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Endocrinology, doi:10.1210/en.2007-1378
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Endocrinology Vol. 149, No. 3 1389-1398
Copyright © 2008 by The Endocrine Society

Corticotropin-Releasing Hormone (CRH) Depresses N-Methyl-D-Aspartate Receptor-Mediated Current in Cultured Rat Hippocampal Neurons via CRH Receptor Type 1

Hui Sheng1, Yanmin Zhang1, Jihu Sun, Lu Gao, Bei Ma, Jianqiang Lu and Xin Ni

Department of Physiology (H.S., Y.Z., J.S., L.G., B.M., X.N.), Changhai Hospital (J.L.), The Key Laboratory of Molecular Neurobiology (H.S., Y.Z., J.S., L.G., B.M., X.N.), Ministry of Education, The Second Military Medical University, Shanghai 200433; and School of Kinesiology (J.L.), Shanghai University of Sport, 650 Qing Yuan Huan Road, Shanghai 200438, People’s Republic of China

Address all correspondence and requests for reprints to: Dr. Xin Ni, Department of Physiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China. E-mail: nxljq2003{at}yahoo.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH, the primary regulator of the neuroendocrine responses to stress, has been shown to modulate synaptic efficacy and the process of learning and memory in hippocampus. However, effects of CRH on N-methyl-D-aspartate (NMDA) receptor, the key receptor for synaptic plasticity, remain unclear. In primary cultured hippocampal neurons, using the technique of whole-cell patch-clamp recordings, we found that CRH (1 pmol/liter to 10 nmol/liter) inhibited NMDA-induced currents in a dose-dependent manner. This effect was reversed by the CRH receptor type 1 (CRHR1) antagonist antalarmin but not by the CRHR2 antagonist astressin-2B, suggesting that CRHR1 mediated the inhibitory effect of CRH. Investigations on the signaling pathways of CRH showed that CRH dose-dependently induced phosphorylated phospholipase C (PLC)-β3 expression and increased intracellular cAMP content in these cells. Blocking PLC activity with U73122 prevented CRH-induced depression of NMDA current, whereas blocking protein kinase A (H89) and adenylate cyclase (SQ22536) failed to affect the CRH-induced depression of NMDA current. Application of inositol-1,4,5-triphosphate receptor (IP3R) antagonist, Ca2+ chelators or protein kinase C (PKC) inhibitors also mainly blocked CRH-induced depression of NMDA currents, suggesting involvement of PLC/IP3R/Ca2+and PLC/PKC signaling pathways in CRH down-regulation of NMDA receptors. Our results suggest that CRH may exert neuromodulatory actions on hippocampus through regulating NMDA receptor function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH, A 41-AMINO-ACID POLYPEPTIDE, functions as the primary regulator of the neuroendocrine responses to stress (1, 2). Apart from hypothalamus, CRH and its receptors are widely distributed throughout the brain. The limbic system, in particular the hippocampus, is rich in neuronal populations that either synthesize CRH or express CRHRs (3, 4, 5, 6). During exposure to stress, CRH can be secreted directly from the nerve terminals located in the hippocampus (7). CRH also produces a number of electrophysiological effects on hippocampal neurons including depolarization of hippocampal neurons in vitro and an increase in the excitability and spontaneous discharge frequency of CA1 and CA3 pyramidal neurons (8, 9, 10). Recently, a number of studies have demonstrated CRH modulation of synaptic efficacy and the process of learning and memory in hippocampus. Application of CRH facilitates the induction and stability of long-term potentiation under defined stimulation conditions in the CA1 region of the hippocampus (11, 12, 13). Injection of CRH into the dorsal hippocampus shortly before the training enhanced context- and tone-dependent fear conditioning in mice (14). Several studies have implicated, in hippocampus, CRH as a neuroprotective factor against glutamate-induced and other oxidative neuronal cell death (15, 16, 17).

Receptors for the amino acid L-glutamate contribute to excitatory synaptic transmission at sites throughout the brain. Presynaptic release of glutamate activates various glutamate receptors that can be broadly divided into ionotropic and metabotropic receptors (18). The N-methyl-D-aspartate (NMDA) receptor is a subclass of ionotropic glutamate receptors that plays a pivotal role in a variety of physiological processes in the central nervous system, including long-term potentiation, with involvement in learning and memory (19, 20). Overactivation or defective regulation of NMDA receptors (NMDARs) is, however, implicated in diverse brain disorders, such as stroke, Alzheimer’s disease, and acute lateral sclerosis (21, 22, 23). NMDARs are heteromeric channels containing two NR1 subunits combined with additional NR2 and NR3 subunits. The remarkable properties of NMDARs are high Ca2+ permeability when the ion channel is open and regulation by phosphorylation (24, 25, 26). Synaptic NMDARs are localized to postsynaptic densities where they are structurally organized in a large macromolecular signaling complex comprised of scaffolding and adaptor proteins, which physically link the receptors to kinase and phosphoprotein phosphatase and downstream signaling proteins (26, 27). Various types of metabotropic receptors are expressed in close proximity to synaptic NMDARs and, in the case of the metabotropic glutamate receptors, have been shown to be physically linked with them via a complex of scaffolding and adaptor proteins (28).

Two major CRHR subtypes, termed CRHR1 and CRHR2, have been identified, which belong to the class II G protein-coupled receptor superfamily (29, 30). In situ hybridization analysis revealed that CRHR1 and -R2 mRNA are localized in the hippocampal CA1 and CA3 region (5). More recently, it has been demonstrated that CRHR1 resides in asymmetric postsynaptic densities of dendritic spines, typical sites for the location of excitatory synapses (7). Therefore, we hypothesized that CRH might act on CRHRs to modulate NMDARs in hippocampus. To test this, we have examined the direct modulation of CRH on NMDA-activated current in cultured rat hippocampal neurons by using the whole-cell patch-clamp technique. Here we showed that CRH depressed NMDAR-mediated current via CRHR1, and these effects are dependent on phospholipase C (PLC)/inositol-1,4,5-triphosphate receptor (IP3R)/Ca2+ and PLC/protein kinase C (PKC) signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
CRH, antalarmin, astressin-2B, phorbol 12-myristate 13-acetate (PMA), 2-aminoethoxydiphenyl borate (2APB), chelerythrine, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), K-D-gluconate, Li-GTP, poly-L-lysine N-methyl-D-aspartic acid (NMDA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and Mg-ATP, were all purchased from Sigma-Aldrich (St. Louis, MO). H89, Gö6976, and U73122 were purchased from Calbiochem (La Jolla, CA). Urocortin II was obtained from Bachem California Inc. (Torrance, CA). DMEM, B27, neurobasal medium, fetal bovine serum (FBS), horse serum, and trypsin were supplied by Life Technologies (Grand Island, NY).

Preparation of hippocampal neuron cultures
Primary hippocampal neurons were cultured according to the protocols from the laboratories of Nelson (31), with some modifications. All animal procedures were approved by the Institutional Animal Care and Use Committee of Second Military Medical University. Briefly, the hippocampi were dissected from neonatal (postnatal d 1) Sprague Dawley rats in ice-cold dissection solution containing sucrose/glucose/HEPES (136 mM NaCl, 5.4 mM KCl, 0.2 mM Na2HPO4, 2 mM KH2PO4, 16.7 mM glucose, 20.8 mM sucrose, 0.0012% phenol red, and 10 mM HEPES, pH 7.4), and then incubated with 0.125% trypsin at 37 C for 15 min. Single-cell suspensions were obtained by mechanical dissociation using a Pasteur pipette with a fire-narrowed tip in DMEM supplemented with 10% heat-inactivated FBS and 10% horse serum. Cells were then plated at a density of 1 x 105 cells/cm2 on poly-L-lysine-coated culture plates or glass coverslips for different experiments. Cultures were maintained in 5% CO2 at 37 C in DMEM containing 10% heat-inactivated FBS and 10% horse serum overnight. The culture media were then changed to serum-free B27/neurobasal medium. Half of the medium was replaced with fresh medium every 3 d. More than 95% of the cells obtained were neurons that were assessed by immunostaining with neuron-specific markers, microtubule-associated protein-2 (MAP2; Neomarkers, Fremont, CA) and neuron-specific enolase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Neurons were cultured 7–10 d before use in experiments.

Immunofluorescence analysis
After 8 d of culture, the cells were fixed in 4% paraformaldehyde for 1 h after washing with PBS. Fixed cells were washed with PBS and incubated with 10% BSA in PBS for 1 h. Then cells were incubated with goat anti-CRHR1 or anti-CRHR2 antibodies at a dilution of 1:500 (Santa Cruz) or mouse anti-MAP2 monoantibody at a dilution of 1:500 overnight at 4 C. The CRHR1 antibody is directed against an epitope between amino acid positions 81 and 109 of CRHR1, where no sequence homology exists to CRHR2. The antibody against CRHR2 was raised against a peptide mapping near the C terminus of CRHR2. All dilutions were made in PBS containing 1% BSA. Subsequently, the specimens were washed with PBS three times and then incubated with rhodamine-conjugated antigoat IgG (1:100) for CRHR1 and CRHR2 or fluorescein isothiocyanate-conjugated antimouse IgG (1:100) for MAP2 at 37 C for 1 h in the dark. For negative controls, the primary antibody was either substituted with a normal IgG in same dilution or preabsorbed with blocking peptide (supplied with antibodies by Santa Cruz). The peptide to antibody ratio was 5:1 (wt/wt). Results were viewed under fluorescent microscope using appropriate filters.

Electrophysiology
Whole-cell patch-clamp experiments were performed at room temperature (22–25 C). A coverslip with cultured neurons was placed in a recording chamber and constantly superfused with the extracellular solution. Whole-cell membrane currents were conventionally recorded under voltage-clamp conditions at a holding potential of –60 mV, using an Axopatch 200B amplifier, Digidata 1322A data acquisition board, and pClamp 9.2 software (Axon Instruments Inc., Foster City, CA). Patch electrodes were pulled from borosilicate glass (Sutter, Novato, CA) with a puller (PC-10, Narishige, Japan), having a resistance of about 3–5 M{Omega} when filled with the pipette solution. Fast capacitance and cell capacitance were canceled by the circuit of the amplifier as much as possible. Forty to eighty percent of the series resistance of the recording electrode was compensated. Current responses were low pass filtered at 1 kHz and sampled at 5 kHz.

The pipette solution was composed of 120 mM K-D-gluconate, 10 mM KCl, 5 mM NaCl, 11 mM EGTA, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 2 mM Mg-ATP, and 1 mM Li-GTP and titrated to pH 7.2 with KOH. The extracellular solution contained 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 10 mM HEPES buffer (titrated to pH 7.4 with NaOH), and 10 mM glucose. Antalarmin, H89, Gö6976, U73122, PMA, 2APB, and chelerythrine were first dissolved in dimethylsulfoxide and then diluted by the extracellular solution to achieve a final concentration of dimethylsulfoxide of less than 0.02%. Other drugs were freshly dissolved in the extracellular solution. The control solutions were delivered by pressure to the soma of each recorded neuron via a 100-µm-diameter tip perfusion pipette controlled by a superfusion system and computer interface (DAD-8VSP; ALA Scientific Instruments Inc., Westbury, NY). The tip of the manifold was positioned about 100 µm away from the recorded neuron. A test solution was exchanged with the control solution to record the current responses in neurons.

NMDA currents were elicited by the application of 3–300 µmol/liter NMDA plus 10 µmol/liter glycine. In all experiments, we monitored rundown and administered the drugs after the NMDA had fully stabilized and rundown had subsided. We monitored the holding current of the cell continuously; this varied by less than 30 pA, indicating that the pharmacological agents used produced little change in resting potential.

Western blotting analysis
Neurons were cultured for 8 d and treated with increasing concentrations of CRH for 3 min. Cells were then scraped off the plate in the presence of lysis buffer consisting of 60 mM Tris-HCl, 2% SDS, 10% sucrose, 2 mM phenylmethylsulfonyl fluoride (Merck, Darmstadt, Germany), 1 mM sodium orthovanadate (Sigma-Aldrich), and 10 µg/ml aprotinin (Bayer, Leverkusen, Germany). The cell lysates were quickly sonicated and centrifuged at 12,000 x g for 5 min at 4 C. The supernatant was collected, and protein concentration was assayed using a modified Bradford assay. The samples were diluted in sample buffer (250 mM Tris-HCl, pH 6.8, containing 4% SDS, 10% glycerol, 2% β-mercaptoethanol, and 0.002% bromophenol blue) and boiled for another 5 min. Aliquots of proteins were separated by SDS-PAGE (10%) and subsequently transferred to nitrocellulose membranes by electroblotting. The membrane was blocked in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 at room temperature for 2 h and then was incubated with antibody raised against phosphorylated PLC-β3 (Cell Signaling Technology, Beverly, MA) at a dilution 1:1000 overnight at 4 C. After three washes with Tris-buffered saline/Tween 20, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody (Santa Cruz) for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz). The light-emitting bands were detected with x-ray film. The resulting band intensities were quantitated using an image scanning densitometer (Furi Technology, Shanghai, China). To control sampling errors, the ratio of band intensities for phosphorylated PLC-β3 to β-actin was obtained to quantify the relative protein expression level.

RIA of cAMP
After 8 d of plating, neurons were treated with increasing concentrations of CRH for 3 min and then terminated by the addition of 0.1 ml 0.3 mol/liter HCl. Cells were frozen overnight, followed by heating of the tubes in boiling water for 5 min. The supernatants were collected by centrifuge and stored at –20 C for later assay for cAMP.

cAMP was assayed using commercially available RIA kits (Shanghai Institute of Biological Products, Shanghai, China). The sensitivity was 0.1 pmol/liter. The mean intra- and interassay coefficients of variation were 4.24 and 5.57%, respectively (manufacturer’s data).

Statistical analysis
The effect of challenging by different drugs on NMDA-induced responses was calculated by dividing the NMDA-evoked response after application of drugs by the control response to gain the magnitude of changes in the modulation of the NMDA-induced currents by the respective drugs.

The results are presented as mean ± SEM. Student’s t test, paired t test, and one-way ANOVA plus least significant difference t test were used to compare the mean values of the NMDA-induced current responses. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of CRHR1 and -R2 in cultured hippocampal neurons
As shown in Fig. 1Go, A and B, numerous cells were extensively immunolabeled either by CRHR1 or CRHR2. It was noteworthy that the somata and processes of these cells were positively labeled by either CRHR1 or CRHR2. Expression of CRHR1 and CRHR2 on neurons was further confirmed by double-immunofluorescence labeling with the antibodies against CRHR1 or -R2 and MAP2. As shown in Fig. 1Go, E and F, almost all of the cells that expressed CRHR1 and CRHR2 were MAP2 positive (yellow).


Figure 1
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  		FIG. 1. Immunofluorescence analysis of CRHRs in cultured hippocampal cells. A and B, Immunostained with antibody against CRHR1 (A) and CRHR2 (B); C and D, the same cells immunostained for MAP2, which was used to confirm the type of neurons; E and F, CRHR1 and MAP2 (E) and CRHR2 and MAP2 (F) overlay; G and H, negative controls: primary antibodies of CRHR1 were preabsorbed with blocking peptide (G) or CRHR2 (H); I and J, negative controls: primary antibody was substituted with either normal goat IgG (I) or normal mouse IgG (J). Original magnifications, x200 (A–J).

 
Suppression of NMDA-evoked currents by CRH in hippocampal neurons
When the holding potential was at –60 mV, application of various concentrations of CRH (0.1 pmol/liter to 10 nmol/liter) on neurons for 3 min did not induce a detectable current in these cells (data not shown). Application of NMDA (100 µmol/liter of NMDA plus 10 µmol/liter glycine in the absence of Mg2+) elicited currents that desensitized within about 5sec to a near steady-state value on nearly all neurons tested. Application of 0.1 nmol/liter CRH significantly depressed the NMDA-evoked currents, and an example is shown in Fig. 2AGo. The peak current was rapidly decreased to 69.6 ± 1.6% of control (n = 8; P < 0.01 vs. control). Steady-state current was also reduced by CRH to 76.1 ± 2.8% of control (n = 8; P < 0.01 vs. control). CRH did not significantly change Isteady-state/Ipeak (Iss/Ip, from 0.57 ± 0.03 to 0.62 ± 0.04). Full recovery of peak and steady-state currents could be obtained after washout with the extracellular solution (Fig. 2AGo). As shown in Fig. 2BGo, administration of increasing concentrations of CRH resulted in depression of NMDA-induced currents in a dose-dependent manner.


Figure 2
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  		FIG. 2. CRH modulates NMDA-elicited currents in hippocampal neurons. A, Upper panel, representative current traces showing that CRH (0.1 nmol/liter) reduced NMDA-elicited (100 µmol/liter) currents from a hippocampal neuron; lower panel, plot of NMDA currents showing that application of CRH for 3 min decreased peak and steady-state currents of NMDA. The NMDA currents after application of CRH were normalized to the control currents induced by NMDA (100 µmol/liter). B, Concentration-response curve of CRH-induced decrease in NMDA currents from eight hippocampal neurons. The NMDA currents affected by CRH were normalized to the control. Data are presented as mean ± SEM. C, The concentration-response relationship of peak current of NMDA in the presence or absence (control, Ctl) of CRH. All responses were normalized to the peak current of 100 µmol/liter NMDA. Each point represents the averaged response of six neurons.

 
The concentration-response relationships of NMDA currents determined in the presence and absence of 0.1 nmol/liter CRH are shown in Fig. 2CGo. CRH decreased the maximum value of the concentration response of NMDA without affecting threshold concentration. The EC50 value (47 µM) of NMDA in the presence of CRH was similar to that of NMDA alone (48 µM), indicating that CRH did not change the affinity of NMDA to its receptor.

CRHR1 but not -R2 mediates the suppressive effect of CRH on NMDA current
To determine the subtype of CRHRs that mediates the inhibitory effect of CRH on NMDA current, the selective CRHR1 antagonist antalarmin (10 nmol/liter) and the CRHR2 antagonist astressin-2B (10 nmol/liter) were used. As shown in Fig. 3Go, A–C, antalarmin completely blocked CRH-induced (0.1 nmol/liter) depression of NMDA currents, whereas astressin-2B failed to block CRH-induced suppression of NMDA currents. Application of antalarmin or astressin-2B alone had no effect on the NMDA-induced (3–300 µmol/liter) currents (data not shown).


Figure 3
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  		FIG. 3. Effect of CRHR antagonists on CRH-induced depression of NMDA currents. A, Upper panel, representative current traces from a neuron showing that CRHR1 antagonist reversed CRH-induced depression of NMDA currents; lower panel, plot of NMDA currents showing that application of CRH (0.1nmol/liter) inhibited NMDA-elicited (100 µmol/liter) currents, and coapplication of CRHR1 antagonist antalarmin (Anta) (10 nmol/liter) and CRH did not affect NMDA currents. B, Upper panel, representative current traces showing that CRHR2 antagonist failed to block inhibition of NMDA (100 µmol/liter) current by CRH; lower panel, plot of NMDA currents showing that NMDA-elicited currents was suppressed by CRH (0.1 nmol/liter), and coapplication of CRHR2 antagonist astressin-2B (Astr) (10 nmol/liter) and CRH (0.1 nmol/liter) reduced NMDA currents. C, Cumulative data showing the effects of CRHR1 and -R2 antagonists on CRH-induced inhibition of NMDA currents (n = 7). Data are presented as mean ± SEM. **, P < 0.01 vs. control. D, One example of the effect of UCN II on NMDA-elicited currents. Application of 0.1nmol/liter urocortin (Uro) II had no effect on NMDA currents. The NMDA currents affected by respective drugs were normalized to the control.

 
To further clarify whether CRHR2 is involved in the CRH-induced modulation of NMDA currents, we examined the effect of the CRHR2 exclusive agonist urocortin II on NMDA current. Application of urocortin II (1 pmol/liter to 10 nmol/liter) did not affect the NMDA-evoked currents (Fig. 3DGo).

CRH induced cAMP production and phosphorylated PLC-β3 expression
It is known that CRHRs could couple to multiple G proteins including Gs, Gi, and Gq/11 and then go on to induce changes in adenylyl cyclase (AC) activity and activation of PLC-β3. Thus, we determined the effect of CRH on cAMP production and expression of phosphorylated PLC-β3, the activated form of PLC-β3, in cultured hippocampal neurons. As shown in Fig. 4AGo, incubation of increasing concentrations of CRH with cells for 3 min resulted in increases in cAMP production in a dose-dependent manner. Expression of phosphorylated PLC-β3 was significantly increased by CRH (1 pmol/liter to 10 nmol/liter) treatment for 3 min. Maximal effect was achieved by the concentration of 0.1 nmol/liter, which caused about 3-fold increase in level of phosphorylated PLC-β3 (Fig. 4BGo).


Figure 4
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  		FIG. 4. CRH induced cAMP production and phosphorylated PLC-β3 expression. A, CRH (1 pmol/liter to 10 nmol/liter) treatment for 3 min stimulated cAMP production in dose-dependent manner. Data are presented as percentage of control ± SEM. *, P < 0.05; **, P < 0.01 vs. control. B, CRH (1 pmol/liter to 10nmol/liter) treatment for 3 min increased phosphorylated PLC-β3 expression. Representative protein bands are presented at the top of histogram. Data of phosphorylated PLC-β3 levels were normalized to the control and presented as mean ± SEM of four experiments. *, P < 0.05; **, P < 0.01 vs. control.

 
The role of AC-PKA signaling pathway in CRH-induced depression of NMDA-evoked currents
We further observed the effects of blocking of AC and PKA on the effects of CRH. Application of the PKA inhibitor H89 (10 µmol/liter) significantly reduced peak and steady-state currents of NMDA (to 80.5 ± 4.1% of control for peak current and to 84.3 ± 3.2% of control for steady-state current; n = 9; P < 0.01 vs. control). Iss/Ip was significantly increased from 0.58 ± 0.01 to 0.64 ± 0.02 (P < 0.05 vs. control). After H89 washout, NMDA responses recovered to near control values. The NMDA-evoked currents were significantly reduced by coapplication of H89 (10 µmol/liter) and CRH (0.1nmol/liter) compared with application of H89 alone (Fig. 5AGo).


Figure 5
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  		FIG. 5. Effects of PKA and AC inhibitors on NMDA currents and CRH-induced depression of NMDA currents. A, Upper panel, plot of normalized NMDA currents showing that CRH (0.1 nmol/liter) and PKA inhibitor H89 (10 µmol/liter) reduced NMDA-evoked (100 µmol/liter) currents, and coapplication of H89 and CRH further decreased NMDA currents; lower panel, cumulative data showing effects of CRH, H89, and H89 plus CRH on NMDA currents (n = 7). The NMDA currents affected by the respective drugs were normalized to the control. Data are presented as mean ± SEM. **, P < 0.01 vs. control; #, P < 0.05; ##, P < 0.01 vs. H89. B, Upper panel, plot of normalized NMDA currents showing that CRH (0.1 nmol/liter), AC inhibitor SQ22536 (10 µmol/liter), and coapplication of SQ22536 and CRH inhibited NMDA-elicited currents; lower panel, cumulative data showing effects of CRH, SQ22536, and SQ22536 plus CRH on NMDA currents (n = 7). The NMDA currents affected by the respective drugs were normalized to the control. Data are presented as mean ± SEM. **, P < 0.01 vs. control.

 
PKA is activated by cAMP, which is produced by AC. Thus, inhibitors of AC would be expected to mimic the effects of PKA inhibitors. In agreement with this idea, the AC inhibitor SQ22536 (10 µmol/liter) decreased NMDA-elicited currents (to 79.9 ± 4.7% of control for peak current and to 85.3 ± 2.1% of control for steady-state current; n = 7; P < 0.01 vs. control). The ratio Iss/Ip was significantly increased from 0.61 ± 0.02 to 0.85 ± 0.02 (P < 0.01 vs. control). Coapplication of SQ22536 and CRH did not further inhibit the NMDA currents compared with the effect induced by application of SQ22536 alone (Fig. 5BGo).

The role of PLC and its downstream signaling pathways in CRH-induced depression of NMDA-evoked currents
Because CRH induced phosphorylated PLC-β3 expression, we first tested the effect of PLC inhibitor U73122 on the effect of CRH. As shown in Fig. 6AGo, application of U73122 (1 µmol/liter) totally prevented the CRH-induced (0.1nmol/liter) decrease of peak and steady-state currents of NMDA. U73122 alone slightly increased the peak current of NMDA to 114 ± 6.7% of control and extensively increased steady-state current to 142.1 ± 7.2% of control (n = 8; P < 0.01 vs. control). Iss/Ip was significantly increased from 0.57 ± 0.03 to 0.73 ± 0.03 (n = 8; P < 0.01 vs. control).


Figure 6
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  		FIG. 6. Inhibition of PLC/IP3/Ca2+ pathways blocked CRH-induced depression of NMDA currents. A, Upper panel, plot of normalized NMDA currents showing that CRH (0.1 nmol/liter) reduced NMDA-evoked (100 µmol/liter) currents, PLC inhibitor U73122 (1 µmol/liter) enhanced NMDA currents, and coapplication of U73122 and CRH failed to inhibit NMDA currents; lower panel, cumulative data showing effects of CRH, U73122, and U73122 plus CRH on NMDA currents (n = 7). The NMDA currents affected by the respective drugs were normalized to the control. Data are presented as mean ± SEM. **, P < 0.01 vs. control; {blacktriangleup}{blacktriangleup}, P < 0.01 vs. CRH; #, P < 0.05; ##, P < 0.01 vs. U73122. B, Upper panel, plot of normalized NMDA currents showing that CRH (0.1 nmol/liter) inhibited NMDA currents, and coapplication of 2APB (1 µmol/liter) and CRH failed to reduce NMDA currents; lower panel, cumulative data showing effects of CRH, 2APB, and 2APB plus CRH on NMDA currents (n = 7). The NMDA currents affected by the respective drugs were normalized to the control. Data are presented as mean ± SEM, **, P < 0.01 vs. control. C, Effect of BAPTA on CRH-induced inhibition of NMDA currents; upper left panel, representative current traces showing effect of CRH on NMDA currents in the presence of BAPTA (20 mmol/liter) in pipette solution; lower left panel, plot of normalized peak NMDA currents showing that dialysis with high concentration of BAPTA (20 mmol/liter) in pipette solution blocked the CRH effect on NMDA currents; right panel, cumulative data from seven neurons. The NMDA currents affected by BAPTA were normalized to the control. Data are presented as mean ± SEM.

 
It is known that PLC activation catalyzes the hydrolysis of membrane phosphoinositol lipids, which leads to the release of IP3 and diacylglycerol. IP3 binding to IP3Rs can trigger the release of Ca2+ from endoplasmic reticulum. Thus, we examined the role of Ca2+ in CRH regulation of NMDA currents. As shown in Fig. 6BGo, 2AGoPB (1 µmol/liter), a membrane-permeable IP3R antagonist, substantially blocked the CRH-induced decrease of NMDA currents. Dialysis with the Ca2+ chelator BAPTA (20 mmol/liter) also mostly prevented CRH-induced depression of NMDA currents (Fig. 6CGo).

PKC is a major downstream signaling molecule that can be activated by the PLC signaling pathway. Thus, we tested whether the PKC signaling pathway is involved in CRH regulation of NMDA currents. Blocking PKC activity with the nonselective inhibitor chelerythrine (5 µmol/liter) totally blocked the CRH-induced depression of NMDA currents (Fig. 7AGo). Application of the PKC{alpha}/β inhibitor Gö6976 (0.1 µmol/liter) partly blocked the CRH-induced decrease of peak and steady-state currents of NMDA (Fig. 7BGo). In addition, application of Gö6976 alone did not change the peak current of NMDA, whereas it significantly increased the steady-state current (to 148.2 ± 1.4% of control; n = 7; P < 0.01 vs. control). Therefore, the Iss/Ip was significantly increased from 0.61 ± 0.03 to 0.75 ± 0.03 (P < 0.01 vs. control).


Figure 7
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  		FIG. 7. Inhibition of PKC signaling pathway blocked CRH-induced suppression of NMDA currents. A, Effect of PKC inhibitor chelerythrine on CRH-induced inhibition of NMDA currents; upper panel, plot of normalized NMDA currents showing that coapplication of chelerythrine (5 µmol/liter) and CRH (0.1 nmol/liter) failed to cause a decrease in NMDA currents; lower panel, cumulative data from seven neurons. **, P < 0.01 vs. control. B, Effect of PKC{alpha}/β inhibitor Gö6976 on CRH-induced suppression of NMDA currents; upper panel, plot of normalized NMDA currents showing that Gö6976 (0.1 µmol/liter) enhanced NMDA currents, and coapplication of Gö6976 and CRH (0.1 nmol/liter) slightly inhibited NMDA currents; lower panel, cumulative data from seven neurons. The NMDA currents affected by the respective drugs were normalized to the control. Data are presented as mean ± SEM. **, P < 0.01 vs. control; {blacktriangleup}, P < 0.05; {blacktriangleup}{blacktriangleup}, P < 0.01 vs. CRH; #, P < 0.05; ##, P < 0.01 vs. Gö6976. C, Effect of PMA on NMDA currents; upper panel, representative current traces showing that PMA inhibited NMDA currents; lower left panel, plot of normalized NMDA showing that application of PMA (1 µmol/liter) reduced NMDA currents; lower right panel, cumulative data from seven neurons. The NMDA currents affected by the respective drugs were normalized to the control. Data are presented as mean ± SEM. **, P < 0.01 vs. control.

 
To further confirm the role of PKC in the modulation of NMDAR function, we observed the effect of the PKC activator PMA on NMDA-evoked current. The results showed that PMA (0.01–1 µmol/liter) significantly decreased both peak and steady-state currents of NMDA in a dose-dependent manner (Fig. 7CGo).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that CRH, in low physiological concentrations, depressed the NMDA-elicited currents in hippocampal neurons, which was independent of the NMDA concentration. These results support the hypothesis that activation of CRHRs is involved in the modulation of NMDAR functions in hippocampus. The activity of NMDARs in hippocampus is regulated by cell signals converging from a variety of G protein-coupled receptors. For instance, activation of serotonergic receptors, metabotropic glutamate receptors, or pituitary adenylate cyclase-activating peptide receptors up-regulate NMDA responses, whereas stimulation of muscarinic receptor reduces NMDA responses in hippocampal pyramidal cells (28, 32, 33, 34, 35).

Previous studies demonstrated that both CRHR1 and -R2 are distributed in hippocampus, but CRHR1 is the predominant subtype of CRHR. However, theses observations were based on the mRNA level of CRHRs (5). Less is currently known about protein expression of CRHR1 and -R2 in hippocampus. In the present study, using the technique of immunofluorescence analysis, we found that the majority of cultured hippocampal neurons were CRHR1 and -R2 positive. To elucidate the subtype of CRHR responsible for the suppressive effect of CRH on NMDARs, we examined actions of the specific antagonists of CRHR1 and -R2 and found that CRHR1 but not -R2 mediated the inhibitory effect of CRH on NMDA-elicited currents. Recently, Ungless et al. (36) found that in the ventral tegmental area, CRH acted on CRHR2 to potentiate NMDARs in dopamine neuron. Thus, it is suggested that in different brain areas, CRHR1 and -R2 exert distinct effect of CRH on NMDARs.

Studies in central nervous system and peripheral tissues indicated that CRH can activate multiple G proteins and lead to activation of multiple signaling pathways. In hippocampus, Blank et al. (12) found that CRHRs coupled to Gs, Gq/11, and Gi in C57BL/6N mice, whereas they coupled only to Gq/11 in BALB/c mice. The study by Elliot-Hunt and co-workers (16) showed that in rat hippocampus, CRH activated PKA and MAPK signaling pathways. In the present study, although we did not demonstrate which subtype of G proteins coupled to CRHRs, the findings that CRH simultaneously activated PKA and PLC-β3 signaling pathways indicate that CRH might couple to multiple G proteins including Gs and Gq/11 in cultured hippocampal neurons. Thereby, we examined the roles of PLC and its downstream signaling pathways as well as the AC-PKA system in CRH regulation of NMDARs. It is found that U73122, the PLC inhibitor, totally prevented CRH-induced suppression of NMDA currents. Activated PLC will catalyze the hydrolysis of phospholipids and release IP3, which mobilizes Ca2+ from intracellular stores, and diacylglycerol, which activates PKC. Our data showed that inhibition of IP3R, [Ca2+]i, or PKC abolished the CRH regulation of NMDA-evoked currents, suggesting the involvement of PLC/IP3R/Ca2+ and PLC/PKC signaling pathways in the action of CRH. We also found that U73122 itself slightly increased peak current and extensively enhanced steady-state current. Thus, we cannot completely exclude the possibility that CRH down-regulation of NMDAR activity is independent of PLC. The role of the PLC signaling pathway in the modulation of NMDARs is controversial. In a number of brain regions, PLC signaling pathways have been shown to be involved in enhancement of NMDAR function (37, 38). Up-regulation of NMDAR function by activation of PKC, a major downstream molecule of PLC, has also been demonstrated in many studies (38, 39, 40, 41). However, some studies showed that activation of PLC signaling pathway appears to down-regulate NMDARs. Gu et al. (42) found that activation of PLC/IP3/Ca2+ signaling pathways inhibit the functions of NMDAR in prefrontal cortex. The study by Grishin and co-workers (35) demonstrated that M1 acetylcholine receptor that couples to Gq protein reduces NMDA responses in CA3 hippocampal pyramidal cells through a Ca2+-dependent signaling pathway.

The PKA signaling pathway has also been shown to regulate NMDAR function in many brain areas including hippocampus (43, 44, 45, 46). In this study, we found that blocking PKA and AC leads to decreases in NMDA currents in hippocampal neurons, suggesting that the AC-PKA signaling pathway has a tonic effect on the activity of NMDARs in these cells. Moreover, we also found that CRH maintained its suppressive effect on NMDA-induced currents in the presence of PKA or AC inhibitor. CRHRs might couple to Gi protein and then go on to inhibit AC. Thus, we measured the cAMP production in these cells and found that CRH treatment increased cAMP production. Taken together, our results suggest that CRH-induced down-regulation of NMDAR is independent of the AC-PKA signaling pathway.

Our study also showed that PKA and AC inhibitors significantly increased Iss/Ip, suggesting that the AC-PKA signaling pathway is involved in the desensitization of NMDARs. These data are consistent with the study of Skeberdis et al. (46), where they showed that H89 and SQ22536 greatly reduced the desensitization of NMDARs in hippocampus. Increasing evidence implicated that PKC activation is associated with desensitization of NMDARs (47). Our findings that application of PLC inhibitor and PKC{alpha}/β inhibitor enhanced the steady state of NMDA current and led to an increase in Iss/Ip suggested that activation of PLC/PKC{alpha}/β signaling pathways increase NMDAR desensitization. Our results demonstrated that CRH decreased both peak and steady-state currents but did not affect Iss/Ip. The effect of CRH on NMDAR desensitization needs to be further investigated.

CRH is a stress-activated neuromodulator in a number of central nervous system regions, including hippocampus. Increasing evidence has implicated involvement of CRH in stress-associated alternations of hippocampal synaptic plasticity, learning, and memory as well as neuroprotection from oxidative insults. Recently, a study by Chen and co-workers (7) demonstrated that in rat hippocampal pyramidal neurons, the CRHR1 was identified at postsynaptic densities of synapse and could be activated by stress-evoked release of CRH. In the present study, it was shown that the effect of CRH on NMDA currents mediated by CRHR1 were present at low physiological concentration (1 pmol/liter), suggesting that modulation of NMDA-mediated responses by activation of CRHR1 in hippocampus would occur under either physiological or stressful conditions.

In summary, CRH acts on CRHR1 to suppress NMDAR-mediated currents in cultured hippocampal neurons, which may be dependent on PLC/IP3R/Ca2+ and PLC/PKC signaling pathways. Our results suggest that CRH may regulate the NMDAR function to exert neuromodulatory actions on hippocampus.


   Footnotes
 
First Published Online December 13, 2007

1 H.S. and Y.Z. contributed equally to this work. Back

Abbreviations: AC, Adenylyl cyclase; 2APB, 2-aminoethoxydiphenyl borate; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CRHR, CRH receptor; FBS, fetal bovine serum; IP3R, inositol-1,4,5-triphosphate receptor; Iss/Ip, Isteady-state/Ipeak; MAP2, microtubule-associated protein-2; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate.

This work was supported by Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0528).

Disclosure Statement: The authors have nothing to disclose.

Received October 9, 2007.

Accepted for publication December 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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