control group or bryostatin plus RO-32-0432 group the control group).
(Student’s t-test, p < 0.001). There is no significant To confirm the synaptic communications between the
difference between the control group and bryostatin plus interneurons and the pyramidal cells in the hippocampal
RO-32-0432 group, which indicates that RO-32-0432 CA1 regions, paired whole-cell recordings were
blocked the memory retention induced by bryostatin-1. performed to detect unitary GABAAergic synaptic
Thus, bryostatin-1 enhanced spatial learning and transmission between the interneurons and pyramidal
memory retention task in Brown Norway rats, indicating cells. As shown in Fig. 4A, the unitary inhibitory
that PKCa or e activation can enhance both learning postsynaptic currents (uIPSCs, lower trace) were
and memory retention. evoked in the postsynaptic pyramidal cells by the
C. Xu et al. / Neuroscience 268 (2014) 75–86 79

spiking of the interneuron (upper trace) in normal resting

conditions. Fig. 4B shows that the interneuron spikes A Control Bryostatin-1(10nM)
were induced by a 10 s depolarization stimulation which
in turn, evoked postsynaptic inhibitory currents in
pyramidal cells. Fig. 4A, B show examples of
synchronized discharges of communication neuron

200 pA
couples. Although there were a few asynchronous
uIPSCs in the pyramidal cell (indicated by X), most of
them are synchronous. These results suggest that 1s
spiking activities in interneurons synchronously evoke

Cumulatve probability

Cumulatve probability
unitary inhibitory postsynaptic currents in the pyramidal 1.0
cells. 0.8 0.8
0.6 Control
0.6 Control
Inhibition of interneurons’ excitation blocked 0.4 0.4 Bryostatin
bryostatin’s effects on sIPSCs 0.2 0.2

To confirm that the enhancement of sIPSCs by bryostatin 0.0 0.0

0 2000 4000 0 100 200 300 400
is through the interneurons’ excitation, we firstly tested Interevent interval (ms) Amplitude (pA)
the effect of bryostatin on sIPSCs after bath-application
of 1 lM Enkephalin, which is a selective l- and
d-receptor agonist and has been shown to selectively C 4
n = 12
n = 12 *

Amplitude (pA)
Frequency (Hz)
suppress the excitability of interneurons (Nakamura
et al., 2007). Secondly, we tested whether the 40
enhancement of sIPSCs induced by bryostatin could be 2
blocked by Enkephalin. 20
As shown in Fig. 5A, B, Enkephalin decreased not
0 0
only the frequency of sIPSCs but also the amplitudes of Bryostatin
Control Bryostatin Control
sIPSCs. In the control condition, the mean frequency
and amplitude of sIPSCs were 2.43 ± 0.88 Hz and
44.39 ± 7.97 pA, whereas the mean sIPSC frequency D TTX TTX + Bryostatin
and amplitude with the presence of Enkephalin were
0.72 ± 0.18 Hz and 34.81 ± 5.91 pA. Enkephalin
significantly decreased the frequency and amplitude of
50 pA

sIPSCs following the bath application of Enkephalin for

5 min (n = 7, p < 0.05, student’s paired t-test).
However, Enkephalin plus bryostatin had no further
effect on the sIPSC frequency (0.65 ± 0.14 Hz, E 3 40
P > 0.05, n = 7) and amplitude (32.3 ± 5.35 pA, n = 7; p > 0.05 n = 7; p > 0.05
Frequency (Hz)

Amplitude (pA)

p > 0.05, n = 7). These results suggested that

inhibiting the interneurons’ excitation occluded
bryostatin’s effect on sIPSCs. As shown in Fig. 5C, D,
the mean frequency and amplitude of sIPSCs were
1.58 ± 0.56 Hz and 35.51 ± 3.06 pA at the control
0 0
condition. The mean frequency and amplitude of TTX +
sIPSCs were 2.51 ± 0.59 Hz and 42.76 ± 4.43 pA with Bryostatin
the presence of bryostatin. Bryostatin significantly
increased the frequency and amplitude of sIPSCs Fig. 2. Bryostatin significantly increased the frequency and ampli-
(p < 0.05, n = 5). When bryostatin was applied tude of sIPSCs but not mIPSCs in CA1 pyramidal neurons. (A)
together with Enkephalin, the mean sIPSC frequency Representative traces show that 10 nM bryostatin-1, a PKC activator,
increased the frequency and amplitude of sIPSCs at 2 min following
and amplitude were 0.45 ± 0.17 Hz and the bath application of bryostatin. (B) Cumulative frequency and
32.98 ± 7.14 pA. Thus, Enkephalin significantly blocked amplitude distributions of sIPSCs based on data shown in (A). Both
the increase of the frequency and amplitude induced by frequency and amplitude distributions were statistically different
bryostatin (p < 0.05, n = 5). These data suggest that under these two experimental conditions (Before vs. after treatment
of bryostatin; p < 0.0001 using K-S test; 100 events analyzed for
the enhancement of sIPSCs by bryostatin is through the
before and 185 events for after treatment of bryostatin). (C) A
interneurons’ excitation. summary of data collected from 12 cells in the absence or presence
of 10 nM bryostatin, showing that bryostatin significantly increased
the frequency and amplitude of sIPSCs. (A paired Student’s t-test,
Bryostatin enhanced GABAergic neurotransmission ⁄⁄
p < 0.001; ⁄p < 0.05; n = 12). (D) Representative traces of
in pyramidal neurons by activating the PKCa mIPSCs obtained before (left) and after the application of 10 nM
pathway bryostatin (right). (E) Bar graphs show the mean frequency or
amplitude of mIPSCs before and after the application of 10 nM
Bryostatin-1, a potent PKC agonist, particularly activates bryostatin. There was no significant difference in the frequency and
PKCa and PKCe (Kazanietz et al., 1994). We want to amplitude of mIPSCs (p > 0.05, Paired Student’s t-test; n = 7).
80 C. Xu et al. / Neuroscience 268 (2014) 75–86

5 *

4 n=6

Fire rate (Hz)


100 pA
At 2 min after 10 nM Bryostatin-1 3s

Control Bryostatin

Fig. 3. Bryostatin excited interneurons of hippocampus. (A) The representative traces showed bryostatin-1 excited interneurons at 2 min following
10 nM bryostatin treatment. (B) Bar chart summarizes the frequencies of the bryostatin excitation in hippocampal interneurons (A paired Student’s t-
test, ⁄p < 0.05; n = 6).

A PKCa inhibitor (20 nM) (Wilkinson et al., 1993), for the

pretreatment with the slice, then tested the effect of
bryostatin on sIPSCs. As shown in Fig. 6B, while
staurosporine did not affect the basic sIPSC activities,
bryostatin did not induce the enhancement of sIPSCs.
In the control condition, the mean frequency and
amplitude of sIPSCs were 1.28 ± 0.35 Hz and
42.71 ± 6.31 pA, whereas the mean sIPSC frequency
and amplitude in the presence of staurosporine were
1.25 ± 0.32 Hz and 42.89 ± 9.2 pA. There were no
B significant changes in frequency and amplitude of
sIPSCs following the bath application of staurosporine
(n = 5, p > 0.05, student’s paired t-test). When
bryostatin was applied together with staurosporine,
PC mean sIPSC frequency and amplitude were
1.57 ± 0.5 Hz and 39.9 ± 5.45 pA. Thus, there were no
significant changes in frequency and amplitude of
sIPSCs when staurosporine was applied either alone or
combined with bryostatin. Like staurosporine, RO-32-
0432 mimed the effect of staurosporine on sIPSCs. As
shown in Fig. 6A, C, the mean frequency and amplitude
of sIPSCs were 2.50 ± 0.62 Hz and 22.22 ± 1.99 pA in
the control condition, whereas the mean sIPSC
frequency and amplitude in the presence of RO-32-0432
Fig. 4. Direct synaptic connections between pyramidal cells and (20 nM) were 2.46 ± 0.79 Hz and 21.27 ± 1.89 pA.
interneurons were recorded in hippocampal CA1 sector. (A) Unitary
There were no significant changes in frequency and
inhibitory postsynaptic currents (uIPSCs) in pyramidal cell evoked by
the interneurons. The upper trace showed that spontaneous firing amplitude of sIPSCs following the bath application of
spikes in interneuron and lower trace shows that inhibitory currents RO-32-0432 (n = 8, p > 0.05, student’s paired t-test).
were recorded in pyramidal neuron. (B) The traces showed that When bryostatin was applied together with RO-32-0432,
synchronous connection. uIPSCs in the pyramidal cell evoked by the mean sIPSC frequency and amplitude were
interneuron which was given a 10-s depolarization stimulation. The
upper trace showed that spikes evoked by a 10-s depolarization 2.10 ± 0.62 Hz and 19.53 ± 2.38 pA. Thus, there were
stimulation in interneuron and lower trace show that inhibitory no significant changes in frequency and amplitude of
currents were recorded in pyramidal neuron. IN, interneuron; PC, sIPSCs when RO-32-0432 was applied either alone or
pyramidal cell. combined with bryostatin. These data suggest that
bryostatin enhanced GABAergic neurotransmission in
pyramidal neurons by activating the PKCa pathway.
explore which isozyme of PKC was involved in the
enhancement of sIPSCs induced by bryostatin. First, we
Bryostatin can switch paired-pulse depression to
used the pretreatment with staurosporine (1 lM), a
broad spectrum and nonselective protein kinase
inhibitor, for 20 min, then tested the effect of bryostatin To further verify the effect of bryostatin on short-term and
on sIPSCs. Second, we used RO-32-0432, a selectively long-term plasticity of GABAergic synapses in
C. Xu et al. / Neuroscience 268 (2014) 75–86 81

Enkephalin +
Control Enkephalin (1µM) Bryostatin
A RO-32-0432 RO-32-0432 +
Control (20 nM) Bryostatin

150 pA

100 pA
B 4
60 n=7
Amplitude (pA)
Frequency (Hz)

3 *
B 4 60 n = 5; p > 0.05
2 n = 5; p > 0.05
20 50
1 *

Amplitude (pA)
Frequency (Hz)
0 0 40

yo in+

yo in+

ke l
En rol

En tro
En hal

En pha

Br hal
Br hal







C Control Bryostatin
Enkephalin 10

0 0

tin e
+ taur ine

Br os e

tin e

sta rin



sta rin


yo po


yo po


Br os
100 pA



+ taur




C 4 30
D 4 n=5 n = 8; p > 0.05 n = 8; p > 0.05
50 n=5 *
Amplitude (pA)
Frequency (Hz)

3 40 3
Amplitude (pA)
Frequency (Hz)

30 20
20 2
1 # 10
0 0 10
ph +

ph +


ke tin

ke tin




En osta

En sta






0 0
sta 32


sta 32


yo -04

Fig. 5. Inhibition of interneurons’ excitation blocked bryostatin’s



yo 04


Br 2

effects on sIPSCs. (A) Representative traces show that 1 lM


Br 2
+ O-3


+ O-3


enkephalin significantly reduced the frequency and amplitude of


sIPSCs. Addition of bryostatin did not induce increase of frequency

and amplitude of sIPSCs. (B) A summary of data collected from
seven pyramidal cells. Enkephalin significantly decreased the fre- Fig. 6. Bryostatin enhanced GABAergic neurotransmission in pyra-
quency and amplitude of sIPSCs (A paired Student’s t-test, midal neurons by activating the PKCa pathway. (A) Representative

p < 0.05; n = 7). Bryostatin had no effect on the frequency and traces show that 20 nM RO-32-0432 had no effect on the frequency
amplitude of sIPSCs. (C) Representative traces show that 10 nM and amplitude of sIPSCs. Addition of bryostatin also had no effect on
bryostatin significantly increased the frequency and amplitude of the frequency and amplitude of sIPSCs, which suggests that RO-32-
sIPSCs. Addition of enkephalin blocked the effect of bryostatin. (D) A 0432 blocked the effect of bryostatin. (B) Bar chart shows that
summary of data collected from five pyramidal cells. Bryostatin staurosporine, a broad-spectrum protein kinase inhibitor, blocked the
significantly increased the frequency and amplitude of sIPSCs (A effect of bryostatin on the frequency and amplitude of sIPSCs (A
paired Student’s t-test, ⁄p < 0.05; n = 7). Enkephalin significantly paired Student’s t-test, p > 0.05; n = 5). (C) Bar chart shows that
blocked the effect of bryostatin (A paired Student’s t-test, #p < 0.05, RO-32-0432, a specific PKCa inhibitor, blocked the effect of bryost-
n = 5). atin on the frequency and amplitude of sIPSCs (A paired Student’s t-
test, p > 0.05, n = 8).
82 C. Xu et al. / Neuroscience 268 (2014) 75–86

A B A Control DCP-LA (100 nM)

25 pA

100 pA
10 ms

1.2 * ** * Bryostatin
Control 1s
** * RO-32 + Bry
1.1 * B
Ratio (p2/p1)

* C **
4 60
n = 14 ** n = 14

Amplitude (pA)
Frequency (Hz)
0.9 40
0.8 20



20 n








Fig. 7. Effect of bryostatin on short-term and long-term GABAergic 0 0

synaptic plasticity in CA1 pyramidal neurons and PKCa pathway. (A)



A paired-pulse depression of IPSCs before 10 nM bryostatin treat-



ment. (B) A paired-pulse facilitation of IPSCs at 5 min following


bryostatin treatment. (C) The ratio of p2/p1 amplitude as a function of

time is shown under control condition (n = 8), in bryostatin treatment
slices (n = 8) and in RO-32-0432 + bryostatin slices (n = 8). Bry- Fig. 8. DCP-LA significantly increased the frequency and amplitude
ostatin significantly increased the ratio of p2/p1 (⁄p < 0.05, of sIPSCs in CA1 pyramidal neurons. (A) Representative traces show
p < 0.01 compared with control group or RO-32-0432 group) and that 100 nM DCP-LA, a PKCe activator, increased the frequency and
RO-32-0432 blocked the increase in the ratio of p2/p1. Points on the amplitude of sIPSCs at 2 min following the bath application of DCP-
plot are mean ± SEM of values obtained from different slices. RO- LA. (B) A summary of data collected from 14 cells in the absence or
32 + Bry group indicates RO-32-0432 plus bryostatin group. presence of 100 nM DCP-LA, showing that DCP-LA significantly
increased the frequency and amplitude of sIPSCs (A paired Student’s
t-test, ⁄⁄p < 0.001; n = 14).
hippocampal CA1 slice. IPSCs induced with paired-pulse
protocol were recorded in hippocampal slices. As shown long-term effect. This further confirmed that bryostatin’s
in Fig. 7, in control condition, second-pulse response effects were through PKCa pathway.
was smaller than the first-pulse response, so PPD
(paired-pulse depression) was induced (n = 8);
PKCe isozyme activity is also involved in GABAergic
however, after the application of bryostatin, the second-
neurotransmission in hippocampal CA1 sector
pulse response was larger than the first-pulse response,
indicating that PPF (paired-pulse facilitation) was To explore if PKCe isozyme is involved in GABAergic
induced. Thus, bryostatin switched PPD to PPF. This neurotransmission in the hippocampus, we tested the
effect continued for 20 min in the presence of bryostatin. effect of DCP-LA, a selective PKCe activator, on
When bryostatin was applied together with RO-32-0432 spontaneous GABAA receptor-mediated inhibitory
(20 nM), the bryostatin switching effect on PPD was postsynaptic currents (sIPSCs). As shown in Fig. 8A, B,
blocked. Generally there was a significant difference in C, the mean frequency and amplitude of sIPSCs were
the paired-pulse ratio among control, bryostatin and 1.63 ± 0.27 Hz and 39.88 ± 3.36 pA in the control
bryostatin + RO-32-0432 groups in different conditions condition. The mean frequency and amplitude of
(F2,20 = 18.48; p < 0.001). There was a significant sIPSCs were 2.92 ± 0.53 Hz and 52.40 ± 6.35 pA at
difference between bryostatin group and control 2 min following the bath application of 100 nM DCP-LA.
(F1,13 = 18.34; p < 0.001) or bryostatin + RO-32-0432 DCP-LA significantly increased the mean frequency and
co-application group (F1,13 = 18.54; p < 0.01). amplitude of sIPSCs (⁄⁄p < 0.01; Paired Student’s t-
However, there was still significant difference in paired- test, n = 14). The representative traces of sIPSCs are
pulse ratio between control and bryostatin + RO-32- shown in Fig. 7A. Therefore, DCP-LA significantly
0432 co-application group (F1,13 = 6.32; p = 0.04568). increased the frequency and amplitude of sIPSCs in
These data suggested an involvement of bryostatin in CA1 pyramidal neurons, indicating PKCe pathway is
the regulation of presynatic function that could have involved in GABAergic neurotransmission.
C. Xu et al. / Neuroscience 268 (2014) 75–86 83

DISCUSSION and amplitude of spontaneous IPSC (sIPSCs) in CA1

pyramidal neurons. The increase of sIPSCs induced by
In this study, we used whole-cell patch-clamp, dual patch- bryostatin is fully blocked by TTX. In addition, we
clamp and extracellular recordings of acute hippocampal preincubated slices with enkephalin to selectively
slices to examine the effect of bryostatin on GABAergic suppress excitability of interneurons (Nakamura et al.,
neurotransmission in the hippocampus in rats. 2007). This blocked the effect of bryostatin on
Bryostatin enhances the GABAergic neurotransmission enhancing GABAergic neurotransmission. The increase
and its modification in pyramidal neurons by a of GABAergic neurotransmission induced by bryostatin
presynaptic mechanism with excitation of GABAergic was also fully blocked by later combined incubation of
interneurons that depends on PKCa & e. bryostatin with enkephalin. All of these results
suggested that bryostatin increases sIPSCs in CA1
Bryostatin enhances GABAergic neurotransmission pyramidal neurons by a presynaptic mechanism (see
in pyramidal neurons by a presynaptic mechanism Fig. 9). Furthermore, we used a dual-recording
that is mediated by PKCa & e activation technique to monitor communicating cell pairs of
interneuron and pyramidal neuron and revealed unique
GABA and glutamate are the most abundant activity-dependent properties of GABAergic synapses in
neurotransmitters in the brain, and their balanced CA1 pyramidal neurons, which demonstrated release of
activation of inhibitory and excitatory functions maintains GABA from the GABAergic terminals of interneurons
the equilibrium of neuronal networks. GABA is the major projecting to CA1 pyramidal neurons. One mechanism
inhibitory neurotransmitter in the central nervous by which DCP-LA, a specific PKCe activator, stimulates
system. A growing body of evidence suggests that GABA release, has been proposed to be by enhancing
plasticity of GABAergic synapses is critical during the activity of pre-synaptic a7 Ach receptors present on
development and aging, and inhibitory plasticity can the GABAergic terminals of interneurons that transmit to
function to drive a perturbed system toward CA1 pyramidal neurons via PKC pathway (Pallas et al.,
homeostasis (Pallas et al., 2006). PKC is an important 2006). Like DCP-LA, bryostatin induces the increase of
signaling molecule pathway in learning and memory sIPSCs in CA1 pyramidal neurons through presynaptic
(Alkon et al., 1998). Reduced PKC activity is associated mechanism.
with Alzheimer’s disease (AD) (Cole et al., 1988). Secondly, pretreatment with staurosporine, a broad-
Bryostatin, a PKC activator, can enhance spatial spectrum protein kinase inhibitor, blocked the increase
memory (Sun and Alkon, 2005) and protect against of sIPSCs induced by bryostatin. Furthermore,
neurodegeneration in AD transgenic mice pretreatment with RO-32-0432, a specific PKCa
(Etcheberrigaray et al., 2004). Here, we investigated inhibitor, mimicked staurosporine’s effect. Finally, we
how bryostatin affects inhibitory neurotransmission in found RO-32-0432 significantly blocked the effect of
the rat hippocampus with short-term plasticity protocols. bryostatin on short-term and long-term GABAergic
Our results showed that bryostatin-1 significantly neurotransmission in hippocampal slices. All of these
increased the frequency and amplitude of spontaneous results suggest that bryostatin enhanced GABAergic
inhibitory postsynaptic currents (sIPSCs). We also found neurotransmission through the PKCa signaling pathway.
that the firing rate of GABAergic interneurons
significantly increased with bath application of
bryostatin. Additionally, simultaneous recordings from
communicating cell pairs of interneuron and pyramidal AL
neurons revealed unique activity-dependent properties
OA _
of GABAergic synapses. Furthermore, methionine Enk _
enkephalin (a selective l- and -opioid receptor agonist),
selectively suppressed excitability of interneurons and
decreased the frequency and amplitude of spontaneous +
IPSCs. Finally, bryostatin enhances the GABAergic Bry PC SP
neurotransmission in pyramidal neurons by activating a Bry
PKCa-dependent pathway and by a presynaptic
mechanism with excitation of GABAergic interneurons.
These results were confirmed by pretreatment with SR
staurosporine, a broad-spectrum protein kinase inhibitor,
and RO-32-0432, a selective PKCa inhibitor. LM
There are several possible mechanisms by which
bryostatin modulates GABAergic neurotransmission. First, Inhibitory Excitatory
bryostatin enhances GABAergic neurotransmission in Fig. 9. Diagram representing the intrinsic circuitry of CA1 region and
CA1 pyramidal neurons by presynaptic mechanism. In our effects of bryostatin or Enkephalin on interneurons. Closed and open
study, the function of GABAergic terminals of interneurons circles denote excitatory (glutamatergic) and inhibitory (GABAergic)
projecting to CA1 pyramidal neurons in the hippocampus synapses, respectively. The arrows indicate the direction of impulse
propagation. +: excitatory. : inhibitory. OA IN: oriens-alveus
via the PKC pathway was demonstrated. Bryostatin interneurons. PC; pyramidal cells. BC: basket cells. Bry: bryostatin.
directly excites interneurons and increases their firing rate, Enk: enkepalin. Al: alveus. SO: stratum oriens. SP: stratum pyrami-
and bryostatin also significantly increases the frequency dale. SR: stratum radiatum. LM: lacunosum-moleculare.
84 C. Xu et al. / Neuroscience 268 (2014) 75–86

However, RO-32-0432 (20 nM) did not completely block 2003). Bryostatin-1 is involved in neuronal functioning
the effects of bryostatin on short-term or long-term and facilitates the induction of LTP via activation of
GABAergic neurotransmissions, which suggests that PKCa and/or PKCe (Kim et al., 2012). However, our
there is an alternative pathway involved in the increase data may reflect a change of GABAergic
of GABAergic transmission. Interestingly, we tested the neurotransmission induced by bryostatin. Inhibitory
effect of DCP-LA, a selectively PKCe agonist, on neurons can shape the excitability and dynamic range of
GABAergic neurotransmission, and found that DCP-LA neuronal circuits (Maffei, 2011). Bryostatin may affect
can increase the frequency and amplitude of sIPSCs in intrinsic properties or synaptic projected by other
the hippocampal CA1 pyramidal neurons. Therefore, neurons in the neuronal circuits.
these results suggest that bryostatin may also activate Finally, PKC activation leads to presynaptic GABA
PKCe to enhance GABAergic neurotransmission. release via synaptic trafficking. Protein phosphorylation is
McGinty et al. reported that PKCa was more expressed an essential regulator of cell function. Presynaptic nerve
in CA3 pyramidal neurons and PKCe was more terminals contain numerous protein kinases whose
concentrated in the dentate granule cells and CA3 activation leads to a facilitation of evoked and
pyramidal neurons. PKCe was also present in the spontaneous neurotransmitter release, which is facilitated
mossy fiber terminals (McGinty et al., 1991). These by vesicle fusion or increase in synaptic vesicle number
PKC subunits’ unique distributions in the different and replenishment (Brager et al., 2002). Phorbol ester,
hippocampal neurons may be involved in bryostatin’s another less potent PKC activator, elevates release
different effect on GABAergic neurotransmission. probability at hippocampal excitatory synapses (Malenka
Thirdly, bryostatin modulates the brain GABAergic et al., 1986; Brager et al., 2002) and also enhances
synaptic plasticity (i.e. modifiability). In normal inhibitory synaptic transmission (Capogna et al., 1995).
hippocampus, paired-pulse stimuli induced PPD of Bryostatin may potentiate GABA release from
IPSCs (Jiang et al., 2000). In our study, bryostatin interneurons via facilitating vesicle mobilization and
switched paired-pulse depression into paired-pulse exocytosis by phosphorylation of a specific protein which
facilitation in IPSCs and this effect continues until is involved in the interaction of synaptic vesicles with the
20 min. These changes in PPF are likely to originate in presynaptic membrane (Robinson et al., 1993).
the presynaptic terminal, so our results may reflect a
change in neurotransmitter release possible by Bryostatin’ effect on enhancing learning and memory
increasing storage, docking or fusion of GABA synaptic
PKC signaling plays an important role in the formation of
vesicles (Thomson, 2000). Paired-pulse facilitation of
learning and memory (Sun and Alkon, 2005), inhibition
neurotransmitter release likely reflects a frequency-
and impairment of PKC functions can lead to deficits in
dependent accumulation of terminal calcium. Bryostatin-
learning and memory, such as AD or aging (Wang
1 increased intracellular calcium levels (Kim et al.,
et al., 1994). It is not surprising, therefore, that the
2012). Both evoked and miniature vesicular release are
therapeutic values of PKC activators on memory
regulated in parallel and the frequency of miniature
deficiency have recently been demonstrated. The non-
synaptic activity can be used as an indicator for evoked
tumorigenic bryostatins are isolated from the marine
release probability (Prange and Murphy, 1999). Our
Bugula neritina with a chemical structure unrelated to
finding of a lack of correlation between evoked release
the tumorigenic phorbol esters. Bryostatin-1, an
probability and miniature IPSCs in the presence of
antineoplastic agent and potent PKC activator, improves
bryostatin bath application was somewhat unexpected.
learning and memory (Sun and Alkon, 2005), as well as
Different mechanisms may exist to independently
enhances mushroom spine formation to restore the
regulate miniature and evoked GABA release.
number of synapse and synaptic responses in aged rats
Bryostatin-1 activates the PKC pathway and directly
(Hongpaisan and Alkon, 2007). Bryostatin-1 promotes
induces a transient increase in the quantal content of
LTP in mice hippocampus (Kim et al., 2012). We used
released GABA or transient terminal calcium increase
RO-32-0432, a more selective PKCa inhibitor, and
by second pulse. Long-term potentiation (LTP), a
found it blocked bryostatin’s effect. We also found that
prolonged increase in synaptic efficacy following the
DCP-LA, a PKCe-specific activator, also improves age-
application of a patterned stimulation, is a cellular model
related learning impairment by enhancing cognitive
of learning and memory (Shimizu et al., 2000).
functions (Yaguchi et al., 2006). PKC activators,
Glutamate receptor-mediated currents (AMPAR- and
especially PKCa and PKCe, promote synaptogenesis
NMDAR-) are involved in synaptic plasticity and learning
(Hongpaisan and Alkon, 2007) and network repair.
and memory (Riedel et al., 2003). LTP is NMDA-
These results suggest that bryostatin enhances
receptor independent, and is at least initially a
GABAergic neurotransmission in age-impaired rats to
presynaptic event (Hussain and Carpenter, 2003). PKC
restore the balance of the excitatory and inhibitory
is known to be essential to the induction of LTP in the
activations and thereby maintains the equilibrium within
hippocampus (Hu et al., 1987). Hussain and Carpenter
the hippocampal neuronal networks.
had tested the effects of several PKC activators and
inhibitors except for bryostatin on glutamatic LTP in the
hippocampus and found the possibility of roles for PKCa
and e on LTP induction, plus a role for PKM f in both This study demonstrates that bryostatin enhances the
induction and maintenance (Hussain and Carpenter, GABAergic neurotransmission in pyramidal neurons by
C. Xu et al. / Neuroscience 268 (2014) 75–86 85

activating PKCa & e-dependent pathway and by a Kim H, Han SH, Quan HY, Jung YJ, An J, Kang P, Park JB, Yoon BJ,
presynaptic mechanism which involves excitation of Seol GH, Min SS (2012) Bryostatin-1 promotes long-term
potentiation via activation of PKCa and PKCe in the
GABAergic interneurons. This study provides some
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Acknowledgments—This study was supported by NIH, United
from oxidative stress-induced cell death. J Neurosci
States grant 1RO3 AGO23937. The authors thank Dr. Thomas
J. Nelson for advising on the use of RO-32-0432 on brain slices Malenka RC, Madison DV, Andrade R, Nicoll RA (1986) Phorbol
and Ms. Kathryn L. Bauman and Ms. Dee DeNuto for their help in esters mimic some cholinergic actions in hippocampal pyramidal
manuscript preparation. neurons. J Neurosci 6(2):475–480.
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(Accepted 6 March 2014)

(Available online 15 March 2014)