Critical involvement of postsynaptic protein kinase activation in LTP at hippocampal mossy fiber synapses on CA3 interneurons

doi:10.1523/JNEUROSCI.5269-09.2010

J Neurosci. Author manuscript; available in PMC 2010 August 24.

Published in final edited form as:

J Neurosci. 2010 February 24; 30(8): 2844–2855.

doi: 10.1523/JNEUROSCI.5269-09.2010

PMCID: PMC2854556

NIHMSID: NIHMS181644

Critical involvement of postsynaptic protein kinase activation in LTP at hippocampal mossy fiber synapses on CA3 interneurons

Emilio J. Galván,^1,⁴ Kathleen E. Cosgrove,^1,³ Jocelyn C. Mauna,² J. Patrick Card,^1,³ Edda Thiels,^2,³ Stephen D. Meriney,^1,³ and Germán Barrionuevo^1,³

¹Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

²Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260

³Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260

Correspondence should be addressed to: Germán Barrionuevo, Department of Neuroscience, A210 Langley Hall, University of Pittsburgh, PA. 15260; Phone: (412) 624-7330; Fax: (412) 624-9198. Email: german/at/pitt.edu

⁴Present address: Departamento de Fisiología Biofísica y Neurociencias, CINVESTAV-IPN, Apartado Postal 14-740, México City 07000, México

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Abstract

Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable of undergoing a Hebbian form of NMDAR-independent LTP induced by the same type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF input to L-M interneurons occurs only at synapses containing mostly calcium impermeable (CI)-AMPARs. Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation of protein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone or in combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapses predominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specific PKA inhibitor peptide PKI_6–22 failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsing onto L-M interneurons loaded with PKI_6–22 induced posttetanic potentation (PTP) but not LTP. Hippocampal sections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strata radiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetate induced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFS delivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significant depression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARs requires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.

Keywords: LTD [Long-Term Depression], Mossy Fiber, Interneurons, Ca3, Kinase, Synaptic plasticity

Introduction

The involvement of protein kinase A (PKA) and protein kinase C (PKC) in the induction and maintenance of LTP in hippocampal pyramidal cells has been studied intensively (Nicoll and Malenka, 1995; Soderling and Derkach, 2000). In area CA3, PKA plays a fundamental role in the induction and maintenance of the N-methyl-D-aspartate receptor (NMDAR)-independent form of LTP at mossy fiber (MF)-to-pyramidal cell synapses (Hopkins & Johnston, 1988; Huang et al., 1994; Weisskopf et al., 1994; Villacres et al., 1998; Yeckel et al., 1999; Calixto et al., 2003). Furthermore, it is well established that PKA modulates the activation of the L-type voltage-gated Ca²⁺ channels (VGCCs) required for the induction of NMDAR-independent LTP at Schaffer collateral-to-pyramidal cell synapses (Grover and Teyler, 1990) as well as MF-to-pyramidal cell synapses (Kapur et al., 2001). PKC also was found to play a role in LTP at MF-to-pyramidal cell synapses. Early evidence showed that presynaptic PKC activation was required for MF LTP induction (Son and Carpenter, 1996; Son et al., 1997; Henze et al., 2000), and more recently, postsynaptic PKC was shown to be necessary for NMDAR-LTP at MF synapses on pyramidal cells (Kwon and Castillo, 2008).

Numerous studies have revealed the extensive heterogeneity in interneuron long-term synaptic plasticity, especially with respect to the mechanisms controlling the induction and polarity of synaptic change (Kullmann and Lamsa, 2007). However, little is known about the intracellular signaling cascades subserving synaptic plasticity at excitatory inputs to hippocampal interneurons. Previous work has shown that postsynaptic activation of the Ca²⁺-calmodulin signaling pathway up-regulates glutamatergic transmission at inputs to interneurons residing in stratum pyramidale of area CA1 (Wang and Kelly, 2001). Potentiation of dendritic Ca²⁺ signaling at inputs to CA1 alveus/oriens interneurons was found to require the co-activation of mGluR5s and PKC (Topolnik et al., 2009). Other studies have focused on the role of protein kinases in activity-dependent plasticity at MF synapses on CA3 interneurons. For example, the presynaptic induction of LTD at the MF input to CA3 interneurons in stratum lucidum is mediated by activation of mGluR7 and PKC at MF terminals (Pelkey et al., 2005). Furthermore, the internalization of mGLuR7s enables a presynaptic form of MF LTP that requires PKA activation (Pelkey et al., 2008). In the dentate gyrus, the postsynaptic induction of MF LTP in basket cells was reported to be blocked by the extracellular application of an inhibitor of PKC (Alle et al., 2001). Therefore, it remains an open question as to whether PKC signaling exerts its action on MF LTP within the pre- or postsynaptic compartment of the MF-to-basket cell synapse.

We previously demonstrated that HFS delivered to MF synapses on CA3 lacunosum-moleculare (L-M) interneurons containing predominantly Ca²⁺-impermeable (CI) AMPARs causes a robust PTP and a NMDAR-independent form of Hebbian LTP at these synapses (Galvan et al., 2008). Here we provide compelling evidence that induction of this form of LTP at the MF-to-L-M interneuron synapse requires postsynaptic action of PKA and PKC, signaling molecules typically associated with several forms of LTP at glutamatergic synapses on hippocampal pyramidal cells.

Materials and Methods

Electrophysiological recordings

Animal use was in accordance with the University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (25 ± 5 days old; Zivic Miller Company) were deeply anaesthetized (Nembutal, IP, 50 mg/kg) and perfused intracardially with a modified artificial cerebrospinal fluid ACSF in which sucrose was substituted for NaCl (in mM): 230 sucrose, 1.9 KCl, 1.2 Na₂PO₄*7H₂O, 25 NaHCO₃, 10 glucose, 1 CaCl₂, 4 MgCl₂, at 4°C. Following 1–2 minutes of perfusion, animals were decapitated and the brains removed. Blocks of hippocampus were glued to the stage of a Leica VT1000S and cut in 385-µm thick sections. Slices were maintained for at least 120 min in an incubation solution of the following composition (in mM): 125 NaCl, 2 KCl, 1.2 NaH₂PO₄, 25 NaHCO₃, 10 glucose, 1 CaCl_2, and 6 MgCl₂. The solution was maintained at pH 7.3 with bubbled with O₂ (95%)/CO₂ (5%) at room temperature. The slices were then transferred to a submersion recording chamber and superfused at a constant flow rate (2.5 ml/min; 24 sec exchange rate including the volume inside the tubing) via a peristaltic pump (Watson Marlow 400) using the following solution (in mM): 125 NaCl, 3 KCl, 1.25 Na₂HPO₄, 25 NaHCO₃, 2 CaCl₂, 1 MgCl₂, 10 glucose, 0.01 (-)-Bicuculline methobromide), and 0.05 D-AP5 (D-2-amino-5-phosphonopentanoic acid). The temperature of the solution in the recording chamber was maintained at 33 ± 1°C. Interneurons were identified visually with infrared video microscopy and DIC optics. Patch pipettes were pulled from borosilicate glass and had resistances of 3–6 MΩ when filled with a solution containing (in mM) 120 K-methylsulphate, 10 NaCl, 10 KCl, 10 HEPES, 0.5 EGTA, 4.5 Mg.ATP, 0.3 Na₂.GTP, and 14 phosphocreatine. Biocytin, (0.1%) was routinely added to the pipette solution to allow subsequent morphological identification and reconstruction of interneurons (Fig. 1A). In experiments aimed at assessing the current-voltage relationship (I–V) of MF AMPA EPSCs, QX-314 (5 mM) was added to the internal solution. Osmolarity was adjusted to 295 – 300 mOsm with a pH 7.2 – 7.3. Access resistance was monitored throughout the duration of the experiments using 10-ms, 5-mV voltage steps. Interneurons were accepted for analysis only when the seal resistance was >1 GΩ and the series resistance (4–9 MΩ) did not change >15% during the experiment.

Figure 1

Selective activation of MF synapses onto the spineless dendrite of L-M interneurons (A) Schematic representation of the hippocampus slice preparation showing the position of the bipolar stimulating electrode (MF_SDG) placed on the tip of the suprapyramidal (more ...)

MF synaptic responses were evoked by extracellular stimulation using concentric bipolar electrodes (12.5 µm inner pole diameter, 125 µm outer pole diameter; FHC Inc., ME) positioned on the suprapyramidal blade of the dentate gyrus (SDG) (Galvan et al., 2008) (Fig. 1A). Stimulation provided by an A.M.P.I. Master 8 connected to a stimulus isolation unit (WPI stimulus isolator A365) consisted of single monopolar pulses (100 to 300 µA; 50–100 µs duration) at ranges of 0.25 Hz to 0.16 Hz. To reduce the probability of antidromic activation of CA3 pyramidal cells via stimulation of their axon collaterals, we used low current intensities that resulted in composite EPSPs with amplitudes less than 30% of the threshold amplitude required to evoke action potentials in the recorded interneurons. Current- and voltage-clamp recordings were obtained with an Axoclamp-1D amplifier (Axon Instruments) in the presence of (-)-bicuculline methiodide (10 µM) and D-2-Amino-5-phosphonovaleric acid (D-AP5; 50 µM), to block GABA_AR- and NMDAR-mediated responses, respectively. Paired pulse facilitation (PPF) was expressed as the paired pulse ratio (PPR) between the average amplitude of the second EPSP to that of the first EPSP in the pair. Loading of the peptides (PKI_6–22, Rp-cAMPs or chelerythrine) was carried out at least 20–25 min before the recording of control MF EPSPs. Signals were low-pass filtered at 5 kHz, digitized at 10 kHz, and stored on disk for off-line analysis. Data acquisition and analysis were performed using customized LabView programs (National Instruments, Austin TX). The group II metabotropic glutamate receptor agonist 2S, 2’R, 3’R)-2-(2’,3’-dicarboxycyclopropyl) glycine (DCG-IV; 1 or 5 µM) was applied at the end of every experiment to confirm the MF origin of the EPSP. Although inhibition of MF transmission by DGC-IV is consistently complete at synapses on pyramidal cells (≥90%); (Kamiya and Yamamoto, 1997), it is variable at MF synapses on interneurons (Alle et al., 2001; Lawrence et al., 2004). Therefore, synaptic responses were classified as of MF origin only if DCG-IV application resulted in inhibition >70% (Lawrence et al., 2004; Galvan et al., 2008). LTP was induced by HFS consisting of 3 trains of 100 pulses delivered at 100 Hz with an intertrain interval of 10 sec. Delivery of each stimulation train to MFs was paired with a simultaneous postsynaptic depolarizing current step (30 ± 0.6 pA; 1 sec). This postsynaptic depolarizing pulse was strong enough to evoke a robust AP burst.

Drugs

D-AP5, (-)-Bicuculline methobromide, and DCG-IV were purchased from TOCRIS (Ellisville, MO). H-89 was from BioMol International LP. Otherwise drugs were purchased from Sigma Chemical (St. Louis, MO). Forskolin, IBMX, H89, Bisindolymaleimide-1, and PDA, were dissolved in DMSO at concentrations of 100, 100, 10, 2, and 25 mM, respectively, and then added to the bath solution. The concentration of DMSO in the final bath solution was 0.1%; otherwise, drugs were dissolved in double distillated H₂O.

Confocal Imaging

L-M interneurons were filled with Lucifer Yellow (1mg / mL) using visually guided whole cell patch clamp. After filling the cell, the slice was loaded with DiI crystals in the SDG (location matched typical placement of stimulating electrode described above) in order to visualize axons from the dentate gyrus granule cells. In a separate set of experiments, DiI crystals were localized in the s. radiatum to label granule cell soma in a retrograde fashion. Slices were returned to the incubation solution for at least 2 hours before being fixed & mounted on slides using Flouromount-G mounting medium. DiI was excited at 514 nm and Lucifer Yellow was excited at 488 nm. Images were collected using an inverted Olympus Fluoview 1000 at the Center for Biologic Imaging at the University of Pittsburgh (3500 Terrace Street, S233 BST, Pittsburgh, PA 15261 USA).

Immunoperoxidase localizations

Rats were anesthetized with an intraperitoneal injection of chloral hydrate (300 mg/kg; dissolved in 0.9% saline) and rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer. The brains were removed following perfusion and placed in a 20% sucrose solution until they sank. The brains were frozen and sectioned at a thickness of 40 µm on a cryostat at −16° C and stored in cryopreservative at −20 °C (Watson et al., 1986) until staining. To probe the sections for PKA immunoreactivity, sections were brought to room temperature over a 30-min period, washed with sequential rinses in 50 mM Tris-buffered saline (TBS) on a rocker table, pretreated with an antigen retrieval citrate buffer at 80°C for 20 min, and rinsed again several times in 50 mM TBS. The tissue was treated to permeabilize and prevent unwanted non-specific staining with 50mM TBS containing 0.4% Triton X-100 and 10% normal goat serum at room temperature for 1 hr. Sections then were incubated with an antibody that selectively recognizes the α isoform of the catalytic subunit of PKA (1:1000; Cell Signaling, Beverly, MA) in 50 mM TBS containing 0.4% Triton X-100 and 10% normal goat serum at 4°C for 48 to 72 hr. To confirm specificity of the immunocytochemical localization, alternate sections were incubated with primary antibody (PKA C-α) that had been pre-incubated with a blocking peptide (Cell Signaling) following the manufacturer’s specifications. Briefly, the antibody was incubated in a solution containing the blocking peptide at 2X concentration of the primary antibody for 1 hr at room temperature before incubation of sections. After washing in 50 mM TBS over 45 min, sections were incubated in biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) in 50 mM TBS containing 0.4% Triton X-100 for 90 min, followed by incubation in ABC complex in 50 mM TBS containing 0.4% Triton X-100 (Vectastain Elite Kit) for 90 min at room temperature on a rotator. After washing the sections in 50 mM TBS, immunostaining was visualized with a substrate solution containing DAB 0.6 mg/ml + 0.03% H₂O₂ + 4 mM NiCl₂ in TBS. Sections were washed several times and then mounted on gelatin-coated slides, dried at room temperature, dehydrated in ethanol, cleared in xylene, and coverslipped with Protocol xylene based medium. Immunocytochemical staining was photographed using an Olympus BX51 photomicroscope equipped with DIC optics. All images were captured using Simple PCI software (C-Imaging Systems; Cranberry, PA). Adobe Illustrator and Adobe Photoshop software were used to assemble illustrations. The brightness and contrast of images was adjusted using Adobe Photoshop, but the color balance was not altered.

Immunofluorescence localizations

To probe the sections for both glutamate decarboxylase (GAD), an interneuron-specific marker, and PKA immunoreactivity, sections were brought to room temperature over a 30-min period, washed with sequential rinses in 0.1M Phosphate Buffered Saline (PBS) on a rocker table, pretreated with an antigen retrieval Tris buffer at 80°C for 20 min, and rinsed again several times in 0.1M PBS. The tissue was pretreated with 0.15M glycine in 0.1M PBS to prevent autofluorescence, and then permeabilized and blocked with 0.1M PBS containing 0.4% Triton X-100 and 10% normal goat serum at room temperature for 1 hr to prevent unwanted non-specific staining. Sections then were incubated with the antibody that selectively recognizes PKA C-α (1:500; Cell Signaling, Beverly, MA) and an antibody that selectively recognizes the 67 isoform of GAD (1:500; Millipore, Temecula, CA) in 0.1M PBS containing 0.4% Triton X-100 and 10% normal goat serum at 4°C for 48 to 72 hr. After washing in 0.1M PBS over 45 min, sections were incubated in biotinylated goat anti-rabbit secondary antibody (Vector Labs) in 0.1M PBS containing 0.4% Triton X-100 for 90 min, followed by sequential washes and incubation in Cy3-Streptavidine and Cy2-anti-mouse secondary antibody in 0.1M PBS containing 0.4% Triton X-100 for 90 min at room temperature on a rotator. After washing the sections in 0.1M PBS, they were mounted on gelatin-coated slides, dried at room temperature, and coverslipped with Fluoromount. Immunofluorescent staining was visualized and captured using a Nikon Diaphot 2000 confocal microscope equipped with a BioRad MRC1024 acquisition system. Improvision Openlab software was used to adjust the brightness and contrast of the images, without altering color balance, and merge the adjusted images for detection of overlap. Adobe Illustrator and Adobe Photoshop software were used to assemble illustrations.

Statistical analysis

Group measures are expressed as means ± S.E.M. Normality of sample distributions was tested with Kolmogorov-Smirnov test, followed by one-way ANOVA and Student-Newman-Keuls pairwise comparisons. In all cases differences were considered significant if P was less than alpha = 0.05. In figures, statistical significance is denoted as follows: * P < 0.05, ** P < 0.01 and *** P < 0.001 (or higher).

Results

Interneurons were identified by visual localization of their soma within L-M (200–300 µm from the boundary between str. pyramidale and str. lucidum, 50–180 µm from tip of dentate gyrus; Ascoli et al., 2009). Recordings were obtained using visually guided whole-cell patch-clamp techniques, as previously described (Calixto et al., 2008; Galvan et al., 2008). CA3 interneurons receive synaptic input from MF small en passant varicosities emerging from the parent axon, and from filopodial extensions of the giant MF bouton on CA3 pyramidal cells (Acsady et al., 1998). Figure 1A shows a schematic representation of the hippocampal slice preparation detailing the typical location of L-M interneurons and the stimulating electrode used to evoke responses from MF synapses. Post-hoc reconstruction of biocytin-labeled L-M interneurons revealed wide-reaching dendritic branches, some of which course near the suprapyramidal blade of the dentate gyrus (SDG; Ascoli et al., 2009), where they are coextensive with MF traveling to s. lucidum or with MF collateral plexuses in the hilus of the dentate gyrus (Claiborne et al., 1986; Acsady et al., 1998) (Fig. 1B). We have previously shown that focal applications of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) restricted to the str. lacunosum-moleculare of area CA3 selectively abolished MF responses in L-M interneurons evoked by stimulation applied to SDG (Cosgrove et al., in press). We surmise from these data that the results presented here were obtained from MF responses that were selectively evoked by synapses near SDG.

Adenylyl cyclase stimulation induces MF potentiation of naïve CI-AMPAR synapses on L-M interneurons

MF synapses on hippocampal interneurons have both CI-AMPARs and CP-AMPARs (Dingledine et al., 1999; Toth et al., 2000; Bischofberger and Jonas, 2002). We previously demonstrated that NMDAR-independent LTP at MF inputs to L-M interneurons is induced exclusively at synapses containing predominantly CI-AMPARs (Galvan et al., 2008). CI-AMPAR EPSCs exhibited a nearly linear current-voltage relationship (I–V) whereas CP-AMPAR EPSCs showed strong inward rectification (Toth and McBain, 1998; Laezza et al., 1999; Galvan et al., 2008). Despite that the large and complex dendritic arbors of L-M interneurons (Ascoli et al. 2009; Fig. 1B) are likely to distort the somatically recorded EPSC waveform due to inadequate space clamp (Williams & Mitchell, 2008), MF synaptic responses can be easily grouped by their non-rectifying or rectifying properties (Fig. 2A).

Figure 2

Forskolin induces MF potentiation only at predominantly CI-AMPAR synapses on L-M interneurons. All experiments were performed in the presence of bicuculline (10 µM) and D-AP5 (50 µM). (A) Representative current-voltage (I–V) relationship (more ...)

Activation of adenylyl cyclase with forskolin (FSK) was found to induce NMDAR-independent long-lasting enhancement of MF synaptic transmission to CA3 pyramidal cells (Hopkins & Johnston, 1988; Huang et al., 1994; Weisskopf et al., 1994; Villacres et al., 1998; Calixto et al., 2003) and dentate gyrus basket cells (Alle et al., 2001). Both types of potentiation were shown to be blocked by inhibition of PKA (Huang et al., 1995). Here we induced plasticity at MF inputs to L-M interneurons by bath application of FSK (50 µM for 10 min) and used the differential current-voltage characteristic of CI-versus CP-AMPAR EPSCs to determine the predominant AMPAR type mediating FSK-induced MF synaptic plasticity.

Consistent with our previous report (Galvan et al., 2008), L-M interneurons with MF synapses expressing mostly non-rectifying AMPARs constituted the majority of the recorded cells (~80%; pie chart in Fig. 2). The slope of the MF EPSP evoked from this significant subpopulation of interneurons exhibited a sustained increase in the EPSP slope after wash-out of FSK (174 ± 29% of baseline 30 min post FSK; P < 0.0001, n = 5). In contrast, the slope of MF EPSPs from interneurons with predominantly rectifying AMPARs showed a persistent depression after FSK treatment (51 ± 23% of baseline 30 min post FSK; P < 0.01, n = 3; Fig 2B, 2C), in agreement with previous observations by others (Maccaferri et al., 1998; McBain et al., 1999). Bath application of the group II mGluR agonist DCG-IV (1 µM) confirmed the MF origin of both CI- and CP-AMPAR-mediated responses (86 ± 2% and 78 ± 5% inhibition 20 min after onset of DCG-IV application, respectively; P < 0.001; Fig 2B, 2C). Unlike FSK, 1,9-dideoxyforskolin (50 µM for 10 min, supplemental Fig. 1), an inactive analogue of FSK, did not affect either the slope of the MF EPSP (104 ± 5% of control at 10 min post application, P > 0.5; DCG-IV sensitivity, 77 ± 29% inhibition, P < 0.001; n = 6) or the paired-pulse ratio (PPR; control PPR: 1.52 ± 0.20; PPR at the end of the 10 min application of 1,9-dideoxyforskolin: 1.51 ± 0.15, P >0.4, n = 6). Together these data show that transient activation of adenylyl cyclase is sufficient to trigger potentiation of MF EPSPs produced by naïve CI-AMPA synapses on L-M interneurons. The data also demonstrate that non-rectifying MF synapses on L-M interneurons are highly responsive to FSK, strongly suggesting that CI-AMPARs may selectively be linked to the activation of the cAMP/PKA cascade.

We also examined the effect of FSK (50 µM) in combination with the cyclic nucleotide phosphodiesterase inhibitor, 1-Methyl-3-isobutylxanthine (IBMX, 50 µM), (Huang et al., 1994; Duffy and Nguyen, 2003), on the isolated MF CI-AMPAR component. Bath perfusion with the polyamine toxin philanthotoxin (PhTx, 5 µM), a selective blocker of CP-AMPARs, minimally decreased the amplitude of MF EPSCs (Vh = −70 mV; PhTx reduction: 9 ± 3% of baseline; P > 0.2; n = 4; Fig. 3A). At the end of PhTx application, the recording was switched to current-clamp conditions (−70 mV), a baseline was recorded for 8 min, and FSK (50 µM) + IBMX (50 µM) were added to the bath perfusion for 10 min. Compared to the effect of application of FSK, co-application of FSK/IBMX induced a fast and sustained potentiation of the slope of the MF EPSP that was larger than the potentiation caused by FSK alone (334 ± 54% of control 10 min after onset of FSK/IBMX; P < 0.0001; Fig. 3A). After wash-out, the slope of the MF EPSP gradually decayed to a plateau level that remained potentiated above control level for at least 30 min post wash-out (170 ± 15.6% of baseline; P < 0.001; Fig. 3A). DCG-IV (5 µM) was applied at the end of the experiments to confirm the MF nature of the responses (84 ± 3% inhibition; P < 0.001; Fig. 3A). We use a higher concentration of DCG-IV because the stimulation of the adenylyl cyclase by FSK produces a PKA-dependent phosphorylation of the mGluR2 receptor, which counteracts the selective inhibitory action of group II mGluR activation on MF boutons (Kamiya and Yamamoto, 1997; Schaffhauser et al., 2000).

Figure 3

The simultaneous inhibition of phosphodiesterases and stimulation of adenylyl cyclase induces potentiation of predominantly CI-AMPAR MF synapses on L-M interneurons. (A) Representative experiment showing the time course of the amplitude of MF-evoked EPSCs (more ...)

The FSK/IBMX-induced potentiation of the MF EPSP was associated with a decrease in paired-pulse facilitation. A decrease in PPR generally is regarded as indicative of a presynaptic effect due to increase in transmitter release (Weisskopf et al., 1994). Figure 3B shows the time course of the PPR in the presence of FSK/IBMX. Bath-application of PhTx (5 µM) did not alter the PPR (control PPR: 1.44 ± 0.1; PPR during PhTx application: 1.42 ± 0.12, P > 0.3; n = 4). However, PPR decreased significantly during application of FSK/IBMX (control PPR: 1.45 ± 0.07; FSK/IBMX PPR: 0.97 ± 0.08; P < 0.01; n = 4), and recovered only partially during wash-out (1.31 ± 0.06, at 30 min post wash-out).

A comparable MF potentiation as described above also was observed in experiments in which the CI-AMPAR component was not isolated (slope of MF EPSP during the FSK/IBMX application, 397 ± 31% of baseline, P < 0.0001; 30 min post wash-out, 185 ± 30% of baseline; P < 0.001; n = 7; DCG-IV sensitivity, 77 ± 29% inhibition; n = 7; Fig. 3C). Given that these cells exhibited a level of potentiation upon FSK/IBMX application similar to that observed after isolation of CI-AMPAR-mediated responses, it seems reasonable to assume that the MF-evoked EPSPs recorded here also were mediated predominantly by CI-AMPARs. Because the magnitude of the FSK/IBMX effect did not differ significantly between the two groups of recorded interneurons, the data were pooled for the analysis shown in Figure 3C. Recordings from cells for which the MF EPSP was contaminated by a polysynaptic component that evoked action potentials during FSK/IBMX application were excluded from all analyses.

The demonstration of FSK-mediated potentiation of MF responses in L-M interneurons is indicative that FSK binding sites are present as previously shown for the giant MF boutons on CA3 pyramidal cells (Worley et al., 1986). Therefore, one possible interpretation of these results is that MF potentiation during FSK application is due to a presynaptic effect (Weisskopf et al., 1994; Nicoll and Malenka, 1995; Lopez-Garcia et al., 1996; Villacres et al., 1998). However, there is also strong evidence for postsynaptic regulation of MF synaptic transmission by cAMP-dependent mechanisms (Yeckel et al., 1999; Sivakumaran et al., 2009). Indeed, our data demonstrating that FSK-mediated potentiation is selective for MF synapses containing predominantly CI-AMPARs are consistent with a postsynaptic locus of PKA activation. To obtain direct evidence on the postsynaptic contribution to the MF potentiation induced by stimulation of the cAMP/PKA pathway, L-M interneurons were loaded with the specific cell-impermeant PKA inhibitor, peptide PKI_6–22 (20 µM) (Duffy and Nguyen, 2003; Skeberdis et al., 2006; Sivakumaran et al., 2009) prior to the application of FSK/IBMX. Bath-application of FSK (50 µM) + IBMX (50 µM) resulted in a similar level of potentiation of the slope of MF EPSPs from control vs. PKI_6–22-loaded cells (maximum slope value in the presence of FSK/IBMX in control cells, 397.4 ± 31% of baseline, n = 10, Fig. 4A, filled circles, 4B filled bars; in cells loaded with PKI_6–22, 394.7 ± 35.3% of baseline; n = 6; Fig. 4A, open circles, 4B open bars; P > 0.5). The effect of FSK/IBMX on PPR also was similar in control vs. PKI_6–22-loaded cells (PPR at 10 min of application of FSK/IBMX in control cells, 0.97 ± 0.08; in PKI_6–22-loaded cells, 0.96 ± 0.1; P > 0.5; data not shown). However, in contrast to the persistence of the FSK/IBMX-induced potentiation in the absence of the inhibitor (filled circles, Fig. 4A; filled bars, Fig. 4B), the FSK/IBMX-induced potentiation in cells loaded with PKI_6–22 was not maintained but decayed rapidly such that the slope of the MF EPSP no longer differed significantly from baseline values 30 min post (116 ± 35% of baseline, P > 0.1; open circles, Fig. 4A; open bars, 4B). Taken together, these data suggest that PKA activation in presynaptic elements is sufficient to produce a robust but transient potentiation of transmission at MF synapses on L-M interneurons. However, increased PKA activation in postsynaptic elements is required for the maintenance of FSK/IBMX-induced MF potentiation at synapses on L-M interneurons, similar to the scenario that was described for FSK/IBMX-induced potentiation at synapses on pyramidal cells (Frey et al., 1993; Huang et al., 1994; Duffy and Nguyen, 2003).

Figure 4

Temporal sequence of projected pre- and postsynaptic PKA contributions to MF potentiation. (A) Time course of the normalized slope of MF-evoked EPSPs before, during, and after a 10-min application of IBMX + FSK (indicated by filled bar), as recorded either (more ...)

To determine the time-course of the isolated postsynaptic component of the FSK-mediated MF potentiation, we performed a mathematical subtraction between the values observed in the presence of FSK/IBMX and those observed in the presence of FSK/IBMX + PKI_6–22. The plot of the residual values suggests that shortly before the beginning of the wash-out, PKA activity in postsynaptic compartments emerges as the critical contributor to the FSK/IBMX-induced MF potentiation at synapses on L-M interneurons (Fig. 4A, blue line). A critical contribution of postsynaptic PKA signaling also was found to apply to the maintenance of the late phase of LTP at hippocampal pyramidal cell synapses (Frey et al., 1993; Huang et al., 1994; Bolshakov et al., 1997; Woo et al., 2002).

Postsynaptic inhibition of PKA prevents HFS-induced LTP at MF synapses on L-M interneurons

The previous results show that postsynaptic PKA activation is necessary for the maintenance of FSK-induced MF potentiation in L-M interneurons. Therefore, we hypothesized that HFS (100 pulses at 100 Hz, 10-sec interpulse interval) that triggers LTP at MF inputs to L-M interneurons is sensitive to inhibition of PKA. In agreement with previous work (Galvan et al., 2008), HFS delivered to the MF input innervating predominately CI-AMPAR synapses on L-M interneurons (confirmed by only a slight PhTx effect: 6 ± 2% of baseline; n = 11; P > 0.11, supplemental Fig. 2A) caused robust PTP of the MF EPSP (203 ± 6% of baseline) followed by sustained LTP of the slope of the MF EPSP (163 ± 17% of baseline 30 min post-HFS; P < 0.001; DCG-IV inhibition, 67 ± 11% of baseline; n = 11; P < 0.01; Supplemental Figs. 2A, 2B). To test the effect of PKA inhibition on MF LTP, hippocampal slices were incubated in ASCF containing the general kinase inhibitor H-89 (10 µM) for 30 – 45 min prior to recording. After obtaining a stable baseline, delivery of HFS induced a robust PTP (181 ± 22% of baseline), but a prolonged potentiation was not observed. Instead, the evoked response returned to baseline level within 30 min (89 ± 16% 30 min post-HFS; n = 7; P > 0.11; DCG-IV (1 µM) inhibition, 70 ± 5%; P < 0.01; Fig. 5A). To determine whether inhibition of PKA may have contributed to this effect, we included in the patch pipette Rp-cAMP (50 µM), a membrane-permeable inhibitor of PKA activation. Baseline MF EPSPs were obtained for at least 10 min to allow Rp-cAMP to diffuse into the cells. Delivery of HFS to the MF input induced PTP (185 ± 14% of baseline) but failed to induce LTP (87 ± 10% at 30 min post-HFS; n = 8; P > 0.1; DCG-IV sensitivity, 82 ± 10% inhibition; P < 0.001; Fig. 5A).

Figure 5

Postsynaptic blockade of PKA prevents LTP at MF synapses on L-M interneurons. (A) Bar graphs summarizing MF-evoked responses after delivery of HFS (3 trains with 100 pulses at 100 Hz, delivered with a 10-sec intertrain interval) to the MFs. LTP was induced (more ...)

Because Rp-cAMP was shown to affect other targets in addition to PKA, its use for determining the role of PKA in LTP has been put into question (Otmakhov and Lisman, 2002; Duffy and Nguyen, 2003). Therefore, to determine the role of postsynaptic PKA in HFS-induced LTP at MF synapses on interneurons, we loaded L-M interneurons with PKI_6–22 (20 µM), as described above. In a first group of experiments (n = 4), we assessed the effect of PKI_6–22 on the isolated CI-AMPAR component of the MF-evoked response by including PhTx (5 µM) in the perfusion bath before delivery of HFS to the MF. In voltage-clamp conditions (Vh = −70 mV), PhTx moderately reduced the amplitude of MF EPSCs (6 ± 1% of baseline; P > 0.12, Fig. 5A, B). At the end of PhTx application, recording was switched to current-clamp conditions (−70 mV) and HFS was delivered to the MF input. Following HFS, the isolated CI-AMPAR component of the MF-evoked EPSP exhibited robust PTP (188 ± 30% of baseline), but the potentiation did not persist and the response declined to control levels over the course of the subsequent 10 to 20 min (102 ± 4% of control level 30 min post-HFS; P > 0.1; DCG-IV (1 µM) sensitivity, 75 ± 4% inhibition; P < 0.0001, Fig. 5A, 5B). With a second group of experiments (n = 9), we omitted PhTx from the perfusion. The effect of postsynaptic PKA inhibition, however, was similar in that we observed robust PTP (182 ± 19% of baseline; P < 0.001), but the response potentiation failed to be maintained (97 ± 11% at 30 min post-HFS; P >0.11; DCG-IV sensitivity, 76 ± 7% inhibition; P < 0.001).

Our observations here and in the foregoing section similarly point to PKA in the postsynaptic elements as a critical player in plasticity at MF synapses. Whereas there is ample evidence for localization of PKA in hippocampal pyramidal cells (Gundlach and Urosevic, 1989; Mucignat-Caretta and Caretta, 2002; Havekes et al., 2007), no evidence exists about the presence of PKA in hippocampal interneurons. We therefore immunostained hippocampal sections for the catalytic subunit of PKA and observed abundant immunoreactivity not only in CA1 and CA3 pyramidal cells, dentate granule cells, and MF (Fig. 6A & B);, but also in interneurons located in strata radiatum and L-M of area CA3 (Fig. 6B). To confirm that PKA immunopositive cells in the dendritic strata of CA3 are indeed interneurons, we carried out double-immunofluorescence staining hippocampal sections for both the catalytic subunit of PKA (Fig. 6C) and the interneuron-specific marker glutamate decarboxylase (GAD; Fig. 6D). Merger of the confocal images revealed that most of the cells in strata radiatum and L-M staining immunopositive for GAD also were immunopositive for PKA (Fig. 6E). These findings provide strong evidence for the presence of PKA in CA3 interneurons, including L-M interneurons. Importantly, these findings are in agreement with the idea that PKA signaling within L-M interneurons contributes to plasticity at MF synapses on these interneurons.

Figure 6

Localization of PKA immunoreactivity within the dorsal hippocampus is illustrated in the coronal plane. (A) Immunoreactivity was observed consistently within the apical dendrites of pyramidal cells in areas CA3 and CA1, within the MF pathway of the s. (more ...)

PKC activation induces potentiation and PKC inhibition prevents HFS-induced LTP at MF synapses on L-M interneurons

Previous work revealed that PKC is present in MF terminals (Worley et al., 1986; Terrian et al., 1991) and is required for induction of LTP at MF synapses on both pyramidal cells (Son and Carpenter, 1996) and basket cell interneurons of the dentate gyrus (Alle et al., 2001). We therefore tested whether direct activation of PKC with phorbol 12,13-diacetate (PDA; 2 µM) affects MF-evoked EPSPs recorded from L-M interneurons. Bath-perfusion of PDA for at least 30 min resulted in a persistent increase in the amplitude of MF EPSPs (200 ± 19% of baseline 20 min after onset of PDA application; n = 6; P < 0.001; Fig. 7A). The enhancement was associated with a decrease in the PPR (PPR control, 1.56 ± 0.1; PPR during PDA application, 1.03 ± 0.04, P < 0.0001; Supplemental Fig. 4) indicating a presynaptic expression. We also examined the effect of PDA on the isolated CI-AMPAR component of MF EPSPs by applying PhTx (5 µM) to the perfusion bath before delivery of HFS to the MF. In voltage-clamp conditions (V_h = −70 mV), PhTx only slightly reduced the amplitude of MF EPSCs (96 ± 2% of baseline; P > 0.10; Fig. 7A, B). At the end of PhTx application, recordings were switched to current-clamp conditions (−70 mV) prior to bath-application of PDA. Similar to our previous observations, PDA induced a pronounced increase in the amplitude of the isolated CI-AMPAR synaptic component MF-evoked EPSPs (200 ± 21% of baseline 30 min after onset of PDA application; P < 0.001; n = 4), and this potentiation effect was associated with a decrease in the PPR (control PPR: 1.57 ± 0.2; PDA PPR: 1.01 ± 0.01; P < 0.0001; n = 4; Supplemental Fig 3A, 3B). Because activation of PKC with PDA reduces the ability of group II mGluRs to suppress MF synaptic transmission (Kamiya and Yamamoto, 1997; Macek et al., 1998), we applied an increased concentration of DCG-IV (5 µM) to confirm the MF nature of the EPSPs (DCG-IV sensitivity: 68 ± 2% inhibition; P < 0.001, Fig. 7A, B).

Figure 7

Postsynaptic PKC activity is required for LTP at MF synapses on L-M interneurons. (A) Bar graphs summarizing the effect of the phorbol ester phorbol 12,13-diacetate (PDA; 2 µM) on the slope of MF-evoked EPSPs (left panel); the effect of preincubation (more ...)

Next, we examined whether PKC activity is necessary for HFS-induced LTP at MF synapses on L-M interneurons. We incubated hippocampal slices in the presence of the PKC inhibitor bisindolylmaleimide (Bin-1, 1 µM) for 45 min to 60 min before the start of recordings. As previously reported (Alle et al., 2001), slices pretreated with Bin-1 showed diminished but significant PTP after delivery of HFS (149 ± 9% of baseline; P < 0.05; n = 9), and this blunted potentiation failed to persist, as indicated by a return of the EPSP amplitude to baseline level in the subsequent minutes (96 ± 10% of control 30 min post-HFS, P >0.1; Fig. 7A). DCG-IV (1 µM) confirmed the MF origin of these responses (82 ± 5% inhibition; n = 9).

Finally, we tested whether postsynaptic PKC activity is required for MF LTP at synapses on L-M interneurons. To that end, we included the PKC inhibitor chelerythrine (10 µM) in the patch pipette (Kwon and Castillo, 2008). Although chelerythrine had little effect on PTP (183 ± 10% of baseline; n = 3), HFS resulted in a weak but significant depression of the isolated CI-AMPAR component of the MF EPSP (87 ± 4% of baseline 30 min post-HFS, n = 3, P < 0.05; DCG-IV sensitivity: 89 ± 10% inhibition; P < 0.001; Fig. 7A, 7C). Recordings from cells in which the CI-AMPAR component was not isolated revealed a similar failure to induce LTP, and the development of a slight depression of the MF EPSP when chelerythrine was included in the patch pipette (PTP: 178 ± 9% of baseline; EPSP amplitude at 30 min post-HFS: 78 ± 6%; n = 8; P < 0.01; DCG-IV sensitivity: 82 ± 5% inhibition, P < 0.001, Fig. 7A). Taken together, our results showing that stimulation of PKC activity with phorbol esters causes an increase in MF-evoked EPSPs of L-M interneurons suggest that PKC can modulate transmission at MF inputs to these interneurons. Our findings that LTP fails to develop in the presence of Bin-1 or chelerythrine indicate that LTP of the CI-AMPAR-mediated component of MF-evoked EPSPs requires PKC. Finally, our observations that postsynaptic application of the PKC inhibitor chelerythrine prevented the development of LTP, despite minimal effect on HFS-evoked PTP, suggest that postsynaptic PKC activity is required for LTP of the isolated CI-AMPAR component of MF synapses on L-M interneurons.

Discussion

Although much is known about the signaling events involved in plasticity at glutamatergic synapses on pyramidal cells, there is considerably paucity of information about the identity and regulation of critical molecular players in plasticity at excitatory synapses on interneurons. The purpose of the present experiments was to assess the role of PKA and PKC in LTP of synaptic transmission at MF synapses on interneurons with soma residing in the str. lacunosum moleculare of hippocampal area CA3. We previously showed that MF synapses on L-M interneurons that contain mainly CI-AMPARs are capable of undergoing LTP induced by the same HFS protocol that induces LTP at MF synapses on pyramidal cells (Galvan et al., 2008). The present results demonstrate for the first time that LTP-inducing activity at naïve MF-to-interneuron synapses leads to the downstream activation of postsynaptic signaling cascades that involve the activation of both PKA and PKC. Specifically, we showed that bath application of the adenylyl cyclase activator FSK causes a lasting potentiation at CI-AMPAR-containing MF synapses on L-M interneurons and that this potentiation, as well as HFS-induced LTP at these synapses, is disrupted by introduction of a membrane-impermeable inhibitor of PKA into the postsynaptic cell. We also showed that bath application of the PKC activator PDA similarly causes a lasting potentiation of transmission at CI-AMPAR-containing MF synapses on L-M interneurons. Postsynaptic injection of a membrane-impermeable inhibitor of PKC interfered with LTP induced by HFS of the MF input to L-M interneurons. Collectively, these findings demonstrate that postsynaptic signaling events shown to play a critical role at glutamatergic synapses on pyramidal cells in areas CA3 and CA1 also are crucial participants in the signaling sequelae underlying long-term modification of transmission at naïve MF synapses that contain primarily CI-AMPARs on L-M interneurons.

Our observations of PKA expression in L-M interneurons are consistent with findings by others showing that the induction of MF LTP in CA3 pyramidal cells requires postsynaptic PKA (Yeckel et al., 1999; Sivakumaran et al., 2009). On the other hand, FSK binding sites are highly expressed at the giant MF bouton on CA3 pyramidal cells (Worley et al., 1986), and previous work demonstrated that presynaptic PKA activity is required for the induction of MF LTP in pyramidal cells (Lonart and Sudhof, 1998; Castillo et al., 2002; Pelkey et al., 2008) and str. lucidum interneurons (Pelkey et al., 2008) as well at Schaffer collateral synapses on CA1 pyramidal cells (Blitzer et al., 1995; Blitzer et al., 1998). Indeed, we found that FSK application produced a robust, albeit transient, presynaptic potentiation in L-M interneurons loaded with the specific PKA inhibitor peptide PKI_6–22. These data would indicate that FSK bindings sites are also present in MF boutons on L-M interneurons. Presynaptic activation of PKA could result in an accelerated vesicle cycling of the readily releasable pool of neurotransmitter (Chavis et al., 1996). Nevertheless, we also show that postsynaptic accumulation of cAMP and PKA activity is necessary for the maintenance of both the chemically-induced potentiation and the electrical-induced MF LTP in L-M interneurons. These findings are consistent with our previous observations indicating that MF LTP in L-M interneurons is postsynaptically induced by a rise in cytosolic [Ca²⁺]_I via activation of L-type VGCCs and mGluR1α, which leads to Ca²⁺ mobilization via IP₃ receptors (IP₃R), and ryanodine receptors (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) from internal stores (IP₃R); (Galvan et al., 2008). We hypothesize that LTP-inducing activities at predominantly CI-AMPAR containing MF synapses lead to PKA activation, most likely via the increase in cAMP production triggered by the Ca²⁺ activation of adenylyl cyclase 1 (Xia et al., 1991; Villacres et al., 1998). In turn, activation of PKA is known to phosphorylate L-type VGCCs (Davare et al., 1999), and enhance the activity of these calcium channels in hippocampal neurons (Kavalali et al., 1997). Furthermore, PKA activation also stimulates Ca²⁺ release from internal stores (Riegel and Williams, 2008). Therefore, the blockade of MF LTP produced by the intracellular injection of PKI_6–22 may be explained by the complete disruption of postsynaptic Ca²⁺ dynamics that follows HFS. Although postsynaptic action of PKA signaling molecules was also shown to be critical for LTP of glutamatergic synapses on hippocampal CA1 pyramidal cells (Duffy and Nguyen, 2003; Esteban et al., 2003; Yang et al., 2008), a postsynaptic role for PKA in hippocampal interneurons does not appear to be a universal principle. For example, examination of LTP at MF synapses on dentate gyrus basket cells revealed that extracellular application of an inhibitor of PKA did not prevent the electrically-induced postsynaptic form of LTP at these synapses, even though bath application of FSK was shown to produce a sustained increase of transmission at these same MF-basket cell synapses (Alle et al., 2001). Furthermore, examination of transmission at naïve MF synapses on interneurons in str. lucidum of area CA3 was revealed to be insensitive to bath application of FSK despite a pronounced potentiating effect of the adenylyl cyclase activator on the MF input to CA3 pyramidal cells (Maccaferri et al., 1998). It was only after L-AP4 mediated internalization of mGLuR7s that MF terminals in the str. lucidum of area CA3 expressed a cAMP/PKA/-mediated presynaptic form of LTP (Pelkey et al., 2008).

Previous work has also shown that the presence of PKC activity in presynaptic MF terminals is necessary for the induction of LTP at MF synapses on pyramidal cells (Terrian et al., 1991; Son and Carpenter, 1996). Furthermore, phorbol ester activation of PKC induces a transient enhancement of glutamate release from the giant MF bouton on CA3 pyramidal cells (Son and Carpenter, 1996; Kamiya and Yamamoto, 1997; Macek et al., 1998; Honda et al., 2000). Here, we show that PDA application also induced a pronounced increase in the amplitude of MF responses from synapses on L-M interneurons. This PDA-induced potentiation was accompanied by a decrease in PPR indicating that an increase in glutamate release due to presynaptic activation of PKC is partly responsible for the synaptic enhancement. This interpretation is consistent with the observations that MF PTP is reduced during bath application of the PKC inhibitor Bin-1. However, postsynaptic PKC blockade prevented the induction of MF LTP while leaving MF PTP intact. A postsynaptic requirement for PKC also was observed for NMDAR-LTP at MF synapses on CA3 pyramidal cells (Kwon and Castillo, 2008), and for NMDAR-dependent LTP in CA1 pyramidal cells (Ling et al., 2002). Similar to the present findings, inhibition of PKC with bath-applied Bin-1 resulted in decreased PTP and blockade of LTP at MF-to-dentate gyrus basket cell synapses (Alle et al., 2001). The observations that PKC activation is involved in LTP at MF synapses on L-M and dentate gyrus basket cell interneurons stand in stark contrast to the findings that PKC activation underlies the HFS-induced LTD at naïve MF synapses on str. lucidum interneurons (Pelkey et al., 2005).

Blockade of L-type VGCCs prevents MF long-term plasticity in L-M interneurons but interference with IP₃R or RyR results in MF LTD induced by the same HFS that induces MF LTP in naive slices (Galvan et al., 2008). This strongly suggests that the rise in postsynaptic [Ca²⁺]_i produced by activation of L-type VGCCs is supplemented by the concomitant activation of IP₃R and RyR-mediated CICR from the internal stores (Nakamura et al., 2000). This interpretation would be consistent with experimental observations suggesting that the mechanisms that regulate the polarity of synaptic efficacy are controlled by the levels of postsynaptic [Ca²⁺]_i (Bear et al., 1987; Artola and Singer, 1993; Hansel et al., 1997; Cormier et al., 2001). Therefore, it is tempting to speculate that the small but persistent post-HFS MF depression in chelerythrine-loaded cells is due to the loss of PKC-mediated facilitatory effects on calcium release from the internal stores (Petersen et al., 1994; Dermitzaki et al., 2004). Presumably this reduction in postsynaptic [Ca²⁺]_i was partially counteracted by the parallel stimulatory actions of PKA activation on L-type VGCCs and CICR, which could have prevented the induction of a full-blown LTD in the absence of PKC activation.

Conclusions

Our current findings provide additional evidence supporting the general notion that the mechanisms underlying MF short-term (Scott et al., 2008) and long-term plasticity (Pelkey et al., 2006) are target-cell-specific and thus, likely to vary across the diversity of interneuron phenotypes receiving input from the dentate granule cells. This diversity shows that any attempt to provide a unified account of the mechanisms underlying interneuron synaptic plasticity from studies of a particular interneuron subtype is problematic. Our data also reveal that the molecular cascades involved in LTP at glutamatergic synapses on some interneuron subtypes may be more similar to those underlying LTP at glutamatergic synapses on pyramidal cells than previously appreciated. Therefore, an intriguing possibility is that other known properties of synaptic plasticity at pyramidal cell synapses, such as MAP kinase signaling (Roberson et al., 1999), de novo synthesis of proteins (Huang et al., 1994; Calixto et al., 2003), cAMP-mediated recruitment of new sites of synaptic transmission (Bolshakov et al., 1997), and synaptic tagging (Frey and Morris, 1998), may also be found to apply to plasticity at MF synapses on L-M interneurons.

Supplementary Material

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Acknowledgements

This work was supported by S24288 (G.B.) and NS046423 (E.T.). The authors wish to thank Dr. Spencer D. Watts for his invaluable assistance in the creation of the confocal images.

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