HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: 93/6/3102    most recent 00939.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Tyszkiewicz, J. P. Articles by Yan, Z. Search for Related Content PubMed PubMed Citation Articles by Tyszkiewicz, J. P. Articles by Yan, Z.

-Amyloid Peptides Impair PKC-Dependent Functions of Metabotropic Glutamate Receptors in Prefrontal Cortical Neurons

Department of Physiology and Biophysics, State University of New York, School of Medicine and Biomedical Sciences, Buffalo, New York

Submitted 9 September 2004; accepted in final form 17 January 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The metabotropic glutamate receptors (mGluRs) have been implicated in cognition, memory, and some neurodegenerative disorders, including the Alzheimer's disease (AD). To understand how the dysfunction of mGluRs contributes to the pathophysiology of AD, we examined the -amyloid peptide (A)-induced alterations in the physiological functions of mGluRs in prefrontal cortical pyramidal neurons. Two potential targets of mGluR signaling involved in cognition, the GABAergic system and the N-methyl-D-aspartate (NMDA) receptor, were examined. Activation of group I mGluRs with (S)-3,5-dihydroxyphenylglycine (DHPG) significantly increased the spontaneous inhibitory postsynaptic current (sIPSC) amplitude, and this effect was protein kinase C (PKC) sensitive. Treatment with A abolished the DHPG-induced enhancement of sIPSC amplitude. On the other hand, activation of group II mGluRs with (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC) significantly increased the NMDA receptor (NMDAR)-mediated currents via a PKC-dependent mechanism, and A treatment also diminished the APDC-induced potentiation of NMDAR currents. In A-treated slices, both DHPG and APDC failed to activate PKC. These results indicate that the mGluR regulation of GABA transmission and NMDAR currents is impaired by A treatment probably due to the A-mediated interference of mGluR activation of PKC. This study provides a framework within which the role of mGluRs in normal cognitive functions and AD can be better understood.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Glutamate, the most abundant excitatory neurotransmitter in the CNS, activates both ligand-gated ion (ionotropic) channels and G-protein-coupled metabotropic (mGluRs) receptors (Nakanishi 1992). The former mediate fast glutamatergic transmission, whereas the latter exert slower and modulatory roles. The eight mGluR subtypes are divided into three groups based on the homology of their amino acid sequences and transduction mechanisms: group I mGluRs (mGluR1 and -5) stimulate phospholipase C and phosphoinositide hydroplysis, group II (mGluR2 and -3) and group III mGluRs (mGlu4, -6, -7, and -8) primarily couple to the inhibition of cAMP formation (Pin and Duvoisin 1995). The differential distribution of mGluR subtypes on pre- and/or postsynaptic terminals allows these glutamate receptors to play important roles in neuronal communication and signal processing underlying higher cognitive functions (Conn 2003). Many forms of synaptic plasticity rely on mGluR-mediated signaling (Anwyl 1999; Bashir et al. 1993; Cho and Bashir 2002). Mice lacking mGluR subtypes show impaired learning and altered synaptic plasticity (Aiba et al. 1994; Lu et al. 1997; Yokoi et al. 1996). Thus mGluRs have been implicated in regulating the molecular targets responsible for learning and memory (Conn and Pin 1997; Nakanishi 1994).

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficiency and memory loss. The pathogenesis of AD is believed to involve the accumulation of -amyloid peptides (A) (Price and Sisodia 1998; Selkoe 2001). Derived from -amyloid precursor protein (APP) via proteolytic processing by - and -secreteases, A has the propensity to aggregate and form neuritic plaques—the main culprit of cognitive decline. In search for a safe and effective pharmacological strategy to ameliorate the A-driven mental impairments, the attention has spread over a number of potential therapeutic targets. The mGluRs, because of their involvement in cognition and neurodegeneration (Nicoletti et al. 1996), offer exciting possibilities for AD drug development (Bruno et al. 2001; Conn and Pin 1997). Elucidating the function and dysfunction of mGluRs under normal and pathological conditions may therefore provide valuable insights into our knowledge of cognition and memory as well as our understanding of senile dementia and AD.

Prefrontal cortex (PFC), the primary brain region involved in cognitive control (Fuster 2001; Miller and Cohen 2001), is one of the main targets of A deposits (Price and Sisodia 1998; Selkoe and Schenk 2003). To understand how the dysfunction of mGluRs contributes to the pathophysiology of AD (Lee et al. 2004), we need to determine whether A interferes with the physiological functions of mGluRs in PFC neurons. PFC neural activity is controlled by GABAergic inhibition and glutamatergic excitation. The GABAergic transmission has been shown to play a key role in "working memory" by shaping the temporal flow of information during cognitive operations (Constantinidis et al. 2002; Rao et al. 2000), whereas the N-methyl-D-aspartate (NMDA)-type glutamate receptor has long been recognized as a major player in synaptic plasticity, learning, and memory (Malenka and Nicoll 1999). Thus the GABAergic system and the NMDA receptor are two potential targets of mGluR signaling involved in cognition. In this study, we examined the A-induced alterations in the mGluR regulation of GABA transmission and NMDA receptor (NMDAR) currents in PFC pyramidal neurons.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Electrophysiological recordings in slices

Young adult rat slices containing PFC were prepared as described previously (Feng et al. 2001; Wang et al. 2003). All experiments were carried out with the approval of State University of New York at Buffalo Animal Care Committee. In brief, animals were anesthetized by inhaling 2-bromo-2-chloro-1,1,1-trifluoroethane (1 ml/100 g, Sigma) and decapitated, and brains were quickly removed, iced, and then blocked for slicing. The blocked tissue was cut in 300–400 µm slices with a Vibrotome while bathed in a low Ca²⁺, HEPES-buffered salt solution (which contained, in mM: 140 sodium isethionate, 2 KCl, 4 MgCl₂, 0.1 CaCl₂, 23 glucose, 15 HEPES, and 1 kynurenic acid, pH = 7.4, 300–305 mosM). Slices were then incubated for 1–6 h at room temperature in a NaHCO₃-buffered saline bubbled with 95% O₂-5% CO₂ (containing, in mM): 126 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 26 NaHCO₃, 1.25 NaH₂PO₄, 10 glucose, 1 pyruvic acid, 0.05 glutathione, 0.1 N^G-nitro-L-arginine, and 1 kynurenic acid, pH = 7.4, 300–305 mosM.

To evaluate the regulation of spontaneous inhibitory postsynaptic current (IPSC) by mGluRs in PFC slices, the whole cell voltage-clamp recording technique (Zhong et al. 2003) was used. Patch electrodes (5–9 M) were filled with the following internal solution (in mM): 100 CsCl, 30 N-methyl-D-glucamine, 10 HEPES, 1 MgCl₂, 4 NaCl, 5 EGTA, 12 phosphocreatine, 2 MgATP, 0.2 Na₃GTP, and 0.1 leupeptin, pH = 7.2–7.3, 265–270 mosM. The slice (300 µm) was placed in a perfusion chamber attached to the fixed-stage of an upright microscope (Olympus) and submerged in continuously flowing oxygenated artificial cerebrospinal fluid. For blocking glutamate transmission, the -amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid/kainate (AMPA/KA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) and N-methyl-D-aspartate receptor antagonist D(–)-2-amino-5-phosphonopetanoic acid (D-APV, 25 µM) were added to the recording solution. Cells were visualized with a x40 water-immersion lens and illuminated with near infrared (IR) light, and the image was detected with an IR-sensitive CCD camera. A Multiclamp 700A amplifier (Axon instruments, Union City, CA) was used for these recordings. Tight seals (2–10 G) from visualized pyramidal neurons were obtained by applying negative pressure. The membrane was disrupted with additional suction and the whole cell configuration was obtained. The access resistances (13–18 M) were compensated 50–70%. Cells were held at –70 mV for the recording of spontaneous IPSCs. All experiments were performed side by side with cells from nontreated versus A-treated slices.

Mini Analysis Program (Synaptosoft, Decatur, GA) was used to analyze synaptic activity. Individual synaptic events with fast onset and exponential decay kinetics were captured with threshold detectors in Mini Analysis software. All quantitative measurements were taken 4–6 min after drug application. IPSCs of 60 s (200–1,000 events) under each different treatment were used for obtaining the cumulative distribution plots. The detection parameters for analyzing synaptic events in each cell in the absence or presence of mGluR agonists were the same. Statistical comparisons of the frequency and amplitude of synaptic currents were made using the Kolmogorov-Smirnov (K-S) test. Numerical values were expressed as means ± SE.

mGluR ligands DHPG, DCG-IV, and (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC), 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), as well as second-messenger reagents calphostin C, myristoylated PKI_14–22, myristoylated PKC_20–28, phorbol 12-myristate 13-acetate (PMA), U1026, and SB203580 (Tocris) were made up as concentrated stocks in water and stored at –20°C. The -amyloid peptide A_25–35, A_1–42, and the control peptide containing the reverse sequence A_40–1 were obtained from Sigma. A_25–35 was resuspended in sterile distilled water at a concentration of 2 mM and incubated at 37°C for 1 h to allow fibril formation (Terzi et al. 1994). A_1–42 and A_40–1 peptides were dissolved in DMSO (2 mM stock) and diluted prior to use.

Whole cell recordings in acutely dissociated neurons

Whole cell recordings of currents in dissociated neurons employed standard voltage-clamp techniques (Tyszkiewicz et al. 2004; Yan et al. 1999). The internal solution consisted of (in mM) 180 N-methyl-D-glucamine (NMG), 40 HEPES, 4 MgCl₂, 0.1 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), 12 phosphocreatine, 2 Na₂ATP, 0.2 Na₃GTP, and 0.1 leupeptin, pH = 7.2–7.3, 265–270 mosM. The external solution consisted of (in mM) 127 NaCl, 20 CsCl, 10 HEPES, 1 CaCl₂, 5 BaCl₂, 12 glucose, 0.001 TTX, and 0.02 glycine, pH = 7.3–7.4, 300–305 mosM. Recordings were obtained with an Axon Instruments 200B patch-clamp amplifier that was controlled and monitored with an IBM PC running pCLAMP (v. 8) with a DigiData 1320 series interface (Axon Instruments, Union City, CA). Electrode resistances were typically 2–4 M in the bath. After seal rupture, series resistance (4–10 M) was compensated (70–90%). Care was exercised to monitor the constancy of the series resistance, and recordings were terminated whenever a significant increase (>20%) occurred. The cell membrane potential was held at –60 mV. NMDA (100 µM) was applied for 2 s every 30 s. Drugs were applied with a gravity-fed "sewer pipe" system. The array of application capillaries (150 µm ID) was positioned a few hundred micrometers from the cell under study. Solution changes were effected by the SF-77B fast-step solution stimulus delivery device (Warner Instrument, Hamden, CT).

Data analyses were performed with AxoGraph (Axon Instruments), Kaleidagraph (Albeck Software, Reading, PA), and StatView (Abacus Concepts, Calabasas, CA). For analysis of statistical significance, Mann-Whitney U tests were performed to compare the current amplitudes in the presence or absence of agonists. Student t-test was performed to compare the differential degrees of current modulation between groups subjected to different treatment.

Western blot analysis

For detecting activated PKC, a phospho-PKC (pan) antibody that recognizes PKC, _I, _II, , , and isoforms only when phosphorylated at a carboxyl-terminal residue homologous to Ser⁶⁶⁰ of PKC_II was used in the Western blot analysis (Gu et al. 2003; Tyszkiewicz et al. 2004). After incubation, slices were transferred to boiling 1% SDS and homogenized immediately. Insoluble material was removed by centrifugation (13,000 g for 10 min), and protein concentration for each sample was measured. Equal amounts of protein from slice homogenates were separated on 7.5% acrylamide gels and transferred to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk for 1 h at room temperature. Then the blots were incubated with the phospho-PKC (pan) antibody (Cell Signaling, 1:2,000) or anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (Cell Signaling, 1:100) for 1 h at room temperature. After being rinsed, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Amersham Biosciences, 1:2,000) for 1 h at room temperature. After three washes, the blots were exposed to the enhanced chemiluminescence substrate. Then the blots were stripped for 1 h at 50°C, followed by saturation in 5% nonfat dry milk and incubated with a PKC antibody recognizing the , , and isoforms (Santa Cruz, 1:2,000) or an antibody recognizing EKR1/2 (Cell Signaling, 1:2000). Quantification was obtained from densitometric measurements of immunoreactive bands on autoradiograms.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Activation of group I mGluRs enhances the spontaneous IPSC amplitude and frequency in rat PFC pyramidal neurons

To investigate the role of mGluRs on GABAergic inhibitory transmission in PFC, we examined the effects of group I and group II mGluR agonists on the spontaneous inhibitory postsynaptic currents (sIPSC) in deep layer PFC pyramidal neurons. Bath application of DHPG (50 µM), a selective group I mGluR agonist, resulted in a marked increase in the amplitude and frequency of sIPSC (Fig. 1A). In contrast, DCG-IV (500 nM), a highly potent group II mGluR agonist, was unable to regulate sIPSC (Fig. 1B). In a sample of cells we tested, DHPG caused a significant increase in the mean amplitude (52.7 ± 5.0%, n = 21; P < 0.001, K-S test) and mean frequency (150.4 ± 19.8%, n = 21; P < 0.001, K-S test) of sIPSC (Fig. 1, C and D), whereas DCG-IV had little effect on either the amplitude (–3 ± 1.5%, n = 3, P > 0.05, K-S test) or the frequency (3.7 ± 5.3%, n = 3; P > 0.05, K-S test) of sIPSC (Fig. 1, C and D). We further recorded the miniature IPSC (mIPSC) in the absence and presence of DHPG. In all cells tested, the amplitude and frequency of mIPSC (recorded in the presence of TTX) were not changed by DHPG (amplitude: –0.3 ± 2.3%; frequency: –4.1 ± 3.1%, n = 8, P > 0.05, K-S test). These results suggest that the modulation of sIPSC amplitude and frequency is specific to group I mGluRs.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Activation of group I, but not group II, metabotropic glutamate receptor (mGluR) enhances the amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSC) in prefrontal cortex (PFC) pyramidal neurons. A: cumulative plots of the distribution of sIPSC amplitude (left) and representative traces (right) before (ctl) and during DHPG (50 µM) application. B: cumulative plots of the distribution of sIPSC amplitude (left) and representative traces (right) before (ctl) and during DCG-IV (500 nM) application. Scale bars: 30 pA, 10 s. C and D: summary histograms comparing the effect of DHPG and DCG-IV on sIPSC amplitude (C) and frequency (D). *, P < 0.001, t-test.

View larger version (40K): [in this window] [in a new window]   FIG. 2. The selective mGluR5 antagonist, but not mGluR1 antagonist, blocks the mGluR enhancement of sIPSC amplitude and frequency. A–C: cumulative plots of the distribution of sIPSC amplitude (top) and representative traces (bottom) before (ctl) and during DHPG (50 µM) application in the absence (A) or presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 5 µM, B) or the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; 50 µM, C). Scale bars: 50 pA, 10 s. D and E: summary histograms comparing the effect of DHPG on sIPSC amplitude (D) and frequency (E) in the absence (–) or presence of MPEP or CPCCOEt. *, P < 0.001, t-test.

We next tried to determine the signaling mechanism underlying the mGluR regulation of sIPSC. Activation of group I mGluRs stimulates the PLC–PKC cascade (Conn and Pin 1997), thus we first examined whether PKC was involved in the regulation of GABA transmission by group I mGluRs.

PFC slices were incubated with the cell-permeable and specific PKC inhibitor, calphostin C (1 µM), for 1 h, followed by recordings of sIPSC in the absence and presence of DHPG. As shown in Fig. 3, A and B, the DHPG enhancement of sIPSC amplitude was abolished in slices treated with calphostin C, suggesting the involvement of PKC. To confirm the role of PKC, we also applied another mechanistically distinct PKC inhibitor, myristoylated PKC_20–28 (20 µM) (Eichholtz et al. 1993). Similarly, the DHPG enhancement of sIPSC amplitude was abolished in slices treated with myristoylated PKC_20–28 (Supplemental Fig. 3 ¹ ). To determine whether the group I mGluR regulation of sIPSC amplitude is PKC-specific, we further examined the DHPG effect in PFC slices pretreated with the cell-permeable PKA inhibitor, myristoylated PKI_14–22 (1 µM) (Glass et al. 1989). As shown in Fig. 3C, after incubation with PKI_14–22, DHPG still induced a robust increase in the amplitude of sIPSC. As summarized in Fig. 3D, DHPG caused little increase in sIPSC amplitude in calphostin-treated cells (5.3 ± 7.2%, n = 7, P > 0.05, K-S test) or PKC_20–28treated cells (3.3 ± 6.1%, n = 4, P > 0.05, K-S test), which was significantly different from the effect of DHPG in nontreated cells (52.6 ± 6.5%, n = 21, P < 0.001, K-S test) or PKI_14–22-treated cells (67.2 ± 17.1%, n = 4; P < 0.001, K-S test).

View larger version (56K): [in this window] [in a new window]   FIG. 3. The DHPG-induced increase in sIPSC amplitude is PKC dependent. A–C: cumulative plots of the distribution of sIPSC amplitude (top) and representative traces (bottom) before and during DHPG (50 µM) application in neurons from the nontreated slice (A) or the slice treated with calphostin C (1 µM, B) or PKI14–22 (1 µM, C). Scale bars: 20 pA, 10 s. D and E: summary histograms comparing the effect of DHPG on sIPSC amplitude (D) and frequency (E) in neurons under different treatments. *, P < 0.001, t-test.

Treatment with -amyloid peptides abolishes the group I mGluR regulation of sIPSC amplitude

-Amyloid peptide (A), derived from APP via proteolytic cleavage, is the primary component of neuritic plaques found in specific brain regions of AD patients (Selkoe and Schenk 2003). To investigate whether A interferes with the group I mGluR modulation of GABAergic transmission in PFC, we incubated PFC slices with A peptides and recorded sIPSC in the absence and presence of DHPG. We first compared the basal properties of sIPSCs in nontreated versus A-treated slices (Supplemental Fig. 2). No significant difference was found between these groups (mean amplitude: control: 31.4 ± 1.9 pA, n = 16; A_25–35-treated: 24.8 ± 3.0 pA, n = 6; A_1–42-treated: 40.6 ± 5.5 pA, n = 10; P > 0.05, t-test; mean event number/min: control: 397.6 ± 50.3, n = 16; A_25–35-treated: 296.7 ± 56.0, n = 6; A_1–42-treated: 374.9 ± 76.5, P > 0.05, t-test). We then examined the effect of DHPG on sIPSCs in A-treated slices.

As shown in Fig. 4, A and B, in the nontreated neuron, DHPG caused a potent enhancement of the sIPSC amplitude, whereas in the neuron treated with the aggregated A_25–35 (1 µM), which represents the biologically active region of A (Pike et al. 1995; Yankner et al. 1990), the effect of DHPG on sIPSC amplitude was abolished. Similarly, in the neuron treated with the full-length A peptide, A_1–42 (1 µM), DHPG failed to increase sIPSC amplitude (Fig. 4C). Lower concentration of A_1–42 (0.5 µM), but not A_1–42 (0.1 µM), also blocked the DHPG effect on sIPSC amplitude (Supplemental Fig. 1). In contrast, in the neuron treated with the control peptide containing the reverse sequence, A_40–1 (1 µM), the enhancing effect of DHPG on sIPSC amplitude was intact (Fig. 4D).

View larger version (54K): [in this window] [in a new window]   FIG. 4. Activation of group I mGluRs fails to increase the sIPSC amplitude in slices treated with A. A–D: cumulative plots of the distribution of sIPSC amplitude (top) and representative traces (bottom) before and during DHPG (50 µM) application in neurons from the nontreated slice (A) or the slice treated with aged A25–35 (1 µM, B) or A1–42 (1 µM, C) or the control peptide A40–1 (1 µM, D). Scale bars: 30 pA, 10 s. E and F: summary histograms comparing the effect of DHPG on sIPSC amplitude (E) and frequency (F) in neurons under different treatments. *, P < 0.001, t-test.

-Amyloid peptides impair the group II mGluR-mediated potentiation of NMDAR currents in PFC neurons

We have recently reported that activation of group II mGluRs increases NMDAR currents in PFC pyramidal neurons via a PKC-dependent mechanism (Tyszkiewicz et al. 2004). Considering that -amyloid peptides impair the group I mGluR-mediated, PKC-dependent regulation of GABAergic transmission, we hypothesized that the group II mGluR-mediated, PKC-dependent regulation of NMDAR currents may also be impaired by A exposure. To investigate this, we incubated PFC slices with A_1–42 (1 µM) for 4 h and recorded NMDAR currents from acutely isolated neurons. We first compared the basal NMDAR currents in nontreated versus A-treated slices (Supplemental Fig. 2). No significant difference on the amplitude of NMDAR currents was found between these groups (control: 1.07 ± 0.19 nA, n = 9; A_1–42-treated: 1.22 ± 0.18 nA, n = 13; A_40–1-treated: 1.24 ± 0.22 nA, n = 4; P > 0.05, t-test). We then examined the effect of group II mGluRs on NMDAR currents in A-treated slices. As shown in Fig. 5, A and B, application of APDC, a selective agonist for group II mGluRs, caused a reversible enhancement of NMDAR currents in the neuron from a nontreated slice but failed to enhance NMDAR currents in the neuron from an A_1–42-treated slice. In contrast, the enhancing effect of APDC on NMDAR currents was intact in the neuron from a slice treated with the control peptide A_40–1 (Fig. 5C). As summarized in Fig. 5D, APDC had little effect on NMDAR currents in A_1–42-treated neurons (1.2 ± 0.8%, n = 13; P > 0.05, Mann-Whitney), which was significantly different from the effect of APDC in nontreated neurons (21.3 ± 1.1%, n = 9, P < 0.001, Mann-Whitney) or A_40–1-treated cells (18.3 ± 1.9%, n = 4; P < 0.001, Mann-Whitney). These data indicate that the group II mGluR regulation of NMDAR currents is disrupted by A.

View larger version (27K): [in this window] [in a new window]   FIG. 5. The group II mGluR-mediated enhancement of NMDAR current is abolished by A treatment. A–C: plots of peak NMDAR currents as a function of time and APDC (50 µM) application in PFC pyramidal neurons isolated from the nontreated slice (A) or the slice treated with A1–42 (1 µM, B) or A40–1 (1 µM, C). Inset: representative NMDA (100 µM)-evoked current traces before and during APDC (50 µM) application (at time points denoted by #). Scale bars: 0.15 nA, 0.5 s. D: cumulative data showing the percentage modulation of NMDAR currents by APDC in neurons under different treatments. *, P < 0.001, t-test.

-Amyloid peptide treatment inhibits the mGluR activation of PKC

We then tried to determine the mechanism underlying the A-induced impairment of mGluR functions. Because both the group I mGluR enhancement of sIPSC amplitude and the group II mGluR potentiation of NMDAR currents are PKC-dependent, we speculated that the impairment of these mGluR functions by A may be caused by the loss of mGluR activation of PKC after A treatment. To examine this, we compared DHPG- and APDC-induced activation of PKC in PFC slices treated with or without A_1–42 (1 µM). As shown in Fig. 6A, application of the group I mGluR agonist DHPG (100 µM) caused a strong increase in PKC activity in the nontreated control slice but failed to do so in the A_1–42 -treated slice. The total level of PKC was not changed by DHPG with or without A_1–42 treatment. As summarized in Fig. 6B, DHPG significantly increased the PKC activity in nontreated slices (1.7 ± 0.03-fold, n = 6, P < 0.001, t-test), but not in A_1–42-treated slices (0.9 ± 0.05-fold, n = 4, P > 0.05, t-test). Similarly, as shown in Fig. 6C, application of the group II mGluR agonist APDC (50 µM) also potently increased PKC activity in the nontreated control slice, but this effect was abolished in the A_1–42-treated slice. Summarized data (Fig. 6D) show that APDC significantly increased PKC activity in control slices (1.9 ± 0.11-fold, n = 6, P < 0.001, t-test) but lost the capability to activate PKC in A_1–42-treated slices (0.8 ± 0.02-fold, n = 4; P > 0.05, t-test). Similarly, the capability of phorbol ester PMA to activate PKC was also impaired in A_1–42-treated slices (Fig. 6E, n = 4), consistent with the previous finding that A directly inhibits PKC activation (Lee et al. 2004).

View larger version (40K): [in this window] [in a new window]   FIG. 6. A treatment blocks the activation of PKC by group I and II mGluRs. A and C: immunoblots of activated PKC autophosphorylated at carboxyl terminus (top) and total PKC (bottom) incubated without or with DHPG (100 µM, 10 min; A) or APDC (50 µM, 10 min; C) in nontreated or A1–42 (1 µM)-treated PFC slices. B and D: quantification of the activated PKC in response to DHPG (B) or APDC (D) in nontreated or A1–42-treated slices. *, P < 0.001, t-test. E: immunoblots of activated PKC (top) and total PKC (bottom) incubated without or with PMA (0.1 µM, 5 min) in nontreated or A1–42 (1 µM)-treated PFC slices. F: immunoblots of p42/44 MAP kinase (p-ERK; top) and total ERK (bottom) incubated without or with DHPG (100 µM, 10 min) in nontreated or A1–42 (1 µM)-treated PFC slices.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The activation of mGluRs has diverse cellular actions that lead to alterations in excitability and synaptic transmission (Anwyl 1999; Mannaioni et al. 2001; Semyanov and Kullmann 2000; Valenti et al. 2003). Because of the important role of GABAergic transmission in "working memory" subserved by PFC (Constantinidis et al. 2002), we first examined the mGluR regulation of GABA transmission in PFC pyramidal neurons. Activation of group I mGluRs produced a strong increase in the amplitude and frequency of sIPSC, which could be caused by the group I mGluR-induced depolarization of GABAergic interneurons as found in the hippocampus (McBain et al. 1994; Miles and Poncer 1993; van Hooft et al. 2000). The DHPG-induced increase in sIPSCs is probably mediated by mGluR5 receptors in PFC neurons. While group II mGluRs have no effect on GABAergic transmission, we found that activation of group II mGluRs significantly potentiated the currents through NMDAR channels in acutely dissociated PFC pyramidal neurons (Tyszkiewicz et al. 2004). It suggests that group II mGluRs not only inhibit evoked glutamate release in PFC via presynaptic mechanisms (Cartmell and Schoepp 2000; Marek et al. 2000) but also regulate postsynaptic NMDA receptor channels, one of the major substrates involved in cognition.

Because mGluRs have been implicated in memory acquisition, learning, and some neurodegenerative disorders (Conn and Pin 1997), growing effort is being directed to understand the relation between mGluR signaling and amyloid accumulation in AD. A strongly inhibits the induction of long-term potentiation (LTP) (Walsh et al. 2002), a synaptic model of learning and memory, and this effect is prevented by a group I/II mGluR antagonist and a selective mGluR5 antagonist, suggesting the involvement of mGluR5 in the A-mediated inhibition of LTP (Wang et al. 2004). Moreover, pharmacologic blockade or activation of different mGluRs produces neuroprotection in models for chronic neurodegenerative disorders. For example, prolonged exposure of cultured cortical cells to A_25–35 induces neuronal apoptosis, and the apoptosis is substantially attenuated by group II or III mGluR agonists (Copani et al. 1995). Selective antagonism of mGluR5 also exhibits neuroprotective function against A toxicity in cortical cultures (Bruno et al. 2000). These results have demonstrated that mGluR signaling exerts an important impact on the cellular actions of A; however, the impact of A deposits on mGluR functions is largely unknown.

The most important finding of the present study is that A treatment impairs two important functions of mGluRs involved in cognition: the group I mGluR regulation of GABA transmission and group II mGluR regulation of NMDA receptor currents. Emerging evidence has suggested that AD is a synaptic failure, with functional—not structural—synaptic changes being responsible for the cognitive deficits prior to frank neuronal degeneration (Hsia et al. 1999; Selkoe 2002). The accumulation of diffuse deposits of A in the brain is an early event in the development of AD, which emphasizes the importance of elucidating the neuronal response to A before clinical symptoms arise. Strong correlations between soluable A levels and the extent of synaptic loss and cognitive impairment have been found in AD brains (Lue et al. 1999; McLean et al. 1999), suggesting that synapses are the initial target in AD (Small et al. 2001). We speculate that the A-induced dysregulation of GABAergic and glutamatergic synapses will cause subtle alterations of cortical synaptic efficacy, leading to impairment of memory as the disease progresses. The aberrant synaptic functions of mGluRs in neurons after A treatment, along with our previous finding on the impaired synaptic functions of muscarinic receptors in A-treated PFC slices and in APP transgenic mice (Zhong et al. 2003), provide a cellular mechanism showing how A could alter the complex regulation of synapses, which may result in the deficient cognition and memory.

Several mechanisms could account for the DHPG effects on GABA transmission. First, mGluRs increase the excitability of GABAergic interneurons by suppressing potassium conductances or potentiating cation currents, therefore leading to the elevated probability of GABA release. Second, mGluRs enhance the probability of action potential-dependent GABA release from axon terminals, therefore leading to the increase of the contribution of large-size (multiquantal) sIPSCs to the overall population of synaptic events. One possible underlying mechanism for this mGluR action is that the mGluRI/phospholipid/PKC signaling regulates the presynaptic protein synaptotagmine that functions as a calcium sensor to trigger synchronous vesicle fusion events, thus facilitating the Ca²⁺ cooperativity of transmitter release. The signaling mechanism mediating the DHPG increase in sIPSC frequency awaits to be elucidated.

Given the PKC dependence of both the group I mGluR enhancement of sIPSC amplitude (this study) and the group II mGluR potentiation of NMDAR currents (Tyszkiewicz et al. 2004), one possible mechanism underlying the A-induced impairment of these mGluR functions is the loss of mGluR activation of PKC after A treatment. Considerable evidence has suggested that many postreceptor signal transduction processes are severely compromised in AD, including the impaired G protein regulation of phospholipase C, the decreased receptor sites for the second messenger IP₃, and the reduced PKC level and activity (Cowburn et al. 2001; Fowler et al. 1995). PKC, the key enzyme in memory processes (Tanaka and Nishizuka 1994), shows deficiency in its translocation and activation in aged rat cortex (Pascale et al. 1998). Anchoring proteins that are involved in PKC signal transduction seem to contribute to this PKC activation deficit (Battaini et al. 1997). Because the stimulation of PKC by mGluRs increases the nonamyloidogenic, secretory pathway of APP processing (Lee et al. 1995), the disrupted mGluR activation of PKC in AD may inhibit the nonamyloidogenic APP processing pathway and lead to increased -amyloid production. Enhanced A generation, in turn, exacerbates mGluR abnormalities, causing the dysfunction of glutamatergic synapses.

Our data have demonstrated that A peptides inhibit only the PKC limb of the mGluR pathway and not the ERK limb, suggesting that mGluR function in general is not affected by A. These data also suggest that other neurotransmitter systems that activate PKC may be inhibited by A as well, like the muscarinic receptors (Zhong et al. 2003). Thus A may have a selective effect on PKC-dependent processes as opposed to general mGluR functioning.

Taken together, this study has revealed that the mGluR regulation of two important targets involved in cognition, GABA transmission and NMDA receptors, are impaired by A treatment, which may be attributable to the A-induced loss of mGluR activation of PKC. Given the important role of mGluRs, GABA transmission, NMDA receptors and PKC in cognition, this finding provides a potentially important clue to the possible ways in which A could act to cause cognitive decline. It supports the notion that elucidation of the effects of A on synaptic function rather than on cell death is critical for understanding the pathogenesis of AD and for finding novel therapeutic targets (Small et al. 2001).

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institutes of Health Grants AG-21923 and MH-63128 and National Science Foundation Grant IBN-0117026 to Z. Yan.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We thank X. Chen for technical support.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The Supplementary Material for this article (3 figures) is available online at http://jn.physiology.org/cgi/content/full/00939.2004/DC1.

Address for reprint requests and other correspondence: Z. Yan, Dept. of Physiology and Biophysics, State University of New York at Buffalo, 124 Sherman Hall, Buffalo, NY, 14214 (E-mail: zhenyan{at}buffalo.edu)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Anwyl R. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Res Rev 29: 83–120, 1999.[CrossRef][Medline]

Aiba A, Kano M, Chen C, Stanton ME, Fox GD, Herrup K, Zwingman TA, and Tonegawa S. Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79: 377–388, 1994.[CrossRef][ISI][Medline]

Bashir ZI, Bortolotto ZA, Davies CH, Berretta N, Irving AJ, Seal AJ, Henley JM, Jane DE, Watkins JC, and Collingridge GL. Induction of LTP in the hippocampus needs synaptic activation of glutamate metabotropic receptors. Nature 363: 347–350, 1993.[CrossRef][Medline]

Battaini F, Pascale A, Paoletti R, and Govoni S. The role of anchoring protein RACK1 in PKC activation in the ageing rat brain. Trends Neurosci 20: 410–415, 1997.[CrossRef][ISI][Medline]

Bolshakov VY, Carboni L, Cobb MH, Siegelbaum SA, and Belardetti F. Dual MAP kinase pathways mediate opposing forms of long-term plasticity at CA3-CA1 synapses. Nat Neurosci 3: 1107–1112, 2000.[CrossRef][ISI][Medline]

Bruno V, Battaglia G, Copani A, D'Onofrio M, Di Iorio P, De Blasi A, Melchiorri D, Flor PJ, and Nicoletti F. Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J Cereb Blood Flow Metab 21: 1013–1033, 2001.[ISI][Medline]

Bruno V, Ksiazek I, Battaglia G, Lukic S, Leonhardt T, Sauer D, Gasparini F, Kuhn R, Nicoletti F, and Flor PJ. Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective. Neuropharmacology 39: 2223–2230, 2000.[CrossRef][ISI][Medline]

Cartmell J and Schoepp DD. Regulation of neurotransmitter release by metabotropic glutamate receptors. J Neurochem 75: 889–907, 2000.[CrossRef][ISI][Medline]

Cho K and Bashir ZI. Cooperation between mglu receptors: a depressing mechanism? Trends Neurosci 25: 405–411, 2002.[CrossRef][ISI][Medline]

Conn PJ. Physiological roles and therapeutic potential of metabotropic glutamate receptors. Ann NY Acad Sci. 1003: 12–21, 2003.[Abstract/Free Full Text]

Conn PJ and Pin JP. Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 7: 205–237, 1997.[CrossRef]

Constantinidis C, Williams GV, and Goldman-Rakic PS. Correlated discharges among putative pyramidal neurons and interneurons in the primate prefrontal cortex. J Neurophysiol 88: 3487–3497, 2002.[Abstract/Free Full Text]

Copani A, Bruno V, Battaglia G, Leanza G, Pellitteri R, Russo A, Stanzani S, and Nicoletti F. Activation of metabotropic glutamate receptors protects cultured neurons against apoptosis induced by beta-amyloid peptide. Mol Pharmacol. 47: 890–7, 1995.[Abstract]

Cowburn RF, O'Neill C, Bonkale WL, Ohm TG, and Fastbom J. Receptor-G-protein signalling in Alzheimer's disease. Biochem Soc Symp 67: 163–175, 2001.

Eichholtz T, de Bont DB, de Widt J, Liskamp RM, and Ploegh HL. A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J Biol Chem 268: 1982–1986, 1993.[Abstract/Free Full Text]

Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623–18632, 1998.[Abstract/Free Full Text]

Feng J, Cai X, Zhao JH, and Yan Z. Serotonin receptors modulate GABA_A receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons. J Neurosci 21: 6502–6511, 2001.[Abstract/Free Full Text]

Fowler CJ, Cowburn RF, Garlind A, Winblad B, and O'Neill C. Disturbances in signal transduction mechanisms in Alzheimer's disease. Mol Cell Biochem 149: 287–292, 1995.[CrossRef][Medline]

Fuster JM. The prefrontal cortex-an update: time is of the essence. Neuron 30: 319–333, 2001.[CrossRef][ISI][Medline]

Glass DB, Cheng HC, Mende-Mueller L, Reed J, and Walsh DA. Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein. J Biol Chem. 264: 8802–8810, 1989.[Abstract/Free Full Text]

Gu Z, Zhong P, and Yan Z. Activation of muscarinic receptors inhibits -amyloid peptide-induced signaling in cortical slices. J Biol Chem 278: 17546–17556, 2003.[Abstract/Free Full Text]

Hsia AY, Masliah E, McConlogue L, Yu GQ, Tatsuno G, Hu K, Kholodenko D, Malenka RC, Nicoll RA, and Mucke L. Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Natl Acad Sci USA 96: 3228–3233, 1999.[Abstract/Free Full Text]

Lee H, Zhu X, O'Neill MJ, Webber K, Casadesus G, Marlatt M, Raina AK, Perry G, and Smith MA. The role of metabotropic glutamate receptors in Alzheimer's disease. Acta Neurobiol Exp 64: 89–98, 2004.

Lee RK, Wurtman RJ, Cox AJ, and Nitsch RM. Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors. Proc Natl Acad Sci USA 92: 8083–8087, 1995.[Abstract/Free Full Text]

Lee W, Boo JH, Jung MW, Park SD, Kim YH, Kim SU, and Mook-Jung I. Amyloid beta peptide directly inhibits PKC activation. Mol Cell Neurosci 26: 222–231, 2004.[CrossRef][ISI][Medline]

Lu YM, Jia Z, Janus C, Henderson JT, Gerlai R, Wojtowicz JM, and Roder JC. Mice lacking metabotropic glutamate receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP. J Neurosci 17: 5196–5205, 1997.[Abstract/Free Full Text]

Lue LF, Kuo YM, Roher AE, Brachova L, Shen Y, Sue L, Beach T, Kurth JH, Rydel RE, and Rogers J. Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease. Am J Pathol 155: 853–862, 1999.[Abstract/Free Full Text]

Malenka RC and Nicoll RA. Long-term potentiation—a decade of progress? Science 285: 1870–1874, 1999.[Abstract/Free Full Text]

Mannaioni G, Marino MJ, Valenti O, Traynelis SF, and Conn PJ. Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function. J Neurosci 21: 5925–5934, 2001.[Abstract/Free Full Text]

Marek GJ, Wright RA, Schoepp DD, Monn JA, and Aghajanian GK. Physiological antagonism between 5-hydroxytryptamine(2A) and group II metabotropic glutamate receptors in prefrontal cortex. J Pharmacol Exp Ther 292: 76–87, 2000.[Abstract/Free Full Text]

McBain CJ, DiChiara TJ, and Kauer JA. Activation of metabotropic glutamate receptors differentially affects two classes of hippocampal interneurons and potentiates excitatory synaptic transmission. J Neurosci 14: 4433–4445, 1994.[Abstract]

McLean CA, Cherny RA, Fraser FW, Fuller SJ, Smith MJ, Beyreuther K, Bush AI, and Masters CL. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol 46: 860–866, 1999.[CrossRef][ISI][Medline]

Miles R and Poncer JC. Metabotropic glutamate receptors mediate a post-tetanic excitation of guinea-pig hippocampal inhibitory neurons. J Physiol 463: 461–473, 1993.[Abstract/Free Full Text]

Miller EK and Cohen JD. An integrative theory of prefrontal cortex function. Annu Rev Neurosci 24: 167–202, 2001.[CrossRef][ISI][Medline]

Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function. Science 258: 597–603, 1992.[Abstract/Free Full Text]

Nakanishi S. Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity. Neuron 13: 1031–1037, 1994.[CrossRef][ISI][Medline]

Nicoletti F, Bruno V, Copani A, Casabona G, and Knöpfel T. Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders? Trends Neurosci 19: 267–271, 1996.[CrossRef][ISI][Medline]

Pascale A, Govoni S, and Battaini F. Age-related alteration of PKC, a key enzyme in memory processes: physiological and pathological examples. Mol Neurobiol 16: 49–62, 1998.[ISI][Medline]

Pike CJ, Walencewicz-Wasserman AJ, Kosmoski J, Cribbs DH, Glabe CG, and Cotman CW. Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25–35 region to aggregation and neurotoxicity. Neurochem 64: 253–265, 1995.

Pin JP and Duvoisin R. The metabotropic glutamate receptors: structure and function. Neuropharmacology 34: 1–26, 1995.[CrossRef][ISI][Medline]

Price DL and Sisodia SS. Mutant genes in familial Alzheimer's disease and transgenic models. Annu Rev Neurosci 21: 479–505, 1998.[CrossRef][ISI][Medline]

Rao SG, Williams GV, and Goldman-Rakic PS. Destruction and creation off spatial tuning by disinhibition: GABA(A) blockage of prefrontal cortical neurons engaged by working memory. J Neurosci 20: 485–494, 2000.[Abstract/Free Full Text]

Rush AM, Wu J, Rowan MJ, and Anwyl R. Group I metabotropic glutamate receptor (mGluR)-dependent long-term depression mediated via p38 mitogen-activated protein kinase is inhibited by previous high-frequency stimulation and activation of mGluRs and protein kinase C in the rat dentate gyrus in vitro. J Neurosci. 22: 6121–6128, 2002.[Abstract/Free Full Text]

Selkoe DJ. Alzheimer's disease: genes, protein, and therapy. Physiol. Rev. 81: 741–766, 2001.[Abstract/Free Full Text]

Selkoe DJ. Alzheimer's disease is a synaptic failure. Science. 298: 789–91, 2002.[Abstract/Free Full Text]

Selkoe DJ and Schenk D. Alzheimer's Disease: molecular understanding predicts amyloid-based therapeutics. Annu Rev Pharmacol Toxicol 43: 545–584, 2003.[CrossRef][ISI][Medline]

Semyanov A and Kullmann DM. Modulation of GABAergic signaling among interneurons by metabotropic glutamate receptors. Neuron 25: 663–672, 2000.[CrossRef][ISI][Medline]

Small DH, Mok SS, and Bornstein JC. Alzheimer's disease and Abeta toxicity: from top to bottom. Nat Rev Neurosci 2: 595–598, 2001.[CrossRef][ISI][Medline]

Tanaka C and Nishizuka Y. The protein kinase C family for neuronal signaling. Annu Rev Neurosci 17: 551–567, 1994.[CrossRef][ISI][Medline]

Terzi E, Holzemann G, and Seelig J. Reversible random coil-beta-sheet transition of the Alzheimer beta-amyloid fragment (25–35). Biochemistry 33: 1345–1350, 1994.[CrossRef][Medline]

Tyszkiewicz JP, Gu Z, Wang X, Cai X, and Yan Z. Group II metabotropic glutamate receptors enhance NMDA receptor currents via a protein kinase C-dependent mechanism in pyramidal neurones of rat prefrontal cortex. J Physiol 554: 765–777, 2004.[Abstract/Free Full Text]

Valenti O, Marino MJ, Wittmann M, Lis E, DiLella AG, Kinney GG, and Conn PJ. Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse. J Neurosci 23: 7218–26, 2003.[Abstract/Free Full Text]

van Hooft JA, Giuffrida R, Blatow M, and Monyer H. Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons. J Neurosci 20: 3544–3551, 2000.[Abstract/Free Full Text]

Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, and Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416: 535–539, 2002.[CrossRef][Medline]

Wang Q, Walsh D, Rowan MJ, Selkoe DJ, and Anwyl R. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24: 3370–3378, 2004.[Abstract/Free Full Text]

Wang X, Zhong P, Gu Z, and Yan Z. Regulation of NMDA receptors by dopamine D₄ signaling in prefrontal cortex. J Neurosci 23: 9852–9861, 2003.[Abstract/Free Full Text]

Yan Z, Hsieh-Wilson L, Feng J, Tomizawa K, Allen PB, Fienberg AA, Nairn AC, and Greengard P. Protein phosphatase I modulation of neostriatal AMPA channels: regulation by DARPP-32 and spinophilin. Nat Neurosci 2: 13–17, 1999.[CrossRef][ISI][Medline]

Yan Z and Surmeier DJ. D5 dopamine receptors enhance Zn²⁺-sensitive GABA_A currents in striatal cholinergic interneurons through a PKA/PP1 cascade. Neuron 19: 1115–1126, 1997.[CrossRef][ISI][Medline]

Yankner BA, Duffy LK, and Kirschner DA. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 250: 279–282, 1990.[Abstract/Free Full Text]

Yokoi M, Kobayashi K, Manabe T, Takahashi T, Sakaguchi I, Katsuura G, Shigemoto R, Ohishi H, Nomura S, Nakamura K, Nakao K, Katsuki M, and Nakanishi S. Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2. Science. 273: 645–647, 1996.[Abstract]

Young PR, McLaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty D, Gallagher TF, Fisher S, McDonnell PC, Carr SA, Huddleston MJ, Seibel G, Porter TG, Livi GP, Adams JL, and Lee JC. Pyridinyl imidazole inhibit