Neuroscience 268 (2014) 75–86

PKC ACTIVATORS ENHANCE GABAERGIC NEUROTRANSMISSION AND PAIRED-PULSE FACILITATION IN HIPPOCAMPAL CA1 PYRAMIDAL NEURONS C. XU, * Q.-Y. LIU AND D. L. ALKON

to be induced by bryostatin. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Blanchette Rockefeller Neurosciences Institute, Morgantown, WV 26506, United States of America

Key words: bryostatin, GABA, PKCa, PKCe, synaptic plasiticity.

Abstract—Bryostatin-1, a potent agonist of protein kinase C (PKC), has recently been found to enhance spatial learning and long-term memory in rats, mice, rabbits and the nudibranch Hermissenda, and to exert profound neuroprotective effects on Alzheimer’s disease (AD) in transgenic mice. However, details of the mechanistic effects of bryostatin on learning and memory remain unclear. To address this issue, whole-cell recording, a dual-recording approach and extracellular recording techniques were performed on young (2–4 months) Brown-Norway rats. We found that bath-applied bryostatin-1 significantly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). The firing rate of GABAergic interneurons significantly was also increased as recorded with a loosely-attached extracellular recording configuration. Simultaneous recordings from communicating cell pairs of interneuron and pyramidal neuron revealed unique activity-dependent properties of GABAergic synapses. Furthermore, the bryostatin-induced increase of the frequency and amplitude of IPSCs was blocked by methionine enkephalin which selectively suppressed the excitability of interneurons. Pretreatment with RO-32-0432, a relatively specific PKCa antagonist, blocked the effect of bryostatin on sIPSCs. Finally, bryostatin increased paired-pulse ratio of GABAergic synapses that lasted for at least 20 min while pretreatment with RO-32-0432 significantly reduced the ratio. In addition, 8-[2-(2-pentyl-cyclopropylmethl)-cyclopropyl]-octanoic acid (DCP-LA), a selective PKCe activator, also increased the frequency and amplitude of sIPSCs. Taken together, these results suggest that bryostatin enhances GABAergic neurotransmission in pyramidal neurons by activating the PKCa & e-dependent pathway and by a presynaptic mechanism with excitation of GABAergic interneurons. These effects of bryostatin on GABAergic transmissions and modifiability may contribute to the improvement of learning and memory previously observed

INTRODUCTION Bryostatin, a potent agonist of protein kinase C (PKC), particularly a & e isozymes, has recently been found to enhance spatial learning and long-term memory in rats, mice, rabbits and the nudibranch Hermissenda (Kazanietz et al., 1994; Sun and Alkon, 2005; Kuzirian et al., 2006; Sun et al., 2008; Wang et al., 2008; Hongpaisan et al., 2013). Bryostatin is also found to increase the levels of synaptic proteins spinophilin and synaptophysin and cause structural changes in synapses (Hongpaisan and Alkon, 2007). Furthermore, bryostatin exerts profound neruoprotective effects on AD transgenic mice (Etcheberrigaray et al., 2004). It has been known that an inhibition or impairment of PKC activity leads to learning and memory disorders (Takashima et al., 1991), therefore, an appropriate activation of PKC isozymes such as PKCa or PKCe results in the restoration of learning and memory (Sun and Alkon, 2010). Bryostatin-1, enhances spatial learning and memory by synaptic or structural remodeling and synaptogenesis in the hippocampus and related cortical areas (Hongpaisan and Alkon, 2007). DHA-CP6, a novel PKCe activator, reduced b-amyloid level by increasing b-amyloid degradation through endothelin-converting enzyme (ECE) (Nelson et al., 2009). PKC activation has been shown to reduce apoptotic neuronal cell death secondary to oxidative stress (Maher, 2001). Additionally, PKC activation enhances Ca2+ currents and elevates cytosolic-free Ca2+, resulting in neurotransmitter release (Swartz et al., 1993; Hussain and Carpenter, 2003), and potassium channel (Hoffman and Johnston, 1998) and sodium channel inhibition (Chen et al., 2005). 8-[2-(2-pentyl-cyclopropylmethl)cyclopropyl]-octanoic acid (DCP-LA), another compound that selectively and directly activates PKCe, enhances the response of presynaptic a7 acetylcholine (Ach) receptors that are involved in glutamate and GABA release (Yamamoto et al., 2005; Kanno et al., 2005).

*Corresponding author. Tel:+1-240-477-3662. E-mail address: [email protected] (C. Xu). Abbreviations: Ach, acetylcholine; aCSF, artificial cerebrospinal fluid; AD, Alzheimer’s disease; AP5, DL-2-amino-5-phosphonovaleric acid; DCP-LA, 8-[2-(2-pentyl-cyclopropylmethl)-cyclopropyl]-octanoic acid; DNQX, 6,7-dinitroquinoxaline-2,3-dione; ECE, endothelin-converting enzyme; EGTA, ethylene glycol tetraacetic acid; HEPES, 2-[4-(2hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; K-S test, Kolmogorov– Smirnov test; LTP, long-term potentiation; PKC, protein kinase C; PPD, paired-pulse depression; PPF, paired-pulse facilitation; sIPSCs, spontaneous inhibitory postsynaptic currents; uIPSCs, unitary inhibitory postsynaptic currents. http://dx.doi.org/10.1016/j.neuroscience.2014.03.008 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 75

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DCP-LA or bryostatin-1 also enhances a transient potentiation and/or long-term potentiation (LTP) in CA1 region of rat hippocampal slices (Yamamoto et al., 2005; Kim et al., 2012). In this study, we explored the effect of bryostatin on GABAergic neurotransmission in rat hippocampal CA1 pyramidal neurons and interneurons by using electrophysiological recordings from hippocampal slices. These experiments were designed to address the following questions: (1) Does bryostatin directly enhance or reduce GABAergic neurotransmission in rat hippocampal CA1 pyramidal neurons? (2) Is the effect of bryostatin on GABAergic neurotransmission through a presynaptic or postsynaptic mechanism? (3) Do interneurons have direct contact on pyramidal neurons in the hippocampal CA1 sector? (4) Which PKC isoforms are involved in PKC activation in GABAergic neurotransmission? (5) How does bryostatin affect short-term and long-term plasticity of GABAergic synapses?

EXPERIMENTAL PROCEDURES Spatial water maze tasks Effects of bryostatin-1 on spatial memory were evaluated in rats in vivo with the Morris water maze task. Male adult Brown Norway rats (2–3 months old; 260–300 g; from National Institute on Aging, Bethesda) were housed in temperature-controlled (20–24 °C) room for a week, allowed free access to food and water, and kept on a 12-h light/dark cycle. All rats were randomly assigned to different groups (10 each except for bryostatin + RO-32-0432 group 7 rats) and swam for 2 min in a 1.5-m (diameter)  0.6-m (depth) pool, filled with water to a depth of 40 cm (24 ± 1 °C). On the following day, rats were trained in a three-trails-per-day task for consecutive days. Each training trial lasted for up to 2 min, during which rats learned to escape from the water by finding a hidden platform that was placed at a fixed location and submerged 1–2 cm below the water surface. The navigation of the rats was viewed on-line by the investigators (Video Monitor BWM9, Javelin Electronics), who were obscured from the rats’ view and tracked by a video-camera. The escape latency and the route of rats’ swimming across the pool to the platform were recorded with a video-tracking system (poly-Track Video Tracking System, San Diego Instruments) for a quantitative analysis. A probe test was used to evaluate retention of the learned navigation experience. The probe test (1 min) was performed after removing the platform, 24 h after the last training trial, by monitoring the distance swum by each rat in the quadrants with the same video-tracking system. At 60 min before water maze training on days 1, 3, and 5, tail-vein injection was used to administer RO-32-0432 (500 lg/kg body weight) or vehicle. After 30 min, bryostatin-1 or vehicle was administered i.p. at 5 lg/kg body weight (60 lg/m2 body surface). Hippocampal slices Young (2–3 months of age) male Brown Norway (BN) rats were obtained from the National Institute on Aging

(Bethesda, MD). After sedation with isoflurane, the rat was decapitated and its brain bisected sagittally and removed, placed into ice-cold sucrose buffer containing the following (in mM): 254 sucrose,10 D-glucose, 26 NaHCO3, 2 CaCl2, 2 MgSO4, 3 KCl, and 1.25 NaH2PO4, saturated with 95% O2/5% CO2, at pH 7.4. Transverse hippocampal slices (250–300 lM thick) were cut with a VT 1000S microtome (Leica, Deerfield, IL). Slices were transferred immediately into a holding chamber and incubated at 32–33 °C for a 30-min recovery period in a mixture of 50% sucrose saline and 50% artificial cerebrospinal fluid (aCSF) containing the following (in mM): 128 NaCl, 10 D-glucose, 26 NaHCO3, 2 CaCl2, 2 MgSO4, 3 KCl and 1.25 NaH2PO4, slices were then placed on a nylon mesh, submerged in normal aCSF bubbled with 95% O2/5% CO2 continuously, and maintained at room temperature (21–24 °C) until whole cell patch clamp recording (30 min to 5 h). Electrophysiological recordings Slices were transferred to a submersion-type recording chamber (Warner Instruments, Hamden, CT) on a Burleigh Gibraltar fixed stage system (Burleigh Instruments, Fisher, NY), secured beneath a nylon harp, and perfused with aCSF heated to 30–33 °C at a rate of 2–3 ml per min. CA1 pyramidal cells and interneurons (oriens-alveus inteneurons of the hippocampus; see Fig. 9) were identified visually by using an Axioskop 2FS microscope (Zeiss) equipped with a 40X waterimmersion objective coupled with an infrared differential interference contrast camera system. Whole-cell patchclamp recordings were established using a dualheadstage MultiClamp 700A amplifier (Axon Instruments, Union City, CA). Membrane current and potential signals were digitized and analyzed with Digidata 1322A and pClamp 8.2 systems (Molecular Devices). Patch pipettes of 5 MO were pulled with a Narishige PP-830 puller (Narishige, Greencale, NY). The pipette solution had the following composition (in mM) unless otherwise stated: 140 KCl, 0.1 CaCl2, 5 EGTA, 10 HEPES, 4 ATP-Mg2+, 0.4 GTP-2Na+, 1 QX314 (Lidocaine N-ethyl bromide), pH 7.2 and 290 mOsm. The diffusion potential (liquid junction potential) was 4 mV calculated by Clampex software. Under these conditions, synaptic currents were acquired at a holding potential of 70 mV, the high concentrations of chloride in the pipette caused the inhibitory postsynaptic current (IPSC) to appear as an inward current. QX314 was added to the pipette solution to block the GABAB-mediated currents and to prevent the generation of Na+-dependent action potential. Spontaneous excitatory amino acid currents (sEPSCs) were excluded from recordings by adding glutamate receptor antagonists DNQX (6,7-dinitroquinoxaline-2,3dione, 20 lM) and AP5 (DL-2-amino-5phosphonovaleric acid, 20 lM); therefore all of the recorded inward currents were spontaneous IPSCs (sIPSCs). The method of recording inhibitory synaptic currents was set up according to Rodriguez-Moreno’s method and with some adjustment (Rodriguez-Moreno et al., 2000).

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Paired-pulse stimulation and dual whole cell patchclamp recordings A paired-pulse stimulation protocol was used to evaluate short-term GABAergic synaptic plasticity in hippocampal slices (more than five animals each group). The pipette solution contained the following (in mM): 90 K+ gluconate, 45 KCl, 1.7 NaCl, 0.1 CaCl2, 2.7 MgCl2, 10 HEPES, 1.1 EGTA, 5 phosphocreatine-Na+, 3.5 ATPK+, 0.3 GTP-Na+, was titrated at pH 7.2 and 290 mOsm. GABAergic, inhibitory postsynaptic currents (IPSCs) are evoked using a concentric bipolar stimulation electrode placed in the stratum pyramidale about 80 lm from the recording cell, which was kept at a holding potential of 70 mV. DNQX and AP-5 were included in the perfusion solution to block glutamatergic currents. Only one cell was recorded from each brain slice. Dual whole-cell patch-clamp recordings were obtained simultaneously from two hippocampal neurons (most pairs of a pyramidal cell and an interneuron) in the hippocampal CA1-isolated slices under visual guidance. Extracellular cell-attached recording Cell-attached recordings were made on interneurons using a dual-headstage MultiClamp 700A amplifier and pClamp 8.2 software. The recording solution included DNQX and AP-5. Cell-attached electrodes were pulled from the walled borosilicate glass capillaries, were filled with 150 mM NaCl, and had resistance of 3–7 MX. While approaching the cell, positive pressure was applied to the patch electrode. The seal (100–300 MX) between the recording pipette and the cell membrane was obtained by applying suction to the electrode. Action potential currents were recorded in voltage-clamp mode (Perkins, 2006).

by changes in noise level or by membrane fluctuations. If the background noise increased during the recording, the data from that cell were discarded. The data generated from these measurements were used to plot cumulative probability amplitude and interevent interval graphs, with each distribution normalized to a maximal value of 1. Cumulative probability plots obtained under different experimental conditions were compared using the nonparametric Kolmogorov–Smirnov (K-S test), which estimates the probability that two cumulative distributions differ from each other by chance alone (Xu et al., 2009). The significance level for the K-S test was set at a value of p < 0.05. All numerical values are expressed as a mean ± standard error of the mean (SEM) and statistic analyses were performed by using a paired Student’s t-test or the analysis of variance (oneway analysis of variance [ANOVA]), whenever appropriate. The differences are considered significant at p < 0.05.

Drugs AP5, DNQX and Bicuculline were purchased from Tocris Cookson Inc, Ellisville, MO. TTX, QX314, Staurosporine aglycone and RO-32-0432 (Bisindolylmaleimide XI hydrochloride) were purchased from Sigma. Bryostatin-1 was purchased from BioMol international, LP. Methionine enkephalin acetate salt was purchased from Bachem Bioscience Inc., PA. Staurosporine aglycone and DNQX were dissolved in DMSO. All stock solutions were stored as frozen aliquots and diluted to test concentration in aCSF by a factor of 1000-fold. Drugs were administered by bath application.

RESULTS

Acquisition and analysis of synaptic currents

Bryostatin enhanced Brown Norway rat water maze spatial learning and memory

GABAA-mediated inward currents were recorded with whole-cell electrodes containing high concentrations of chloride and 1 mM QX314 using the continuous single-electrode voltage-clamp mode. Access resistance (15% during the experiment. If the access resistance increased during the course of the experiment and caused significant reductions in the synaptic current amplitudes, efforts were made to improve access (such as applying additional suction or slight positive pressure); if this failed, the experiment was discontinued. Spontaneously occurring synaptic currents were filtered at 2 kHz, and digitized at 10 kHz using Digidata 1322A. Synaptic currents were collected at 60 s for each experimental condition. Off-line analysis of synaptic currents was performed using the Minianalysis software (Version 6.0; Synaptosoft, Decatur, GA). Synaptic currents were screened automatically using an amplitude threshold of 3 pA. Events were then visually screened to ensure that the analysis was not distorted

Effects of bryostatin-1 on spatial learning and memory were previously evaluated in Male adult Wistar rats, using a hidden-platform water maze and it was found that bryostatin enhanced rat water maze spatial learning and memory (Sun and Alkon, 2005). Here we want to explore the effect of bryostatin-1 on Brown Norway rats. As shown in Fig. 1A, the latency of escape to the hidden platform in the three groups of rats decreased gradually during the training sessions, indicating that all rats were able to learn the task through training and the learning was progressive. However, there was a significant group difference in escape latency (F(2,14) = 12.86, p < 0.001), indicating difference in learning between the treatments. The difference between rats that received bryostatin-1 or vehicle was significant (F(1,9) = 16.16, p < 0.05), indicating that spatial learning in rats that were injected with bryostatin1 was faster than that of the rats injected with vehicle. The difference between rats that received bryostatin-1 or co-administration of bryostatin-1 and RO-32-0432 was significant (F(1,9) = 9.15, p < 0.05), indicating RO-32-0432, a more selective PKCa inhibitor, blocked

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100

Escape latency (s)

A

Bryostatin significantly increased the frequency and amplitude of sIPSCs but not mIPSCs in CA1 pyramidal neurons

Control

80

Bryostatin-1

60

Bryostatin-1 + RO-32-0432

40 20 0

1

2

3

4

5

Training days

Swimming distance in quadrant (% of total)

B

#

60

Control

**

50

Bryostatin-1

40

Bryostatin-1 + RO-32-0432

# #

30 20 10 0

Opp

Aj-R

Aj-R

Target

Quadrant Fig. 1. Effects of bryostatin-1 on rat performance in the hidden platform water maze task. (A) Escape latency (means ± SEM) in water maze training for 5 days in control. Bryostatin (n = 10) and 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 compared with other quadrants). Opp, opposite to target quadrant; Aj-r, adjacent right quadrant; Aj-L, adjacent left quadrant.

the enhancement of learning induced by bryostatin-1. As shown in Fig. 1B, quadrant tests 24 h after the last training trial showed that all three groups showed a target quadrant preference (Control group: F(3,39) = 3.93, P < 0.05; Bryostatin-1 group: F(3,39) = 47.85, P < 0.001; Bryostatin plus RO-32-0432 group: F(3,27) = 11.45, P < 0.001). The difference in the target quadrant preference between groups was significant (F(3,11) = 9.91, P < 0.01), indicating that the bryostatin-1 rats spent significantly more time than the control group or bryostatin plus RO-32-0432 group (Student’s t-test, p < 0.001). There is no significant difference between the control group and bryostatin plus RO-32-0432 group, which indicates that RO-32-0432 blocked the memory retention induced by bryostatin-1. Thus, bryostatin-1 enhanced spatial learning and memory retention task in Brown Norway rats, indicating that PKCa or e activation can enhance both learning and memory retention.

To explore the effect of bryostatin on spontaneous GABAA receptor-mediated inhibitory postsynaptic currents (sIPSCs) and miniature GABAA receptormediated 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 were confirmed by the application of GABAA receptor antagonist bicuculline. As shown in Fig. 2A, C, the mean frequency and amplitude of sIPSCs were 1.77 ± 0.29 Hz and 39.11 ± 2.46 pA at the control 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. Bryostatin significantly increased the mean frequency and amplitude of sIPSCs (⁄⁄p < 0.01 or ⁄p < 0.05; Paired Student’s t-test, n = 12). The representative traces of sIPSCs are shown in Fig. 2A. The cumulative 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 (n = 7). There was no significant difference in the frequency and amplitude of mIPSCs between the control group and bath application bryostatin group (P > 0.05; Paired Student’s t-test, n = 7). Therefore, bryostatin significantly increased the frequency and amplitude of sIPSCs but not mIPSCs in CA1 pyramidal neurons. Simultaneous recordings from communicating cell pairs of interneuron and pyramidal neuron revealed unique activity-dependent properties of GABAergic synapses Bryostatin significantly increased the inhibitory postsynaptic currents, so we tested the effect of bryostatin on interneurons in hippocampal CA1 sector using a loosely-attached extracellular recording configuration. Under these recording conditions, bryostatin significantly increased the firing rate of interneurons. As shown in Fig. 3A, B, the mean frequency of firing rate of interneurons was 2.1 ± 0.5 Hz in control. When 10 nM bryostatin was bath-applied at 2 min, the mean frequency of firing rate of interneurons increased to 3.75 ± 0.84 Hz (⁄p < 0.05 comparing with the control group). To confirm the synaptic communications between the interneurons and the pyramidal cells in the hippocampal CA1 regions, paired whole-cell recordings were performed to detect unitary GABAAergic synaptic transmission between the interneurons and pyramidal cells. As shown in Fig. 4A, the unitary inhibitory postsynaptic currents (uIPSCs, lower trace) were evoked in the postsynaptic pyramidal cells by the

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A

200 pA

B

Bryostatin enhanced GABAergic neurotransmission in pyramidal neurons by activating the PKCa pathway Bryostatin-1, a potent PKC agonist, particularly activates PKCa and PKCe (Kazanietz et al., 1994). We want to

Cumulatve probability

1s

Inhibition of interneurons’ excitation blocked bryostatin’s effects on sIPSCs

1.0 0.8 0.6

Control

0.4

Bryostatin

0.2 0.0 0

2000

1.0 0.8 0.6

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0.4

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0.2 0.0

4000

0 100 200 300 400

4

n = 12

3

Amplitude (pA)

Amplitude (pA)

C

Frequency (Hz)

Interevent interval (ms)

**

2 1 0

D

60

n = 12

*

40 20 0

Control

Control

Bryostatin

Bryostatin

TTX + Bryostatin

50 pA

TTX

1s 3

n = 7; p > 0.05

2 1

40

Amplitude (pA)

E Frequency (Hz)

To confirm that the enhancement of sIPSCs by bryostatin is through the interneurons’ excitation, we firstly tested 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 suppress the excitability of interneurons (Nakamura et al., 2007). Secondly, we tested whether the enhancement of sIPSCs induced by bryostatin could be blocked by Enkephalin. As shown in Fig. 5A, B, Enkephalin decreased not only the frequency of sIPSCs but also the amplitudes of 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 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 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, P > 0.05, n = 7) and amplitude (32.3 ± 5.35 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 condition. The mean frequency and amplitude of sIPSCs were 2.51 ± 0.59 Hz and 42.76 ± 4.43 pA with the presence of bryostatin. Bryostatin significantly increased the frequency and amplitude of sIPSCs (p < 0.05, n = 5). When bryostatin was applied together with Enkephalin, the mean sIPSC frequency and amplitude were 0.45 ± 0.17 Hz and 32.98 ± 7.14 pA. Thus, Enkephalin significantly blocked the increase of the frequency and amplitude induced by bryostatin (p < 0.05, n = 5). These data suggest that the enhancement of sIPSCs by bryostatin is through the interneurons’ excitation.

Bryostatin-1(10nM)

Control

Cumulatve probability

spiking of the interneuron (upper trace) in normal resting conditions. Fig. 4B shows that the interneuron spikes 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 couples. Although there were a few asynchronous uIPSCs in the pyramidal cell (indicated by X), most of them are synchronous. These results suggest that spiking activities in interneurons synchronously evoke unitary inhibitory postsynaptic currents in the pyramidal cells.

n = 7; p > 0.05

20

0

0

TTX

TTX + Bryostatin

TTX

TTX + Bryostatin

Fig. 2. Bryostatin significantly increased the frequency and amplitude of sIPSCs but not mIPSCs in CA1 pyramidal neurons. (A) Representative traces show that 10 nM bryostatin-1, a PKC activator, increased the frequency and amplitude of sIPSCs at 2 min following the bath application of bryostatin. (B) Cumulative frequency and amplitude distributions of sIPSCs based on data shown in (A). Both frequency and amplitude distributions were statistically different under these two experimental conditions (Before vs. after treatment of bryostatin; p < 0.0001 using K-S test; 100 events analyzed for before and 185 events for after treatment of bryostatin). (C) A 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, ⁄⁄ p < 0.001; ⁄p < 0.05; n = 12). (D) Representative traces of mIPSCs obtained before (left) and after the application of 10 nM bryostatin (right). (E) Bar graphs show the mean frequency or amplitude of mIPSCs before and after the application of 10 nM bryostatin. There was no significant difference in the frequency and amplitude of mIPSCs (p > 0.05, Paired Student’s t-test; n = 7).

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A

B 5

At 2 min after 10 nM Bryostatin-1

3s

Fire rate (Hz)

100 pA

4

* n=6

3 2

1

0

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 ttest, ⁄p < 0.05; n = 6).

A IN PC

B IN PC

Fig. 4. Direct synaptic connections between pyramidal cells and interneurons were recorded in hippocampal CA1 sector. (A) Unitary inhibitory postsynaptic currents (uIPSCs) in pyramidal cell evoked by the interneurons. The upper trace showed that spontaneous firing spikes in interneuron and lower trace shows that inhibitory currents were recorded in pyramidal neuron. (B) The traces showed that synchronous connection. uIPSCs in the pyramidal cell evoked by the interneuron which was given a 10-s depolarization stimulation. The upper trace showed that spikes evoked by a 10-s depolarization stimulation in interneuron and lower trace show that inhibitory currents were recorded in pyramidal neuron. IN, interneuron; PC, pyramidal cell.

explore which isozyme of PKC was involved in the enhancement of sIPSCs induced by bryostatin. First, we used the pretreatment with staurosporine (1 lM), a broad spectrum and nonselective protein kinase inhibitor, for 20 min, then tested the effect of bryostatin on sIPSCs. Second, we used RO-32-0432, a selectively

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 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, 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-320432 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 (20 nM) were 2.46 ± 0.79 Hz and 21.27 ± 1.89 pA. There were no significant changes in frequency and amplitude of sIPSCs following the bath application of RO-32-0432 (n = 8, p > 0.05, student’s paired t-test). When bryostatin was applied together with RO-32-0432, mean sIPSC frequency and amplitude were 2.10 ± 0.62 Hz and 19.53 ± 2.38 pA. Thus, there were no significant changes in frequency and amplitude of sIPSCs when RO-32-0432 was applied either alone or combined with bryostatin. These data suggest that bryostatin enhanced GABAergic neurotransmission in pyramidal neurons by activating the PKCa pathway. Bryostatin can switch paired-pulse depression to facilitation To further verify the effect of bryostatin on short-term and long-term plasticity of GABAergic synapses in

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Enkephalin + Control Enkephalin (1µM) Bryostatin

RO-32-0432 (20 nM)

Control

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150 pA

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Frequency (Hz)

n En tro ke l En pha lin ke p Br hal yo in+ sta tin Bryostatin+ Enkephalin

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-3

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04 32 R + O-3 Br 2 yo -04 sta 32 tin

l tro Co n

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0

0 Fig. 5. Inhibition of interneurons’ excitation blocked bryostatin’s effects on sIPSCs. (A) Representative traces show that 1 lM 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 frequency and amplitude of sIPSCs (A paired Student’s t-test, ⁄ p < 0.05; n = 7). Bryostatin had no effect on the frequency and amplitude of sIPSCs. (C) Representative traces show that 10 nM bryostatin significantly increased the frequency and amplitude of sIPSCs. Addition of enkephalin blocked the effect of bryostatin. (D) A summary of data collected from five pyramidal cells. Bryostatin significantly increased the frequency and amplitude of sIPSCs (A paired Student’s t-test, ⁄p < 0.05; n = 7). Enkephalin significantly blocked the effect of bryostatin (A paired Student’s t-test, #p < 0.05, n = 5).

10

2-

0

2

l

10

-3

20

20

tro

30

3

n = 8; p > 0.05

Co n

Co

nt ro l Br yo sta tin Br y En osta ke tin ph + al in

0

30 n = 8; p > 0.05

Amplitude (pA)

#

40

Br yo sta tin Br yo En sta ke tin ph + al in

1

4

*

ro l

2

n=5

Frequency (Hz)

50

*

Amplitude (pA)

3

n=5

Co nt

Frequency (Hz)

4

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D

S in + taur Br os e yo po sta rin tin e

0

sp ro

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au

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nt

ro

l

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or S + taur ine Br os yo po sta rin tin e

Bryostatin

n = 5; p > 0.05

50

Co

nt

60

n = 5; p > 0.05

3

Co

Control

4

20 0

0

C

B

Co

*

40

au

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n=7

60

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En rol ke p En hal in ke p Br hal yo in+ sta tin

Frequency (Hz)

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B

100 pA

1s

Fig. 6. Bryostatin enhanced GABAergic neurotransmission in pyramidal neurons by activating the PKCa pathway. (A) Representative traces show that 20 nM RO-32-0432 had no effect on the frequency and amplitude of sIPSCs. Addition of bryostatin also had no effect on the frequency and amplitude of sIPSCs, which suggests that RO-320432 blocked the effect of bryostatin. (B) Bar chart shows that staurosporine, a broad-spectrum protein kinase inhibitor, blocked the effect of bryostatin on the frequency and amplitude of sIPSCs (A paired Student’s t-test, p > 0.05; n = 5). (C) Bar chart shows that RO-32-0432, a specific PKCa inhibitor, blocked the effect of bryostatin on the frequency and amplitude of sIPSCs (A paired Student’s ttest, p > 0.05, n = 8).

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A

B

A

DCP-LA (100 nM)

100 pA

25 pA

Control

10 ms

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hippocampal CA1 slice. IPSCs induced with paired-pulse protocol were recorded in hippocampal slices. As shown in Fig. 7, in control condition, second-pulse response was smaller than the first-pulse response, so PPD (paired-pulse depression) was induced (n = 8); however, after the application of bryostatin, the secondpulse response was larger than the first-pulse response, indicating that PPF (paired-pulse facilitation) was induced. Thus, bryostatin switched PPD to PPF. This effect continued for 20 min in the presence of bryostatin. When bryostatin was applied together with RO-32-0432 (20 nM), the bryostatin switching effect on PPD was blocked. Generally there was a significant difference in the paired-pulse ratio among control, bryostatin and bryostatin + RO-32-0432 groups in different conditions (F2,20 = 18.48; p < 0.001). There was a significant difference between bryostatin group and control (F1,13 = 18.34; p < 0.001) or bryostatin + RO-32-0432 co-application group (F1,13 = 18.54; p < 0.01). However, there was still significant difference in pairedpulse ratio between control and bryostatin + RO-320432 co-application group (F1,13 = 6.32; p = 0.04568). These data suggested an involvement of bryostatin in the regulation of presynatic function that could have

1

n = 14

40 20

D

CP

-L

ro nt

A

l

l

A

0

0

ro

Fig. 7. Effect of bryostatin on short-term and long-term GABAergic synaptic plasticity in CA1 pyramidal neurons and PKCa pathway. (A) A paired-pulse depression of IPSCs before 10 nM bryostatin treatment. (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). Bryostatin significantly increased the ratio of p2/p1 (⁄p < 0.05, ⁄⁄ p < 0.01 compared with control group or RO-32-0432 group) and RO-32-0432 blocked the increase in the ratio of p2/p1. Points on the plot are mean ± SEM of values obtained from different slices. RO32 + Bry group indicates RO-32-0432 plus bryostatin group.

2

nt

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2

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** * *

Bryostatin Control RO-32 + Bry

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Fig. 8. DCP-LA significantly increased the frequency and amplitude of sIPSCs in CA1 pyramidal neurons. (A) Representative traces show that 100 nM DCP-LA, a PKCe activator, increased the frequency and amplitude of sIPSCs at 2 min following the bath application of DCPLA. (B) A summary of data collected from 14 cells in the absence or 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).

long-term effect. This further confirmed that bryostatin’s effects were through PKCa pathway. PKCe isozyme activity is also involved in GABAergic neurotransmission in hippocampal CA1 sector To explore if PKCe isozyme is involved in GABAergic neurotransmission in the hippocampus, we tested the effect of DCP-LA, a selective PKCe activator, on spontaneous GABAA receptor-mediated inhibitory postsynaptic currents (sIPSCs). As shown in Fig. 8A, B, C, the mean frequency and amplitude of sIPSCs were 1.63 ± 0.27 Hz and 39.88 ± 3.36 pA in the control condition. The mean frequency and amplitude of sIPSCs were 2.92 ± 0.53 Hz and 52.40 ± 6.35 pA at 2 min following the bath application of 100 nM DCP-LA. DCP-LA significantly increased the mean frequency and amplitude of sIPSCs (⁄⁄p < 0.01; Paired Student’s ttest, n = 14). The representative traces of sIPSCs are shown in Fig. 7A. Therefore, DCP-LA significantly increased the frequency and amplitude of sIPSCs in CA1 pyramidal neurons, indicating PKCe pathway is involved in GABAergic neurotransmission.

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DISCUSSION In this study, we used whole-cell patch-clamp, dual patchclamp and extracellular recordings of acute hippocampal slices to examine the effect of bryostatin on GABAergic neurotransmission in the hippocampus in rats. Bryostatin enhances the GABAergic neurotransmission and its modification in pyramidal neurons by a presynaptic mechanism with excitation of GABAergic interneurons that depends on PKCa & e. Bryostatin enhances GABAergic neurotransmission in pyramidal neurons by a presynaptic mechanism that is mediated by PKCa & e activation GABA and glutamate are the most abundant neurotransmitters in the brain, and their balanced activation of inhibitory and excitatory functions maintains the equilibrium of neuronal networks. GABA is the major inhibitory neurotransmitter in the central nervous system. A growing body of evidence suggests that plasticity of GABAergic synapses is critical during development and aging, and inhibitory plasticity can function to drive a perturbed system toward homeostasis (Pallas et al., 2006). PKC is an important signaling molecule pathway in learning and memory (Alkon et al., 1998). Reduced PKC activity is associated with Alzheimer’s disease (AD) (Cole et al., 1988). Bryostatin, a PKC activator, can enhance spatial memory (Sun and Alkon, 2005) and protect against neurodegeneration in AD transgenic mice (Etcheberrigaray et al., 2004). Here, we investigated how bryostatin affects inhibitory neurotransmission in the rat hippocampus with short-term plasticity protocols. Our results showed that bryostatin-1 significantly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). We also found 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 neurons revealed unique activity-dependent properties of GABAergic synapses. Furthermore, methionine 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 neurotransmission in pyramidal neurons by activating a PKCa-dependent pathway and by a presynaptic mechanism with excitation of GABAergic interneurons. These results were confirmed by pretreatment with staurosporine, a broad-spectrum protein kinase inhibitor, and RO-32-0432, a selective PKCa inhibitor. There are several possible mechanisms by which bryostatin modulates GABAergic neurotransmission. First, bryostatin enhances GABAergic neurotransmission in CA1 pyramidal neurons by presynaptic mechanism. In our study, the function of GABAergic terminals of interneurons projecting to CA1 pyramidal neurons in the hippocampus via the PKC pathway was demonstrated. Bryostatin directly excites interneurons and increases their firing rate, and bryostatin also significantly increases the frequency

and amplitude of spontaneous IPSC (sIPSCs) in CA1 pyramidal neurons. The increase of sIPSCs induced by bryostatin is fully blocked by TTX. In addition, we preincubated slices with enkephalin to selectively suppress excitability of interneurons (Nakamura et al., 2007). This blocked the effect of bryostatin on enhancing GABAergic neurotransmission. The increase of GABAergic neurotransmission induced by bryostatin was also fully blocked by later combined incubation of bryostatin with enkephalin. All of these results suggested that bryostatin increases sIPSCs in CA1 pyramidal neurons by a presynaptic mechanism (see Fig. 9). Furthermore, we used a dual-recording technique to monitor communicating cell pairs of interneuron and pyramidal neuron and revealed unique activity-dependent properties of GABAergic synapses in CA1 pyramidal neurons, which demonstrated release of GABA from the GABAergic terminals of interneurons projecting to CA1 pyramidal neurons. One mechanism by which DCP-LA, a specific PKCe activator, stimulates GABA release, has been proposed to be by enhancing the activity of pre-synaptic a7 Ach receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via PKC pathway (Pallas et al., 2006). Like DCP-LA, bryostatin induces the increase of sIPSCs in CA1 pyramidal neurons through presynaptic mechanism. Secondly, pretreatment with staurosporine, a broadspectrum protein kinase inhibitor, blocked the increase of sIPSCs induced by bryostatin. Furthermore, pretreatment with RO-32-0432, a specific PKCa inhibitor, mimicked staurosporine’s effect. Finally, we found RO-32-0432 significantly blocked the effect of bryostatin on short-term and long-term GABAergic neurotransmission in hippocampal slices. All of these results suggest that bryostatin enhanced GABAergic neurotransmission through the PKCa signaling pathway.

AL

+

OA IN

Bry

_

Enk _

SO

BC PC

+

SP Bry SR LM

Inhibitory

Excitatory

Fig. 9. Diagram representing the intrinsic circuitry of CA1 region and effects of bryostatin or Enkephalin on interneurons. Closed and open circles denote excitatory (glutamatergic) and inhibitory (GABAergic) synapses, respectively. The arrows indicate the direction of impulse propagation. +: excitatory. : inhibitory. OA IN: oriens-alveus interneurons. PC; pyramidal cells. BC: basket cells. Bry: bryostatin. Enk: enkepalin. Al: alveus. SO: stratum oriens. SP: stratum pyramidale. SR: stratum radiatum. LM: lacunosum-moleculare.

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However, RO-32-0432 (20 nM) did not completely block the effects of bryostatin on short-term or long-term GABAergic neurotransmissions, which suggests that there is an alternative pathway involved in the increase of GABAergic transmission. Interestingly, we tested the effect of DCP-LA, a selectively PKCe agonist, on GABAergic neurotransmission, and found that DCP-LA can increase the frequency and amplitude of sIPSCs in the hippocampal CA1 pyramidal neurons. Therefore, these results suggest that bryostatin may also activate PKCe to enhance GABAergic neurotransmission. McGinty et al. reported that PKCa was more expressed in CA3 pyramidal neurons and PKCe was more concentrated in the dentate granule cells and CA3 pyramidal neurons. PKCe was also present in the mossy fiber terminals (McGinty et al., 1991). These PKC subunits’ unique distributions in the different hippocampal neurons may be involved in bryostatin’s different effect on GABAergic neurotransmission. Thirdly, bryostatin modulates the brain GABAergic synaptic plasticity (i.e. modifiability). In normal hippocampus, paired-pulse stimuli induced PPD of IPSCs (Jiang et al., 2000). In our study, bryostatin switched paired-pulse depression into paired-pulse facilitation in IPSCs and this effect continues until 20 min. These changes in PPF are likely to originate in the presynaptic terminal, so our results may reflect a change in neurotransmitter release possible by increasing storage, docking or fusion of GABA synaptic vesicles (Thomson, 2000). Paired-pulse facilitation of neurotransmitter release likely reflects a frequencydependent accumulation of terminal calcium. Bryostatin1 increased intracellular calcium levels (Kim et al., 2012). Both evoked and miniature vesicular release are regulated in parallel and the frequency of miniature synaptic activity can be used as an indicator for evoked release probability (Prange and Murphy, 1999). Our finding of a lack of correlation between evoked release probability and miniature IPSCs in the presence of bryostatin bath application was somewhat unexpected. Different mechanisms may exist to independently regulate miniature and evoked GABA release. Bryostatin-1 activates the PKC pathway and directly induces a transient increase in the quantal content of released GABA or transient terminal calcium increase by second pulse. Long-term potentiation (LTP), a prolonged increase in synaptic efficacy following the application of a patterned stimulation, is a cellular model of learning and memory (Shimizu et al., 2000). Glutamate receptor-mediated currents (AMPAR- and NMDAR-) are involved in synaptic plasticity and learning and memory (Riedel et al., 2003). LTP is NMDAreceptor independent, and is at least initially a presynaptic event (Hussain and Carpenter, 2003). PKC is known to be essential to the induction of LTP in the hippocampus (Hu et al., 1987). Hussain and Carpenter 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 induction and maintenance (Hussain and Carpenter,

2003). Bryostatin-1 is involved in neuronal functioning and facilitates the induction of LTP via activation of PKCa and/or PKCe (Kim et al., 2012). However, our data may reflect a change of GABAergic neurotransmission induced by bryostatin. Inhibitory neurons can shape the excitability and dynamic range of neuronal circuits (Maffei, 2011). Bryostatin may affect intrinsic properties or synaptic projected by other neurons in the neuronal circuits. Finally, PKC activation leads to presynaptic GABA release via synaptic trafficking. Protein phosphorylation is an essential regulator of cell function. Presynaptic nerve terminals contain numerous protein kinases whose activation leads to a facilitation of evoked and spontaneous neurotransmitter release, which is facilitated by vesicle fusion or increase in synaptic vesicle number and replenishment (Brager et al., 2002). Phorbol ester, another less potent PKC activator, elevates release probability at hippocampal excitatory synapses (Malenka et al., 1986; Brager et al., 2002) and also enhances inhibitory synaptic transmission (Capogna et al., 1995). Bryostatin may potentiate GABA release from interneurons via facilitating vesicle mobilization and exocytosis by phosphorylation of a specific protein which is involved in the interaction of synaptic vesicles with the presynaptic membrane (Robinson et al., 1993). Bryostatin’ effect on enhancing learning and memory PKC signaling plays an important role in the formation of learning and memory (Sun and Alkon, 2005), inhibition and impairment of PKC functions can lead to deficits in learning and memory, such as AD or aging (Wang et al., 1994). It is not surprising, therefore, that the therapeutic values of PKC activators on memory deficiency have recently been demonstrated. The nontumorigenic bryostatins are isolated from the marine Bugula neritina with a chemical structure unrelated to the tumorigenic phorbol esters. Bryostatin-1, an antineoplastic agent and potent PKC activator, improves learning and memory (Sun and Alkon, 2005), as well as enhances mushroom spine formation to restore the number of synapse and synaptic responses in aged rats (Hongpaisan and Alkon, 2007). Bryostatin-1 promotes LTP in mice hippocampus (Kim et al., 2012). We used RO-32-0432, a more selective PKCa inhibitor, and found it blocked bryostatin’s effect. We also found that DCP-LA, a PKCe-specific activator, also improves agerelated learning impairment by enhancing cognitive functions (Yaguchi et al., 2006). PKC activators, especially PKCa and PKCe, promote synaptogenesis (Hongpaisan and Alkon, 2007) and network repair. These results suggest that bryostatin enhances GABAergic neurotransmission in age-impaired rats to restore the balance of the excitatory and inhibitory activations and thereby maintains the equilibrium within the hippocampal neuronal networks.

CONCLUSION This study demonstrates that bryostatin enhances the GABAergic neurotransmission in pyramidal neurons by

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

activating PKCa & e-dependent pathway and by a presynaptic mechanism which involves excitation of GABAergic interneurons. This study provides some functional basis that bryostatin protects against memory impairment in AD and aging, and provides further physiological support for treating AD in a new direction with non-toxic PKC activators. Acknowledgments—This study was supported by NIH, United States grant 1RO3 AGO23937. The authors thank Dr. Thomas J. Nelson for advising on the use of RO-32-0432 on brain slices and Ms. Kathryn L. Bauman and Ms. Dee DeNuto for their help in manuscript preparation.

REFERENCES Alkon DL, Nelson TJ, Zhao W, Cavallaro S (1998) Time domains of neuronal Ca2+ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K+ channel cascade. Trends Neurosci 21(12):529–537. Brager DH, Capogna M, Thompson SM (2002) Short-term synaptic plasticity, simulation of nerve terminal dynamics, and the effects of protein kinase C activation in rat hippocampus. J Physiol 541(Pt. 2):545–559. Capogna M, Ga¨hwiler BH, Thompson SM (1995) Presynaptic enhancement of inhibitory synaptic transmission by protein kinases A and C in the rat hippocampus in vitro. J Neurosci 15(2):1249–1260. Chen Y, Cantrell AR, Messing RO, Scheuer T, Catterall WA (2005) Specific modulation of Na+ channels in hippocampal neurons by protein kinase C epsilon. J Neurosci 25(2):507–513. Cole G, Dobkins KR, Hansen LA, Terry RD, Saitoh T (1988) Decreased levels of protein kinase C in Alzheimer brain. Brain Res 452(1–2):165–174. Etcheberrigaray R, Tan M, Dewachter I, Kuipe´ri C, Van der Auwera I, Wera S, Qiao L, Bank B, Nelson TJ, Kozikowski AP, Van Leuven F, Alkon DL (2004) Therapeutic effects of PKC activators in Alzheimer’s disease transgenic mice. Proc Natl Acad Sci U S A 101(30):11141–11146. Hoffman DA, Johnston D (1998) Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC. J Neurosci 18(10):3521–3528. Hongpaisan J, Alkon DL (2007) A structural basis for enhancement of long-term associative memory in single dendritic spines regulated by PKC. Proc Natl Acad Sci U S A 104(49):19571–19576. Hongpaisan J, Xu C, Sen A, Nelson TJ, Alkon DL (2013) PKC activation during training restores mushroom spine synapses and memory in the aged rats. Neurobiol Dis 55:44–62. Hu GY, Hvalby O, Walaas SI, Albert KA, Skjeflo P, Andersen P, Greengard P (1987) Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation. Nature 328(6129):426–429. Hussain RJ, Carpenter DO (2003) The effects of protein kinase C activity on synaptic transmission in two areas of rat hippocampus. Brain Res 990(1–2):28–37. Jiang L, Sun S, Nedergaard M, Kang J (2000) Paired-pulse modulation at individual GABAergic synapses in rat hippocampus. J Physiol 523(Pt. 2):425–439. Kanno T, Yaguchi T, Yamamoto S, Yamamoto H, Fujikawa H, Nagata T, Tanaka A, Nishizaki T (2005) 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors. J Neurochem 95(3):695–702 (epub 2005 Aug 31). Kazanietz MG, Lewin NE, Gao F, Pettit GR, Blumberg PM (1994) Binding of [26-3H]bryostatin 1 and analogs to calcium-dependent and calcium-independent protein kinase C isozymes. Mol Pharmacol 46(2):374–379.

85

Kim H, Han SH, Quan HY, Jung YJ, An J, Kang P, Park JB, Yoon BJ, Seol GH, Min SS (2012) Bryostatin-1 promotes long-term potentiation via activation of PKCa and PKCe in the hippocampus. Neuroscience 226:348–355. Kuzirian AM, Epstein HT, Gagliardi CJ, Nelson TJ, Sakakibara M, Taylor C, Scioletti AB, Alkon DL (2006) Bryostatin enhancement of memory in Hermissenda. Biol Bull 210(3):201–214. Maffei A (2011) The many forms and functions of long term plasticity at GABAergic synapses. Neural Plast 2011:1–9. Maher P (2001) How protein kinase C activation protects nerve cells from oxidative stress-induced cell death. J Neurosci 21(9):2929–2938. Malenka RC, Madison DV, Andrade R, Nicoll RA (1986) Phorbol esters mimic some cholinergic actions in hippocampal pyramidal neurons. J Neurosci 6(2):475–480. McGinty JF, Couce ME, Bohler WT, Ways DK (1991) Protein kinase C subspecies distinguish major cell types in rat hippocampus: an immunocytochemical and in situ hybridization histochemical study. Hippocampus 1(3):293–301. Nakamura M, Sekino Y, Manabe T (2007) GABAergic interneurons facilitate mossy fiber excitability in the developing hippocampus. J Neurosci 27(6):1365–1373. Nelson TJ, Cui C, Luo Y, Alkon DL (2009) Reduction of beta-amyloid levels by novel protein kinase C (epsilon) activators. J Biol Chem 284(50):34514–34521. Pallas SL, Wenner P, Gonzalez-Islas C, Fagiolini M, Razak KA, Kim G, Sanes D, Roerig B (2006) Developmental plasticity of inhibitory circuitry. J Neurosci 26(41):10358–10361. Perkins KL (2006) Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices. J Neurosci Methods 154(1–2):1–18. Prange O, Murphy TH (1999) Correlation of miniature synaptic activity and evoked release probability in cultures of cortical neurons. J Neurosci 19(15):6427–6438. Riedel G, Platt B, Micheau J (2003) Glutamate receptor function in learning and memory. Behav Brain Res 140(1–2):1–47. Robinson PJ, Sontag JM, Liu JP, Fykse EM, Slaughter C, McMahon H, Su¨dhof TC (1993) Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 365(6442):163–166. Rodriguez-Moreno A, Lopez-Garcia JC, Lerma J (2000) Two populations of kainate receptors with separate signaling mechanisms in hippocampal interneurons. Proc Natl Acad Sci USA 97:1293–1298. Shimizu E, Tang YP, Rampon C, Tsien JZ (2000) NMDA receptordependent synaptic reinforcement as a crucial process for memory consolidation. Science 290(5494):1170–1174. Sun MK, Alkon DL (2005) Dual effects of bryostatin-1 on spatial memory and depression. Eur J Pharmacol 512:43–51. Sun MK, Alkon DL (2010) Pharmacology of protein kinase C activators: cognition-enhancing and antidementic therapeutics. Pharmacol Ther 127(1):66–77. Sun MK, Hongpaisan J, Nelson TJ, Alkon DL (2008) Poststroke neuronal rescue and synaptogenesis mediated in vivo by protein kinase C in adult brains. Proc Natl Acad Sci U S A 105:13620–13625. Swartz KJ, Merritt A, Bean BP, Lovinger DM (1993) Protein kinase C modulates glutamate receptor inhibition of Ca2+ channels and synaptic transmission. Nature 361(6408):165–168. Takashima A, Yokota T, Maeda Y, Itoh S (1991) Pretreatment with caerulein protects against memory impairment induced by protein kinase C inhibitors in the rat. Peptides 12(4):699–703. Thomson AM (2000) Facilitation, augmentation and potentiation at central synapses. Trends Neurosci 23(7):305–312 (review). Wang HY, Pisano MR, Friedman E (1994) Attenuated protein kinase C activity and translocation in Alzheimer’s disease brain. Neurobiol Aging 15(3):293–298. Wang D, Darwish DS, Schreurs BG, Alkon DL (2008) Analysis of long-term cognitive-enhancing effects of bryostatin-1 on the rabbit (Oryctolagus cuniculus) nictitating membrane response. Behav Pharmacol 19(3):245–256.

86

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

Wilkinson SE, Parker PJ, Nixon JS (1993) Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J 294(Pt. 2):335–337. Xu C, Cui C, Alkon DL (2009) Age-dependent enhancement of inhibitory synaptic transmission in CA1 pyramidal neurons via GluR5 kainate receptors. Hippocampus 19(8):706–717. Yaguchi T, Nagata T, Mukasa T, Fujikawa H, Yamamoto H, Yamamoto S, Iso H, Tanaka A, Nishizaki T (2006) Linoleic acid

derivative DCP-LA improves learning impairment in SAMP8. Neuroreport 17(1):105–108. Yamamoto S, Kanno T, Nagata T, Yaguchi T, Tanaka A, Nishizaki T (2005) The linoleic acid derivative FR236924 facilitates hippocampal synaptic transmission by enhancing activity of presynaptic alpha7 acetylcholine receptors on the glutamatergic terminals. Neuroscience 130(1):207–213.

(Accepted 6 March 2014) (Available online 15 March 2014)