| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China; 2International Cooperative Research Project/Solution Oriented Research for Science and Technology Cell Mechanosensing, JST, Nagoya; 3Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya; 4Department of Aging Intervention, National Institute for Longevity Sciences, Oobu; 5Department of Anatomy and Neurobiology, Nagasaki University School of Medicine, Nagasaki; and 6Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan
Submitted 30 October 2007; accepted in final form 3 July 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
7-nicotinic acetylcholine receptor (7nAChR). We herein demonstrate that the LLPPREGS could be separated into two independent processes: the above-mentioned early presynaptic-origin short-term potentiation (STPPREGS) and a delayed postsynaptic N-methyl-D-aspartate receptor (NMDAr)-dependent long-term potentiation termed LTPPREGS. This study focused on the analysis of the signaling mechanism underlying the LTPPREGS. PREGS increased the tyrosine phosphorylation of NR2B, a subunit of NMDAr, and the NMDAr-mediated Ca2+ influx in the granule cells. The enhanced Ca2+ influx was largely attenuated by the NR2B subunit inhibitor ifenprodil and the Src kinase family inhibitor PP2. PREGS also triggered a persistent phosphorylation of extracellular signal-regulated kinase 2 (ERK2) followed by an ERK-dependent phosphorylation of cAMP-response element-binding protein (CREB), which was crucial for the LTPPREGS induction and was sensitive to ifenprodil. These results suggest that PREGS induces an acute increase in the NR2B tyrosine phosphorylation which enhances the Ca2+ influx through NMDAr, followed by an activation of the ERK/CREB signaling cascade that leads to LTPPREGS. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
; Vallee et al. 2001). The deterioration in the cognitive performance of aged rats has been suggested to be linked to the low levels of PREGS in the hippocampus (Flood et al. 1995). A significant negative correlation has been found in the levels between 
-amyloid and PREGS in the brain of Alzheimer's disease patients (Weill-Engerer et al. 2002). Anti-amnesic effects of PREGS have also been demonstrated in animal amnestic models produced by competitive or noncompetitive NMDA receptor antagonists (Cheney et al. 1995; Mathis et al. 1994, 1996), and 25–35-amyloid peptide (Maurice et al. 1998). These observations strongly support that PREGS is an important endogenous factor for enhancing both memory and learning; however, the exact underlying mechanisms of this action still remain to be elucidated.
Long-term potentiation (LTP) is widely accepted as a candidate cellular mechanism for the storage of information (Bliss and Collingridge 1993). In immature hippocampal CA1 neurons (younger than postnatal day 6), PREGS has been observed to induce a long-lasting strengthening of AMPA-mediated miniature excitatory postsynaptic currents via retrograde modulation of presynaptic NMDAr (Mameli et al. 2005). We previously reported that in the mature hippocampal slices (>6 wk) an acute application of PREGS for 10 min triggered a persistent potentiation at the perforant path-granule cell synaptic transmission in the hippocampal dentate gyrus (DG) (Chen et al. 2002). We also have provided evidence that PREGS transiently increases the probability of glutamate release by sensitizing the presynaptic 7nAChR (Chen and Sokabe 2005). In addition, PREGS has been shown to exert acute effects through the positive actions to ionotropic glutamate receptors including the potentiation of Ca2+ conductivity and Ca2+ influx via NMDAr (Mukai et al. 2000; Takebayashi et al. 1998; Wu et al. 1991). PREGS also antagonizes the GABA/glycine-activated Cl– currents (Paul and Purdy 1992). In spite of such a variety of studies concerning the molecular targets of PREGS, the mechanism by which PREGS induces a long-lasting potentiation in the adult rat is still not fully understood.
Activity-dependent LTP at glutamatergic synapses in the adult hippocampus is typically divided into two stages, namely an early labile phase dependent on covalent modifications of existing proteins and a late stable phase requiring synthesis of new proteins (Nguyen and Kandel 1996). Increasing evidence indicates that the long-lasting potentiation of synaptic efficacy requires an activation of ERKs in mammals (Atkins et al. 1998; Blum et al. 1999; Impey et al. 1999; Kelleher et al. 2004; Schmitt et al. 2005; Shalin et al. 2006). The phosphorylation of CREB, as a downstream target of ERKs, is also a necessary component for hippocampus-dependent memory formation in mammals (Bourtchulasze et al. 1994; Impey et al. 1996). It would therefore be interesting to clarify whether the PREGS-induced synaptic potentiation shares similar mechanisms with those observed in the activity-dependent LTP.
The present study demonstrates that an acute application of PREGS exerts two independent effects, one is on the presynaptic 7nAChR, whereas the other is on postsynaptic NMDAr, thus leading to an early presynaptic-origin short-term potentiation (STPPREGS) and a delayed postsynaptic-origin long-term potentiation (LTPPREGS), respectively. The induction of the LTPPREGS requires a potentiation of the NR2B subunit of postsynaptic NMDAr followed by an activation of the ERK/CREB signaling cascade.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Slice preparation and staining with voltage-sensitive dye
The rats were decapitated under deep anesthesia with ethyl ether. The brains were taken quickly after removing the skulls and then placed in ice-cold artificial cerebrospinal fluid (ACSF)for around 10 min. ACSF of the following composition (in mM) 126 NaCl, 2.7 KCl, 1.24 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, and 10 glucose. ACSF was oxygenated with a gas mixture of 95% O2-5% CO2, and the pH was adjusted to 7.4. Horizontal slices (350 µm in thickness) of the brain were cut using a vibrating microtome (Microslicer DTK 1500, Dousaka EM, Kyoto, Japan). The slices were incubated in a rest chamber containing fresh oxygenated ACSF at room temperature for more than 1 h to recover the slices from damage.
Electrophysiological recordings
All the slices used in this study were stained with voltage-sensitive dye RH482 (0.1 mg/ml, Nippon Kanko Shikiso Kenkyujo, Okayama, Japan) for 15 min. The stained slices were kept in the fresh oxygenated ACSF for 
30 min for recovery. The slices were then transferred to a recording chamber and placed on the stage of an Olympus inverted microscope (IMT-2, Nikon, Tokyo, Japan) and was perfused continuously with ACSF at a flow rate of 1–2 ml/min. The experiments were performed at room temperature (28 ± 1°C).
The electrical and optical recordings, as well as the evaluation of the electrical and the optical signals, were performed as described previously by us (Chen et al. 2006a). Briefly, orthodromic stimuli were delivered using an electrically polished bipolar tungsten electrode or a glass electrode filled with 0.9% NaCl (tip resistance of 5 M
) that was placed in the outer third of stratum moleculare to stimulate the lateral perforant path (Lin et al. 2006). Constant current pulses (0.1 ms, 0.06 Hz) were supplied by a stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan). The intensity of the test stimuli was adjusted to evoke 
50% of the maximum value of optical excitatory postsynaptic potential (op-EPSP). Light from a tungsten-halogen lamp (type JC-24v/200W, Kondo Philips, Tokyo, Japan) was collimated, and an interference filter with a transmission maximum at 700 ± 10 nm (Olympus Optical) was used. Changes in the light absorption associated with membrane potential changes were measured with an optical recording system equipped with a high speed diode camera with metal oxide (MOS) image sensors (128 x128 pixels, each of which receives light from a 25 x 25 µm sample area) and a data processing unit (HR Deltaron-1700 Fujix; Fuji Photo Film, Tokyo, Japan). To analyze the change in the neuronal activities over time at a given region, the data from each pixel were stored, and retrieved, and then the amplitude of optical signal (indicated as a percent change in optical absorbency) was plotted as a function of time.
In some experiments, field potential recordings from str. moleculare were simultaneously performed with optical recordings to ensure that the optical response was consistent with the electrophysiological response. After a slice was submerged in a recording chamber containing ACSF, a bipolar tungsten electrode was placed in str. moleculare to stimulate the perforant path, using a hydraulic micromanipulator (MMW204, Narishige, Tokyo, Japan) mounted on the microscope stage. The field excitatory postsynaptic potentials (f-EPSPs) were recorded from s. moleculare with a 4–5 M resistance glass microelectrode filled with 0.9% NaCl, which was connected to a home-made, neutralized, high-input-impedance preamplifier.
Western blot analysis and immunoprecipitation assay
After a series of electrophysiological experiments, the slices were harvested immediately and then were frozen on dry ice. The hippocampal DG regions from the experimental slices were micro-dissected on dry ice and stored at –80°C until use. The hippocampus lysates from the individual slices were homogenized in a lysis buffer containing (in mM) 50 Tris-HCl (pH 7.5), 150 NaCl, 5 EDTA, 10 NaF, 1 sodium orthovanadate, 1% Triton X-100, 0.5% sodium deoxycholate, 1 phenylmethylsulfonyl fluoride and either a protease inhibitor cocktail (Complete; Roche, Mannheim, Germany) or a modified lysis buffer containing 0.1% SDS for immunoprecipitation assays.
The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). Total proteins (20 µg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred to a polyphorylated difluoride (PVDF) membrane. The membranes were incubated with 5% bovine serum albumin or skim milk in tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature, and then they were incubated with a primary antibody diluted in a solution at 4°C overnight. After being washed with TBST three times, the membranes were then incubated with an HRP-labeled secondary antibody and were developed using an ECL detection Kit (Amersham Biosciences, Piscataway, NJ). The membranes were treated with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) at 50°C for 30 min and then were re-proved with an antibody recognizing different proteins.
For immunoprecipitation assays, total proteins (500 µg) were incubated with an appropriate antibody and then protein-G sepharose was added and further incubated. Resulting immunoprecipitates were recovered by centrifugation and resuspended in a sample buffer. The samples (10 µl) were separated by electrophoresis and blotted onto a PVDF membrane. The membranes were incubated with primary antibodies, and the proteins were detected by HRP-conjugated secondary antibodies using an ECL detection kit (Amersham Biosciences).
[Ca2+]i measurements by multi-photon laser microscopy
Ca2+ indicator, Indo-1, was loaded into hippocampal granule cells in the slice using a local ester loading method as described previously (Regehr and Tank 1991). The main advantage of this method is generally noninvasive, and it can simultaneously measure the intracellular Ca2+ ([Ca2+]i) in multiple neurons. Only a small area of the slice was labeled with the local perfusion of a labeling solution using a delivery pipette and a suction pipette. The labeling solution was prepared as follows: 5 µl of 1 mM indo-1 AM in 5 µl of Cremophor EL (Sigma) were mixed and dispersed with 500 µl of balanced salt solution (BSS). BSS consisting of Mg2+-free contains (in mM) 145 NaCl, 2.5 KCl, 1 CaCl2, 10 glucose, and 10 HEPES (oxygenated with a gas mixture of 95% O2-5% CO2, adjusted to pH 7.4 with NaOH and to an osmolatity of 315–325 mosM with sucrose). A delivery pipette with a large tip (25 µm), which was filled with the labeling solution, was positioned below the slice surface in s. moleculare and a suction pipette was positioned near the slice surface to suck a stream of the labeling solution so that the stream hits only a certain small area of the slice surface. A steady thin stream of labeling solution was provided by fine-tuning both the pressure of an injector (transjector 5246, Eppendorf) and the vacuum of the suction pipette. Because most of the labeling solution is immediately washed out of the bath, the absorption of Indo-1 AM occurred very locally in the slice. Therefore Indo-1AM was absorbed specifically by the dendrites of granule cells and accumulated in the granule cells. The AM dye was cleaved to Indo-1 and diffused to the cell bodies within 30–45 min. As some interneurons located in granule cell layer extend their dendrites to s. moleculare (Freund and Buzsáki 1988), they may also be labeled by this method. In practice, three-dimensional reconstructed images of the brain slices showed that some labeled interneuron-like cells were in granule cell layer but mostly at the boundary with s. moleculare. In addition, many of them were distributed in the optical layers different from those rich in labeled granule cells. Based on this information, we could exclude the images of interneuron-like cells and minimize the possible contamination by their signals. The slice labeled with Indo-1 was mounted in a recording chamber and observed under an upright microscope (Olympus BX51WI) through a water immersion lens (x20 Olympus XLUMPlanFl). Indo-1 fluorescence was observed by a multi-photon laser scanning microscope (BioRad Radiance 2000 MP) with a direct detector system. Indo-1 was excited at 710–730 nm, and the resulting fluorescence was measured at 390 nm and 495 nm every 5 s. The average of fluorescence intensities (F390 and F495) from individual cells in a ROI were calculated and the ratio, (F390*b)/(F495*a), was used as an indicator of [Ca2+]i in the cells, where a (10–20) and b (128–160) were arbitrary constants.
This local loading approach has an advantage, namely the [Ca2+]i changes are reported from a population of healthy cells located below the slice surface. The measurements were made in s. moleculare distant from the loading site. The slices were perfused with BSS containing (in mM) 130 NaCl, 5.4 KCl, 2.0 CaCl2, 5.5 glucose, and 10 HEPES (adjusted to pH 7.3 with NaOH and to an osmolatity of 315–325 mosM with sucrose). BSS was oxygenated with a gas mixture of 95% O2-5% CO2. In all experiments, the neurons were exposed to NMDA (5 µM) for 1 min. Ca2+ signals were averaged for 20–30 cells under the same microscopic field.
Data analysis/statistics
All data were retrieved and processed using the Microcal Origin 6.1 software program (OriginLab, Northampton, MA). 1) The synaptic efficacy was estimated from the op-EPSP amplitude that was repeatedly measured in the same area (pixel) of tissue before, during, and after the administration of drugs. The degree of potentiation was expressed as the percent increase in either the amplitude of op-EPSP or the slope of f-EPSP. When the op-EPSP amplitude or f-EPSP slope was kept at the levels >10 or 20% or larger, respectively, of the basal level for >60 min, it was considered to be a long-lasting potentiation (LTP). 2) The activation levels of ERK1/2, CREB, and NR2A/2B were expressed as their phosphorylation levels normalized by the respective protein amounts. The Western blot bands were scanned and analyzed with the image analysis software package, National Institutes of Health Image. Most summarized data of densitometry represent the average from at least four experiments. 3) [Ca2+]i changes were measured by LaserSharp 2000 (BioRad) and then were analyzed by LaserPix 4.0 (BioRad). We used the ratio of Indo-1 fluorescence intensities at two emission wavelengths 390 and 495 nm as an indicator of [Ca2+]i change. The group data were expressed as the means ± SE. Statistical analyses were performed using the STATA 7.0 software program (STATA). LTPPREGS data were analyzed using two-way ANOVA, which examined all the data recorded between 55 and 60 min after washing PREGS. In the analysis of immunoblot and Ca2+ imaging data, statistical differences among the values for the individual groups were determined by a one-way analysis (ANOVA), followed by the Bonferroni post hoc test. A P value of <0.05 was considered to indicate a statistically significant difference.
Chemicals and antibodies
NMDA, PREGS, ifenprodil and 4-diamino-2,3-dicyano-1-4-bis [2-aminophynylthio] butadiene (U0126) were purchased from Sigma (St. Louis, MO). Indo-1 AM was purchased from Dojin (Kumamoto, Japan). NMDAr channel blocker (+)-MK-801 hydrogen maleate (MK-801) was obtained from Tocris Cookson (Bristol, UK). PD98059 was from Calbiochem (San Diego, CA). These drugs were dissolved in dimethyl sulfoxide (DMSO) for stock solution and then were diluted to the superfusing solution at a final concentration of 0.1% DMSO. The treatment with DMSO alone at 0.1% concentration had no effect on the basal levels of synaptic transmission. Other chemicals of special grade were obtained from Wako (Osaka, Japan).
Antibodies against phospho-ERK (Thr202/Tyr204) (9101), ERK (9102), phospho-CREB (Ser133) (9191), and CREB (9192) were obtained from Cell Signaling Technology (Beverly, MA). Anti-phosphotyrosine antibody (clone 4G10; 05–321) was purchased from Upstate Biotechnology (Charlottesville, VA). Anti-NR2A (clone 5; N17320) and -NR2B (clone 13; N38120) antibodies were purchased from Transduction Laboratories (San Diego, CA). For the immunoprecipitation assays, antibodies against NR2A (sc-9056) and NR2B (sc-9057) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
In an initial experiment, we sought to confirm our previous results (Chen et al. 2002), namely that acute PREGS exposure of hippocampal slices from adult rats triggered a rapid and long-lasting potentiation of the perforant path-granule cell synaptic transmission. As shown in Fig. 1 A, the amplitude of op-EPSP was instantly increased in response to the perfusion of 50 µM PREGS, reaching its peak (138 ± 4.3%) within 10 min, and then maintained a potentiated level (126 ± 7.2%) over 80 min even after the cessation of PREGS perfusion (34 slices from 22 rats). On the application of a lower concentration (15 µM) of PREGS, we could see a rapid increase of the op-EPSP amplitude (119 ± 3.55% just after post-PREGS, P < 0.05); however, the increased level declined soon after the washout of PREGS from the slices (101 ± 5.4%, n = 8 slices/4 rats). This suggests that the PREGS-induced long-lasting potentiation (LLPPREGS) critically depends on the concentration of PREGS. Figure 1B is a plot of the amplitude of op-EPSP measured at 60 min after the cessation of PREGS perfusion versus PREGS concentration, thus showing a sigmoidal shape with an apparent EC50 of 22.1 µM.
|
Two independent processes in LLPPREGS
We previously reported that an acute application of PREGS transiently increases the probability of glutamate release from the perforant path terminal by sensitizing the presynaptic 7nAChR (Chen and Sokabe 2005). It is therefore natural to think that the increased glutamate release by PREGS is responsible for the induction of LLPPREGS. To assess the feasibility of this hypothesis, the selective 7nAChR antagonist MLA was used to prevent the effect of PREGS on the glutamate release (Chen and Sokabe 2005). Unexpectedly, the pretreatment of slices with MLA (10 µM) attenuated only the early component of LLPPREGS, while not affecting the persistent potentiation of op-EPSP. As shown in Fig. 2 A, in the presence of MLA, op-EPSP slowly increases and reaches a stable plateau at 20 min after the washout of PREGS, thus keeping its level for over 40 min (122.74 ± 5.81% in 60 min post-PREGS, n = 5 slices/5 rats).
|
). The result in Fig. 2B clearly shows that MK-801 at 10 µM almost perfectly abolishes the late component of LLPPREGS (99.33 ± 6.1% at 60 min post-PREGS, n = 6 slice/5 rats; Fig. 2B), whereas it leaves an early transient component of which the amplitude and time course are essentially the same as the component sensitive to MLA (see the inset showing a time course of the component obtained by subtracting the area of the LLPPREGS in the presence of MLA from that of LLPPREGS in Fig. 2A). However, MK-801 had no effect on the established LLPPREGS (Fig. 2C). These findings demonstrate that the LLPPREGS can be decomposed into two independent processes, an early presynaptic 7nAChR-sensitive short-term potentiation termed STPPREGS and a delayed postsynaptic NMDAr-dependent long-term potentiation termed LTPPREGS. PREGS enhances NMDAr-mediated Ca2+ influx
The results from the preceding experiments therefore suggest that the LTPPREGS induction requires NMDAr-mediated Ca2+ influx. To confirm this idea, we measured changes in the intracellular-free calcium concentration ([Ca2+]i) on an application of NMDA in the granule cells in the hippocampal slices used to induce LTPPREGS. To mimic the depolarization-induced Mg2+-release from NMDAr in optical recordings, the experiments were done with Mg2+-free medium (Chen et al. 2006b; Mukai et al. 2000; Takebayashi et al. 1998). In the absence of NMDA, Mg2+ removal per se or an application of 50 µM PREGS in the absence of external Mg2+ induced no detectable [Ca2+]i elevation (data not shown).
Figure 3 B shows a pair of typical time series of images for NMDA (5 µM)-induced [Ca2+]i changes in the absence (top) and presence (bottom) of PREGS, where significantly larger increases in [Ca2+]i are visible in the presence of PREGS. Actually, as shown in Fig. 3C, the application of 50 µM PREGS significantly enhanced the NMDA-induced [Ca2+]i increase up to approximately 3-fold of that before PREGS application (column 2, P < 0.01). After the washout of PREGS from the slices, the effect of PREGS disappeared (column 3 vs. column 2, P < 0.01). Both the NMDA-induced [Ca2+]i increases and the enhanced [Ca2+]i increases by PREGS were completely canceled by the NMDAr blocker MK-801 (column 4). Furthermore, the enhancing effect of PREGS on the NMDA-induced [Ca2+]i increases was completely abolished by the specific NR2B inhibitor ifenprodil (5 µM) (column 5 vs. column 2, P < 0.01), but ifenprodil (5 µM) itself did not affect the levels of [Ca2+]i induced by NMDA (column 6, P > 0.05), thus supporting the notion that PREGS enhances the NMDAr-mediated Ca2+ influx through a modulation of the NMDAr subunit NR2B.
|
PREGS enhances phosphorylation of NR2B subunit
One possible mechanism for the PREGS-regulating NMDAr function may be tyrosine phosphorylation of certain sites on NR2B subunit as suggested by previous studies (Jiang et al. 2004; Mukai et al. 2000). We therefore examined the tyrosine phosphorylation of NR2A and NR2B subunits (phospho-NR2A/2B) by applying anti-phospho-NR2A/2B antibodies to the immunoprecipitate of neuronal homogenates from micro-dissected DGs. Figure 4 A illustrates a representative gel pattern of phosphorylated NR2A/2B bands in various pharmacological conditions. A densitometry analysis revealed that the tyrosine phosphorylation levels of NR2B immediately after the cessation of the 10-min PREGS application showed a significant increase (4.2-fold, column 2, n = 12 slices/8 rats) in comparison with the control (column 1, P < 0.01). In contrast, the level of phospho-NR2A showed no significant alteration. One notable finding was the fact that neither 5 µM ifenprodil (column 3) nor 10 µM MLA (column 4) affected the PREGS-increased tyrosine phosphorylation of NR2B subunit (P > 0.05). However, the inhibition of NR2B subunit by ifenprodil was able to prevent the induction of LTPPREGS (Fig. 4B) as in the case of the PREGS-enhanced [Ca2+]i increase (column 3, Fig. 3C). Furthermore, the pretreatment with the Src kinase family inhibitor PP2 perfectly prevented the induction of LTPPREGS (Fig. 4C), thus supporting the hypothesis that the increase in the NR2B tyrosine phosphorylation is crucial for LTPPREGS induction.
|
There is evidence pointing that the Ca2+ influx across NMDAr can activate ERK (Chen et al. 2006b; Kurino et al. 1995), which is important for multiple forms of synaptic plasticity and memory behavior (Blum et al. 1999; Impey et al. 1999). To determine whether the LTPPREGS induction involves ERK-CREB signaling, an experiment was designed to estimate the phosphorylation of ERK1/2 (phospho-ERK1/2) using an immunoblot analysis on the micro-dissected hippocampal DG. Expectedly, PREGS triggered obvious increases in the phospho-ERK1/2 levels in a dose-dependent manner, whereas the total protein amounts of ERK1/2 were not altered by PREGS as can be seen in Fig. 5 A. Their dose-response curves shown in the right panel exhibit sigmoidal shape with apparent EC50 of 16.41 and 19.2 µM for phospho-ERK1 and -ERK2, respectively (at each dose, n = 12 slices from 6 rats). These curves strikingly resemble to the dose-response curve of LTPPREGS amplitude (Fig. 1B), thus implying that LTPPREGS induction is closely coupled with ERK1/2 activation. However, when examined, the time courses of phospho-ERK1 and -ERK2, in which samples were prepared before (0 min) and after 5, 10, 30, and 60 min of PREGS perfusion for 10 min, we noticed a big difference between them. As shown in Fig. 5B, phospho-ERK1 showed a transient increase in response to 10-min PREGS application, while phospho-ERK2 had a biphasic profile (n = 12 slices/6 rats, P < 0.01). Conceivably, the decreases in the levels of phospho-ERK1 and -ERK2 at 20 min may reflect some reversible responses of ERK1/2 to PREGS because PREGS was washed out at 10 min after PREGS application. Only the phospho-ERK2 seems to involve another slowly going irreversible process, resulting in a biphasic activation with time. This time profile of the phospho-ERK2, but not of ERK1, roughly accords with that of the op-EPSP potentiation induced by PREGS (Fig. 1), suggesting that the phospho-ERK2 is more closely related to LTPPREGS. In addition, the PREGS-increased phospho-ERK2 at 30 min after the start of PREGS perfusion was significantly attenuated by 5 µM ifenprodil (column 3 vs. column 2, P < 0.01), but not by 10 µM MLA (column 4 vs. column 2, P > 0.05; Fig. 5C). Taken together with the results in Fig. 2A, this pharmacological result thus supports the proposed close association between LTPPREGS and the PREGS-increased phospho-ERK2, although we cannot exclude the possibility of the phospho-ERK1 involvement in LTPPREGS. Elucidation of differential roles and underlying mechanisms of ERK1 and ERK2 activations in LTPPREGS awaits future studies.
|
7nAChR antagonist MLA (10 µM) did not alter the increase of phospho-CREB by PREGS (column 4, P > 0.05), thus implying that Ca2+ influx across NMDAr cascades the ERK/CREB signaling pathway leading to LTPPREGS.
Finally, to confirm whether the activation of ERK/CREB signaling by PREGS plays a crucial role in the LTPPREGS induction, slices were pretreated with the MEK inhibitor PD98059 (50 µM) or U0126 (30 µM) for 30 min prior to PREGS delivery. We found that both PD98059 and U0126 completely blocked the induction of LTPPREGS. The blocked levels of the op-EPSP by PD98059 (108.75 ± 8.3%, n = 7 slices from 5 rats; Fig. 6 A) and by U0126 (98.33 ± 4.3%, n = 10 slices from 6 rats; Fig. 6B) at 60 min after the start of PREGS perfusion showed no significant difference from the control levels before PREGS delivery. The PREGS-induced transient potentiation of op-EPSP was intact in either case with PD98059 or U0126, thus clearly demonstrating that the ERK/CREB signaling regulates the induction of LTPPREGS without affecting the early transient potentiation of op-EPSP that should be under the control of the presynaptic potentiation mediated by PREGS-potentiated 7nAChR.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
7nAChR-dependent STPPREGS and a delayed postsynaptic NMDAr-dependent LTPPREGS. As the STPPREGS has recently been analyzed in detail (Chen and Sokabe 2005), this study focused on the clarification of the molecular mechanisms of the LTPPREGS. Particular attention has been paid to the analysis of PREGS-induced alteration of NMDAr functions and its downstream factors because the induction of LTPPREGS was totally dependent on NMDAr. Our results have clearly demonstrated that PREGS facilitates the NMDAr-mediated Ca2+ influx, which is caused by an increased tyrosine-phosphorylation of NR2B subunit. The resulting elevation of [Ca2+]i triggers a sustaining activation of ERK/CREB, which is needed for the induction of LTPPREGS.
The first question we should address may be whether the longevity of the LTPPREGS reflects either an enduring modification of the synaptic machinery or the inability to completely washout the applied PREGS from the slices after ACSF re-perfusion. Considering the result that a relatively low dose of PREGS exhibited a reversible action on the synaptic transmission (Fig. 1B), it is unlikely that LTPPREGS is caused by an enduring effect of putative residual PREGS after its washout. In addition, as described in RESULTS, after the washout of PREGS from the slices, the facilitating effect of PREGS on the NMDAr-mediated Ca2+ influx disappeared, thus indicating that the action of PREGS on the NMDAr is reversible. Furthermore, the NMDAr blocker MK-801 prevented the induction of LTPPREGS, whereas it had no effect when applied after LTPPREGS was established, thus supporting that the nature of the long-term potentiation in the synaptic efficacy by PREGS is attributable to a sustained intracellular process triggered by PREGS rather than the continual effect caused by residual PREGS. We also noticed that pregnenolone (PREG), a nonsulfated form of PREGS, did not show a similar effect to that of PREGS on slice preparations (Chen and Sokabe 2005), thus indicating that the PREGS effects should not arise from a nonspecific membrane-perturbing effect of a lipophilic steroid.
One of the major findings in the present study is that the induction of LTPPREGStotally depends on NMDAr, more specifically on its subunit NR2B. The requirement of the activation of NR2B subunit in PREGS-induced plasticity has also been established in immature hippocampal synapses (Mameli et al. 2005). In addition, Mukai et al. (2000) reported that the NR2B subunit inhibitor ifenprodil perfectly inhibited the PREGS-enhanced Ca2+ influx mediated by a recombinant NMDAr expressed in CHO cells, and this finding closely correlated with our result, namely that the PREGS-potentiated Ca2+ influx via NMDAr was nearly completely inhibited by ifenprodil (Fig. 3C). NR2B subunit, a major functional component of the hippocampal NMDAr (Monyer et al. 1994), thus regulates the conductance of the NMDAr channel through the phosphorylation on its tyrosine residues (Yu et al. 1997). Consistent with this mechanism, we observed that the acute exposure to PREGS increased the tyrosine phosphorylation of NR2B subunit as shown in Fig. 4A. A variety of mechanisms for the upregulation of NMDAr channel via the tyrosine phosphorylation of NR2B subunit have been proposed as follows: slowing down the decay kinetics of NMDAr-mediated currents; increasing the open probability of the NMDAr channel (Yu et al. 1997); stabilizing NMDAr on the cell surface (Grosshans et al. 2002); and preventing the removal of signaling molecules from the NMDAr complex by protecting the NR2B subunit against the degradation caused by the calcium-activated protease, calpain (Salter and Kalia 2004). Considering the result that PREGS exerts a relatively rapid effect in a reversible manner, the former two mechanisms may therefore be mainly responsible for the PREGS-enhanced Ca2+ influx across NMDAr channels. Of course direct measurements of NMDA-induced currents in the presence of PREGS are required before making any definitive conclusions regarding this hypothesis.
Another interesting mechanism has been proposed regarding the effect of PREGS on NR2B subunit, namely, PREGS acts as a positive allosteric modulator of NMDAr, probably via a direct interaction with NR2B subunit, to facilitate the action of glutamate to NMDAr (Gibbs et al. 1999; Jiang et al. 2004). The latter study proposes a hydrophobic pocket selective to PREGS binding at the NR2B trans-membrane domain facing to the lipid bilayer based on a sophisticated site direct mutagenesis along this domain. However, they did not discuss how the PREGS-induced changes in NR2B conformation relates to its tyrosine phosphorylation. We do not know how to reconcile the two different mechanisms for the functional potentiation of NR2B by PREGS; tyrosine phosphorylation and direct binding leading to a conformational change in NR2B subunit. One possible idea may be that the conformational change of NR2B by the direct binding of PREGS makes NR2B more susceptible to its tyrosine phosphorylation; however, this question remains to be answered in the future studies.
It may be natural here to consider the potential kinase(s) responsible for the PREGS-induced tyrosine phosphorylation of NR2B. Src kinase family, a member of the NMDAr channel complex, increases the open probability of NMDAr channel (Yu et al. 1997) through the tyrosine phosphorylation of NR2B (Salter and Kalia 2004), which is crucial for the induction of the activity-dependent LTP in various types of synapses (O'Dell et al. 1991). The activation of Src kinase family by tetanic stimulation (Lu et al. 1998) causes a long-lasting facilitation of NMDAr currents in the hippocampal CA1 (Lu et al. 1999; Malenka and Nicoll 1999). Our preliminary observation that the PREGS-enhanced Ca2+ influx via NMDAr was significantly attenuated by the Src kinase family inhibitor PP2 (not shown), thus suggesting that Src kinase family is one of the most probable candidates responsible for the PREGS-induced tyrosine phosphorylation of NR2B. A question remains, however, regarding which mechanisms regulate the activity of Src kinase family. Although little information is available on this question, one possible upstream factor of the Src activation may be the sigma 1 (
1) receptor. Meyer et al. (2002) reported that PREGS regulates the glutamate release in hippocampal neurons through the activation of 1 receptor. Furthermore, we recently reported that the neurosteroid dehydroepiandrosterone sulfate (DHEAS) increases the phosphorylation of Src via 1 receptor (Chen et al. 2006b) while recovering the reduction in the tyrosine phosphorylation of NR2B after cerebral ischemia also via 1 receptor (Li et al. 2006). Therefore PREGS might act as a 1 receptor activator to indirectly regulate NR2B subunit. Experiments are now in progress in our laboratory to address this important issue. Finally it should be mentioned that the tyrosine phosphorylation of NR2B had already been increased significantly at the time when the 10-min administration of PREGS was ceased (Fig. 4A) and LTPPREGS started to develop (Fig. 2A), thus clearly demonstrating that the NR2B phosphorylation is an indispensable upstream event for the induction of LTPPREGS.
The striking resemblance of the dose- and time-dependent profiles between the activation of ERK2 and LTPPREGS (compare Figs. 1B and 2A with 5, A and B, respectively) strongly supports that ERK2 is a critical signal molecule situated at the very end of the signal cascade responsible for LTPPREGS induction. It is well established that ERK pathway is an essential component of NMDAr signal transduction controlling the neuronal synaptic plasticity (Thomas and Huganir 2004) through regulating the AMPAr trafficking (Qin et al. 2005; Zhu et al. 2002). Therefore ERK has emerged as an important Ca2+-dependent constituent of the final common pathway leading to the induction of multiple forms of synaptic plasticity. It is also noteworthy that the ERK2 activation in LTPPREGS lasted a relatively longer period over 1 h in comparison to that in the activity-dependent LTP. In the activity-dependent LTP only an early and transient ERK2 activation has been noted (Kelleher et al. 2004), probably because the MAPK phosphatase is upregulated quickly by a negative feedback loop to deactivate MAPK/ERK (Davis et al. 2000). In contrast, ligand-induced LTP seems to involve a long-lasting ERK2 activation. For example, Ying et al. (2002) reported that brain-derived neurotrophic factor (BDNF) induced an LTP associated with a persistent ERK2 activation as long as 3 h in the hippocampal dentate gyrus. The induction of late LTP by BDNF is associated with translocation of ERK2 to the nucleus in which it activates the transcription of immediate early genes (Rosenblum et al. 2002). Hippocampal NMDAr is coupled to the ERK pathway by a direct interaction between NR2B subunit and RasGRF1 (Krapivinsky et al. 2003). A certain class of small GTP-binding proteins may contribute to the sustained ERK2 activation. York et al. (1998) reported that ERK can persistently be activated after the formation of a stable up-stream complex between the small G proteins, Rap-1 and B-raf, which are known as MEK activators. The present study has demonstrated that LTPPREGS appears to develop immediately after the washout of PREGS (Fig. 2A) and that the MEK inhibitor PD98059 could inhibit LTPPREGS induction even though this drug was washed away before the development LTPPREGS (Fig. 6). Therefore it may not be unreasonable to think that PD98059 disrupts the formation of a stable up-stream complex to prevent the long-lasting activation of ERK2 that is indispensable for the induction of LTPPREGS.
Our results have indicated that PREGS, through the tyrosine phosphorylation of NR2B subunit followed by ERK/CREB signaling, induces the long-lasting potentiation of synaptic efficacy, which may exert a "cognitive" effect in the adult rat brain. The PREGS-regulated synaptic plasticity as a result of the activation of this signal pathway fits well with a number of previous reports (Atkins et al. 1998; Blum et al. 1999), thus indicating that this pathway plays an important role in both learning and memory. However, the relatively high concentration of PREGS used in the present study appears to be nonphysiological. Bulk concentrations of PREGS in tissue homogenates have been estimated at the nanomolar level, yet the micromolar concentrations of PREGS are required to modulate the NMDAr function as demonstrated in the present and other studies (Gibbs et al. 1999). Mameli et al. (2005) found that a PREGS-like neurosteroid is synaptically released in a retrograde manner from depolarized postsynaptic CA1 neurons, which could be mimicked by 17 µM of exogenously applied PREGS. A significant net synthesis of PREGS induced by NMDA stimulation suggests that a relatively high concentration of PREGS is produced in the hippocampus under physiological conditions (Kimoto et al. 2001). In addition, because P450scc and hydroxysteroid sulfotransferase are highly localized in hippocampal neurons, the local concentration of PREGS around the neurons, particularly around synapses, may be 10- to 20-fold greater than the bulk concentration (57 nM) observed after NMDA stimulation. Collectively, it may not be unreasonable to think that the relatively high concentration (50 µM) of PREGS employed in this study would be within a physiological range and that PREGS may therefore function as a physiological potentiator for neuronal communication.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: M. Sokabe, Dept. of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan (E-mail: msokabe{at}med.nagoya-u.ac.jp)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361: 31–39, 1993.[CrossRef][Medline]
Blum S, Morre AN, Adams F, Dash PA. Mitogen-activated protein kinase cascade in the AC1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory. J Neurosci 19: 3535–3544, 1999.
Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva AJ. Deficient long-term memory in mice with a targeted mutation of the camp-responsive element-binding protein. Cell 79: 59–68, 1994.[CrossRef][Web of Science][Medline]
Chen L, Dai XN, Sokabe M. Chronic administration of dehydroepiandrosterone sulfate (DHEAS) primes for facilitated induction of long-term potentiation via sigma 1 (sigma1) receptor: optical imaging study in rat hippocampal slices. Neuropharmacology 50: 380–392, 2006a.[CrossRef][Web of Science][Medline]
Chen L, Miyamoto Y, Furuya K, Dai XN, Mori N, Sokabe M. Chronic DHEAS administration facilitates hippocampal long-term potentiation via an amplification of Src-dependent NMDA receptor signaling. Neuropharmacology 51: 659–670, 2006b.[CrossRef][Web of Science][Medline]
Chen L, Momiyama S, Sokabe M. Synaptic mechanisms of neurosteroid-induced long-term potentiation at hippocampus in rat brain slices. Neurosci Res Abstr II-B-019, 2002.
Chen L, Sokabe M. Presynaptic modulation of synaptic transmission by pregnenolone sulfate as studied by optical recordings. J Neurophysiol 94: 4131–4144, 2005.
Cheney DL, Uzunov D, Guidotti A. Pregnenolone sulfate antagonizes dizocilpine amnesia, role for allopregnanolone. Neuroreport 6: 1697–1700, 1995.[Web of Science][Medline]
Darnaudery M, Koehl M, Piazza PV, Le Moal M, Mayo W. Pregnenolone sulfate increases hippocampal acetylcholine release and spatial recognition. Brain Res 852: 173–179, 2000.[CrossRef][Web of Science][Medline]
Davis S, vanhoutte P, Pages C, Caboche J, Laroche S. The MAPK/ERK cascade targets both Elk-1, camp response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J Neurosci 20: 4563–4572, 2000.
Freund TF, Buzsáki G. Alterations in excitatory and GABAergic inhibitory connections in hippocampal transplants. Neuroscience 27: 373–385, 1988.[CrossRef][Web of Science][Medline]
Flood JF, Morley JE, Roberts E. Pregnenolone sulfate enhances post-training memory processes when injected in very low doses into limbic system structures: the amygdala is by far the most sensitive. Proc Natl Acad Sci USA 92: 10806–10810, 1995.
Gibbs TT, Yaghoubi N, Weaver CE Jr, Park-Chung M, Russek SJ, Farb DH. Modulation of ionotropic glutamate receptors by neuroactive steroids, In: Neurosteroids: A New Regulatory Function in the Nervous System, edited by Baulieu EE, Robel P, Schumacher M. Totowa, NJ: Humana, 1999, p. 167–190.
Grosshans DR, Clayton DA, Coultrap SJ, Browning MD. LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat Neurosci 5: 27–33, 2002.[CrossRef][Web of Science][Medline]
Impey S, Mark M, Villacres EC, Chavkin C, Storm DR. Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus. Neuron 16: 973–982, 1996.[CrossRef][Web of Science][Medline]
Impey S, Obrietan K, Storm DR. Making new connections: role of ERK/MAP kinase signaling in neuronal plasticity. Neuron 23: 11–14, 1999.[CrossRef][Web of Science][Medline]
Jiang MK, Mierke DF, Russek SJ, Farb DH. A steroid modulatory domain on NR2B controls N-methyl-D-aspartate receptor proton sensitivity. Proc Natl Acad Sci USA 101: 8198–8203, 2004.
Kelleher RJ, Govindarajan A, Jung HY, Kang H, Tonegawa S. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116: 467–479, 2004.[CrossRef][Web of Science][Medline]
Kimoto T, Tsurugizawa T, Ohta Y, Makino J, Tamura Ho, Hojo Y, Takata N, Kawato S. Neurosteroid synthesis by cytochrome p450-containing systems localized in the rat brain hippocampal neurons: N-methyl-D-aspartate and calcium-dependent synthesis. Endocrinology 142: 3578–3589, 2001.
Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE, Medina I. The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1. Neuron 40: 775–784, 2003.[CrossRef][Web of Science][Medline]
Kurino M, Fukunaga K, Ushio Y, Miyamoto E. Activation of mitogen activated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors. J Neurochem 65: 1282–1289, 1995.[Web of Science][Medline]
Li Z, Zhou R, Cui SZ, Xie GQ, Cai WY, Sokabe M, Chen L. DHEAS prevents ischemia-induced LTP impairment in rat hippocampal CA1 by up-regulating tyrosine phosphorylation of NMDA receptor. Neuropharmacology 51: 958–966, 2006.[CrossRef][Web of Science][Medline]
Lin YW, Yang HW, Wang HJ, Gong CL, Chiu TH, Min MY. Spike-timing-dependent plasticity at resting and conditioned lateral perforant path synapses on granule cells in the dentate gyrus: different roles of N-methyl-D-aspartate and group I metabotropic glutamate receptors. Eur J Neurosci 23: 2362–2368, 2006.[CrossRef][Web of Science][Medline]
Lu WY, Xiong ZG, Lei S, Orser BA, Dudek E, Browning MD, MacDonald JF. G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors. Nat Neurosci 2:331–338, 1999.[CrossRef][Web of Science][Medline]
Lu YM, Roder JC, Davidow J, Salter MW. Src activation in the induction of long-term potentiation in CA1 hippocampal neurons. Science 279: 1363–1367, 1998.
Malenka RC, Nicoll RA. Long-term potentiation–a decade of progress? Science 285: 1870–1874, 1999.
Mameli M, Carta M, Partridge LD, Valenzuela CF. Neurosteroid-induced plasticity of immature synapses via retrograde modulation of presynaptic NMDA receptors. J Neurosci 25: 2285–2294, 2005.
Mathis C, Paul SM, Crawley JN. The neurosteroid pregnenolone sulfate blocks NMDA antagonist-induced deficits in a passive avoidance memory task. Psychopharmacology 116: 201–206, 1994.[CrossRef][Medline]
Mathis C, Vogel E, Cagniard B, Criscuolo F, Ungerer A. The neurosteroid pregnenolone sulfate blocks deficits induced by a competitive NMDA antagonist in active avoidance and lever-press learning tasks in mice. Neuropharmacology 35: 1057–1064, 1996.[CrossRef][Web of Science][Medline]
Maurice T, Su TP, Privat A. Sigma () receptor agonists and neurosteroids attenuate -amyloid peptide-induced amnesia in 25–35 mice through a common mechanism. Neuroscience 83: 413–428, 1998.[Web of Science][Medline]
Meyer DA, Carta M, Partridge LD, Covey DF, Valenzuela CF. Neurosteroids enhance spontaneous glutamate release in hippocampal neurons. J Bio Chem 277: 28725–28732, 2002.
Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12: 529–540, 1994.[CrossRef][Web of Science][Medline]
Mukai H, Uchino S, Kawato S. Effects of neurosteroids on Ca2+ signaling mediated by recombinant N-methyl-D-aspartate receptor expressed in chinses hamster ovary cells. Neurosci Letter 282: 93–96, 2000.[CrossRef]
Nguyen PV, Kandel ER. A macromolecular synthesis-dependent late phase of long-term potentiation requiring camp in the medial perforant pathway or rat hippocampal slices. J Neurosci 16: 3189–3198, 1996.
O'Dell TJ, Kandel ER, Grant SG. Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors. Nature 353: 558–560, 1991.[CrossRef][Medline]
Paul SM, Purdy RH. Neuroactive steroids. FASEB 6: 2311–2322, 1992.[Abstract]
Qin Y, Zhu Y, Baumgart JP, Stornetta RL, Seidenman K, Mack V, van Aelst L, Zhu JJ. State-dependent Ras signaling and AMPA receptor trafficking. Genes Dev 19: 2000–2015, 2005.
Regehr WG, Tank DW. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci 37: 111–119, 1991.
Rosenblum K, Futter M, Voss K, Erent M, Skehel PA, French P, Obosi L, Jones MW, Bliss TV. The role of extracellular regulated kinases I/II in late-phase long-term potentiation. J Neurosci 22: 5432–5441, 2002.
Salter MW, Kalia LV. Src kinases: a hub for NMDA receptor regulation. Nat Rev Neurosci 5: 317–328, 2004.[CrossRef][Web of Science][Medline]
Schmitt JM, Guire ES, Saneyoshi T, Soderling TR. Calmodulin-dependent kinase kinase/calmodulin kinase I activity gates extracellular-regulated kinase-dependent long-term potentiation. J Neurosci 25: 1281–1290, 2005.
Shalin SC, Hernandez CM, Dougherty MK, Morrison DK, Sweatt JD. Kinase suppressor of Ras1 compartmentalizes hippocampal signal transduction and subserves synaptic plasticity and memory formation. Neuron 50: 765–779, 2006.[CrossRef][Web of Science][Medline]
Takebayashi M, Kagaya A, Uchitomi Y, Yokota N, Horiguchi J, Yamawaki S. Differential regulation by pregnenolone sulfate of intracellular Ca2+ increase by amino acids in primary cultured rat cortical neurons. Neurochem Int 32: 205–211, 1998.[CrossRef][Web of Science][Medline]
Thomas GM, Huganir RL. MAPK cascade signaling and synaptic plasticity. Nat Rev Neurosci. 5: 173–83, 2004.[CrossRef][Web of Science][Medline]
Vallee M, Mayo W, Le Moal M. Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. Brain Res Rev 37: 301–312, 2001.[CrossRef][Medline]
Weill-Engerer S, David JP, Sazdovitch V, Liere P, Eychenne B, Pianos A, Schumacher M, Delacourte A, Baulieu EE, Akwa Y. Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented patients. J Clinical Endocrinol Metab 87: 5138–5143, 2002.
Wu FS, Gibbs TT, Farb DH. Pregnenolone sulfate: a positive allosteric modulator at the NMDA receptor. Mol Pharmacol 40: 333–336, 1991.[Abstract]
Ying SW, Futter M, Rosenblum K, Webber MJ, Hunt SP, Bliss TV, Bramham CR. Brain-derived neurotrophic factor induces long-term potentiation in intact adult hippocampus: requirement for ERK activation coupled to CREB and upregulation of arc synthesis. J Neurosci 22: 1532–1540, 2002.
York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ. Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392: 622–626, 1998.[CrossRef][Medline]
Yu XM, Askalan R, Keil GJ 2nd, Salter MW. NMDA channel regulation by channel-associated protein tyrosine kinase Src. Science 275: 674–678, 1997.
Zhu JJ, Qin Y, Zhao M, Van Aelst L, Malinow R. Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell 110: 443–455, 2002.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  G. Sadri-Vakili, G. C. Janis, R. C. Pierce, T. T. Gibbs, and D. H. Farb Nanomolar Concentrations of Pregnenolone Sulfate Enhance Striatal Dopamine Overflow in Vivo J. Pharmacol. Exp. Ther., December 1, 2008; 327(3): 840 - 845. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
), but not 15 µM (
), induces a long-lasting increase of op-EPSP amplitude. Top: traces represent the typical recordings at 5 min before (a and c) and 60 min after (b and d) the application of PREGS, the same as in the following C. B: concentration-response curve of percentage increase of op-EPSP induced by PREGS. The changes in the op-EPSP amplitude were measured at 60 min after the cessation of PREGS vs. various concentrations of PREGS (1–100 µM). C: effect of PREGS (50 µM) on the f-EPSP slope. The experiment was carried out with the same preparations as those used in A. *P < 0.05; **P < 0.01.
