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* Central Institute of Mental Health Mannheim, University of Heidelberg, Mannheim, Germany;
Department of Pharmacological Sciences, University of Milan, Milan, Italy;
Institute of Pharmacology, Charité-University Medicine, Berlin, Germany; and
Max-Planck-Institute for Medical Research, Heidelberg, Germany
1Correspondence: Central Institute of Mental Health, J 5, D-68159 Mannheim, Germany. E-mail: peter.gass{at}zi-mannheim.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: learned helplessness • serotonin • hippocampus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This hypothesis predicts that major depression results from a dysregulation of neurotransmission by the monoamines serotonin, norepinephrine, and dopamine, and that pharmacological treatments act by normalizing depressed levels of these neurotransmitters (2)
. However, a substantial fraction of patients with depression do not respond adequately to pharmacological treatments aimed at normalization of the monoaminergic systems. Moreover, although common antidepressant drugs affect the monoamine metabolism within hours, the clinical effects of these compounds are usually observed only after a few weeks, indicating that other biological systems may be involved in the pathophysiology of, or at least in the recovery from, depression.
Recent evidence indicates that glutamate homeostasis and neurotransmission are also altered in major depressive disorder, but the nature of the disruption and the mechanisms by which it contributes to the syndrome are unclear (3)
. Magnetic resonance spectroscopy studies revealed increased levels of glutamate in the brain of patients with depression (4)
. Clinical observations that glutamate-reducing substances like lamotrigine and riluzole can be effective in patients with severe depression resistant to standard pharmacotherapy further support this concept (5
, 6)
. Glutamate can act via ligand-gated ion channels, such as 
-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors, or by metabotropic receptors (7)
. With respect to substances that act via ionotropic glutamate receptors, there is evidence that compounds reducing NMDA receptor activity and compounds improving AMPA receptor activity (so-called AMPAkines) have antidepressive effects (8
, 9)
.
Rodent models can provide important tools for elucidating the pathophysiology of psychiatric diseases (10)
. In the context outlined here, knockout mice with altered expression of specific glutamate receptor subtypes could be used to study the role of these receptors for emotional behaviors. The antidepressant effects of AMPAkines would suggest that mice with compromised AMPA receptor signaling show a depressive phenotype (9)
. This hypothesis was tested in the present work using a knockout mouse strain lacking a major component of the AMPA receptor complex, the AMPA receptor subunit GluR-A, which has been shown to play an important role for synaptic plasticity in the hippocampus, a limbic structure important for emotional behavior (11)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Since then, GluR-A–/– mice were backcrossed for more than 10 generations onto the C57BL/6 genetic background. The genotype was tested and confirmed in each individual mouse used in this study. Prior to all experiments, 3- to 6-mo-old male mice were single-housed for 2 wk in a reversed dark-light cycle with lights on at 6 PM. Animals were supplied with food and water ad libitum. German animal welfare authorities approved all experiments.
Behavioral experiments
Behavioral experiments were essentially carried out as described earlier (12)
. All behavioral tests were conducted during the dark cycle, i.e., in the animals’ active phase. Prior to each test, mice were acclimatized to the experimental room for at least 15 min. Control animals were always wild-type littermates of GluR-A–/– mice. In all of the experiments, the investigator was masked to the genotype of the mice during behavioral testing.
Learned helplessness
In the learned helplessness paradigm, the animals were exposed to a transparent plexiglas shock chamber (18x18x30 cm3), equipped with a stainless steel grid floor (Coulborn precision-regulated animal shocker; Coulborn Instruments, Düsseldorf, Germany), through which they received 360 foot shocks (0.150 mA) on two consecutive days, respectively. The foot shocks applied were unpredictable with varying shock (1–3 s) and interval episodes (1–15 s), amounting to a total session duration of 
52 min. Twenty-four hours after the second shock procedure, learned helplessness was assessed by testing shuttle box performance (Graphic State Notation; Coulborn Instruments). The shuttle box consisted of two equal-sized compartments (18x18x30 cm3) that were separated by a small gate (6 cm wide and 7 cm high). The shuttle box also contained a grid floor, through which current could be applied, and a signaling light at the top of both compartments. Spontaneous initial shuttles from one compartment to the other were counted during the first 2 min by red light beams at the bottom of each of the two divisions. Performance was analyzed according to the behavior during 30 shuttle escape trials. Each trial started with a light stimulus of 5 s, announcing a subsequent foot shock of maximum 10 s duration. The intertrial interval was 30 s. The following behavioral reactions were defined: escape latency, time needed for shuttling to the other section in reaction to the electric shock; and failure, when no attempt to escape was made. Total time of testing for helplessness was 20 min, the exact time period depending on the animal’s ability to learn the paradigm.
Open-field test
Activity monitoring was conducted in a square white open field, measuring 50 x 50 cm2 and illuminated from above by 25 lux. Mice were placed individually into the arena and monitored for 10 min by a video camera (Sony CCD IRIS; Sony, Tokyo, Japan). The resulting data were analyzed using the image processing system EthoVision 2.3 (Noldus Information Technology, Wageningen, The Netherlands). For each sample, the system recorded position, object area, and status of defined events. Parameters assessed for the present study were total distance moved, velocity, and time in the center, which was defined as the area 10 cm distant from the walls.
Novel cage test
The number of rearings in a novel home cage was analyzed for 5 min as an indicator for exploration
Hot plate test
To exclude altered pain sensitivity as a confounding factor for learned helplessness, the mice were tested on the hot plate test (ATLab, Vendargues, France). Temperature was set at 53 ± 0.3°C, and a 45-s cutoff was introduced to prevent injury to mice. Latency to first reaction, i.e., licking hind paws or jumping, was assessed.
Statistical analysis was performed in the XLStat 7.5 statistics program (Addinsoft, Andernach, Germany). Intergroup comparisons were calculated by 1-way ANOVAs. For analysis of the learned helplessness parameters, a Mann-Whitney U test was performed because samples are not normally distributed.
Neurochemical experiments
Preparation of protein extracts
Hippocampus was dissected from the whole brain, immediately frozen on dry ice, and stored at –80°C. Different subcellular fractions were prepared as described previously, with some modifications (13)
. Tissues were homogenized in a Teflon-glass potter in ice-cold 0.32 M sucrose containing 1 mM HEPES, 1 mM MgCl2, 1 mM EDTA, 1 mM NaHCO3, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), at pH 7.4, in the presence of a complete set of protease and phosphatase inhibitors. The homogenized tissue was centrifuged at 1000 g for 10 min, in order to separate a pellet (P1) enriched in nuclear components from the supernatant (S1). The resulting supernatant S1 was centrifuged at 13,000 g for 15 min to obtain a clarified fraction of cytosolic proteins (S2). The pellet (P2), corresponding to a crude membrane fraction, was resuspended in buffer containing 150 mM KCl and 1% Triton X-100 in a glass-glass potter and centrifuged at 100,000 g for 1 h. The resulting supernatant (S3), referred to as Triton X-100 soluble fraction (TSF), was stored at –20°C; the pellet (P3), referred to as Triton X-100 insoluble fraction (TIF), was homogenized in a glass–glass potter in 20 mM HEPES, protease and phosphatase inhibitors and stored at –20°C in the presence of glycerol 30%. Total protein content was measured in the subcellular fractions by the Bio-Rad Protein Assay (Bio-Rad, Milan, Italy).
Western blot analysis
Western blot analyses were performed in the TIF fraction. Equal amounts of proteins (5 µg) were electrophoretically run on a sodium dodecyl sulfate (SDS)/8% polyacrylamide gel under reducing conditions. Nitrocellulose membranes (Bio-Rad) were blocked with 10% nonfat dry milk in TBS/0.1% Tween-20 buffer and then incubated with primary antibody. The conditions of the primary antibodies were the following: NR1 (Zymed; 1:1000 in 3% nonfat dry milk), β-actin (Sigma-Aldrich; 1:10,000 in 3% nonfat dry milk). After 3 washes of 10 min in TBS/Tween-20, the blots were incubated 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody, and immunocomplexes were visualized by chemiluminescence using the ECL Western blot analysis kit (Amersham Life Science, Milan, Italy) according to the manufacturer’s instructions. Results were standardized to a β-actin control protein detected by evaluating the band density at 43 kDa.
Determination of monoamines and glutamate
Serotonin (5-HT) tissue levels were analyzed as described previously, using high-performance liquid chromatography (HPLC) with electrochemical detection (14)
. Noradrenaline (NA) was measured by HPLC with electrochemical detection after extraction to alumina, according to Felice et al. (15)
with minor modifications (16)
. For determination of glutamate, amino acids were precolumn derivatized with o-phthalaldehyde/2-mercaptoethanol using a refrigerated autoinjector and then separated on an HPLC column (ProntoSil C18 ace-EPS, 50 mm x 3 mm i.d.; VDS Optilab, Berlin, Germany) at a flow rate of 0.6 ml/min and a column temperature of 40°C. The mobile phase was 50 mM sodium acetate, pH 5.7, in a linear gradient from 5 to 21% acetonitrile in 20 min. Derivatized amino acids were detected by their fluorescence at 450 nm after excitation at 330 nm (17)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 18)
. Thus, these animals were well suited to study the role of GluR-A-containing AMPA receptors in behavior during adulthood. The learned helplessness paradigm evaluates the coping capabilities of mice in an aversive test situation after 2 days of intense stress evoked by exposure to a series of unpredictable and uncontrollable foot shocks. When tested for helpless behavior, GluR-A–/– mice made significantly more escape failures (20.89±3.06 vs. 1.33±0.37; P<0.001; Fig. 1
A) and had significantly increased escape latencies (7.81±3.0.85 vs. 2.23±0.24; P<0.001; Fig. 1B
) compared to their wild-type littermates. When mice were scored individually, the most helpless individuals were exclusively GluR-A–/– (Fig. 1C
), indicating that the deficiency in AMPA signaling results in increased depression-like behavior.
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These results reflect true coping deficits, as they were not caused by an altered pain sensitivity, as shown in a hot plate test (Fig. 2
A), nor by hypolocomotion, which is a confounding factor in the learned helplessness paradigm, potentially causing false-positive results (i.e., increased learned helplessness). Indeed, in accordance with earlier experiments, GluR-A–/– revealed a hyperlocomotoric phenotype in the novel cage test (Fig. 2B
) and the open-field test (Fig. 2C, D
). Furthermore, when only tested in the 2-way avoidance task of the learned helplessness paradigm, i.e., without preceding foot-shock exposure, GluR-A–/– mice are indistinguishable from controls with respect to both failures (0 vs. 0, i.e., both genotypes do not make failures without stress exposure; Fig. 1D
), as well as escape latencies (1.22±0.14 vs. 1.49±0.14; Fig. 1E
). This demonstrates that GluR-A–/– mice are principally capable of performing the avoidance task as well as wild-type animals.
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GluR-A–/– mice show alterations in glutamatergic and monoaminergic signaling
On the neurochemical level, GluR-A–/– mice showed a significant increase of total glutamate levels in the hippocampus (12.11±0.17 vs. 11.35±0.29 pmol/mg tissue; P<0.01; Fig. 3
A). To address the question of whether GluR-A–/– mice may also have changes in NMDA receptor-mediated signaling, we investigated the expression of the main subunit of NMDA receptors, i.e., NR-1, which is present in all NMDA receptors. Indeed, compared to wild-type animals, GluR-A–/– mice exhibited a significant 30% up-regulation of hippocampal NR-1 protein levels in a subcellular fraction with enriched postsynaptic compartment (Fig. 3B
).
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According to the monoamine deficiency hypothesis of depression, we also investigated the tissue levels of serotonin and norepinephrine in the hippocampus of GluR-A deficient animals. GluR-A–/– mice demonstrate a strong and significant reduction of both serotonin (692.13±13.39 vs. 847.53±40.86 pg/mg tissue; P<0.05; Fig. 4
A) and norepinephrine (413.76±12.94 vs. 498.58±25.58 pg/mg tissue; P<0.05; Fig. 4B
) in the hippocampus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 19
, 20)
. This model has already been successfully used to demonstrate the role of other genes in the pathogenesis of depression, such as the glucocorticoid receptors BDNF and CREB (12
, 21
, 22)
. In the present study, GluR-A–/– mice revealed significantly increased learned helplessness when compared to their wild-type littermates (Fig. 1)
. This most likely reflects the depression-like coping deficit attributed to this model, because alterations in pain sensitivity, hypolocomotion, or learning deficits in the avoidance task could be excluded as confounding factors (Fig. 2)
. Furthermore, the reduction of serotonin and norepinephrine levels observed in the hippocampus of GluR-A–/– mice is in strong agreement with the monoamine deficiency hypothesis of depression. Indeed, almost all current drugs used to treat depression in humans act by altering serotonergic and noradrenergic neurotransmission (Fig. 4)
.
Depression is often concurrent with anxiety, and there is also evidence that glutamate is dysregulated in the central nervous system of patients with anxiety disorders (23
24
25)
. In the present study, GluR-A–/– mice spent less time in the anxiety-related compartment of the open field; however, this difference was not statistically significant (P>0.1). This finding is in line with earlier experiments showing only a weak increase in anxiety-related behaviors in male GluR-A–/– mice (18)
.
The depression-like phenotype of animals with compromised AMPA signaling is in good agreement with deficiencies in AMPA receptor-related transduction pathways reported in patients with depression (9)
. The results of our hypothesis-directed approach also correspond well with current concepts regarding the role of AMPA and NMDA receptors in antidepressive therapy, postulating that compounds that augment AMPA receptor signaling or that decrease NMDA receptor functions have antidepressant effects (8
, 9
, 24
, 26)
. The disturbed glutamate homeostasis with increased glutamate levels displayed by GluR-A–/– mice (Fig. 3)
is in good agreement with these concepts, and it is in line with the observation that cortical glutamate levels may be elevated in medication-free subjects with depression (4)
. The up-regulation of glutamate levels in GluR-A–/– mice may be biologically interpreted as a compensatory mechanism in response to the lack of GluR-A-containing AMPA receptors. However, increased glutamate levels could also further explain the depressive phenotype of GluR-A–/– mice, because antidepressants have been shown to reduce extracellular glutamate release (27)
.
We also found that enhanced glutamate levels are associated with increased synaptic expression of the essential NR-1 subunit of the NMDA receptor. Although the precise relevance of this effect in GluR-A–/– mice remains to be established, these changes may also contribute to the "depressive" phenotype by amplifying the toxic effects set in motion under stressful adverse conditions (28
, 29)
. The increased expression of the NR-1 subunit may also provide support to the notion that NMDA receptor antagonists have antidepressant effects in animal models (30
, 31)
as well as in humans (26)
. However, it must be mentioned that NR-1 up-regulation has not been reported in array studies from postmortem tissue (32)
, and one postmortem study reported a trend toward reduced NR-1 protein (33)
. Likewise, NR-1 up-regulation has so far not been seen after induction of depression-like behavioral states in experimental animals, although the effect of these experimental conditions on synaptic NMDA receptor expression remains to be established. Moreover, we cannot exclude that NR-1 up-regulation in GluR-A–/– mice represents a developmental compensation process during ontogenesis in mice with a classical, i.e., lifelong, knockout of the GluR-A subunit of AMPA receptors.
Current concepts also have suggested that depression is related to a relative imbalance between the synaptic and extrasynaptic glutamatergic tone via NMDA receptors (for a review, see refs. 8
, 34
, 35
). Extrasynaptic NMDA receptors are thought to decrease BDNF, reduce "cell health" and perhaps impair neurogenesis, and functionally reduce activation of synaptic NMDA receptors (36)
. The latter are positively coupled to cell survival and BDNF production. The trophic effects of synaptic NMDA activation would thus be reduced at the same time as the toxic effects of extrasynaptic NMDA activation are increased. However, so far most of the experiments underlying these concepts have been done in neuronal cell cultures. In the present study, only a subcellular fraction enriched of the intrasynaptic compartment was investigated. Thus, it is not clear whether there is also an increase of NR-1 in extrasynaptic compartments of GluR-A–/– mice. A potential dysbalance in favor of the extrasynaptic fraction would additionally explain the depressive phenotype of the mice.
In summary, GluR-A–/– mice seem to represent a good model to further investigate the pathophysiology underlying the depressive phenotype and to identify changes in neural plasticity and resilience evoked by the genetic alterations in glutamatergic function. In addition, GluR-A–/– mice may be a valuable tool to study biological mechanisms of AMPA receptor modulators as well as the efficacy of NMDA antagonists in reducing behavioral or biochemical changes that correlate with increased helplessness. Future experiments with regional-specific or inducible/conditional GluR-A-deficient mice will help to identify those brain areas in which AMPA receptors are crucial, as well as sensitive periods during development or in adulthood for the depression-like alterations to occur. In conclusion, this animal model will be useful in the future to study the role of glutamatergic signaling in the pathophysiology and therapy of depression.
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ACKNOWLEDGMENTS |
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Received for publication January 30, 2008. Accepted for publication April 24, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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