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Human Molecular Genetics, 2003, Vol. 12, No. 10 1079-1086
DOI: 10.1093/hmg/ddg128
© 2003 Oxford University Press

Genetic dissection of anxiety in autoimmune disease

Kazuhiro Nakamura1, Yan Xiu1, Mareki Ohtsuji1, Gen Sugita1,2, Masaaki Abe1, Naomi Ohtsuji1, Yoshitomo Hamano1, Yi Jiang1,3, Noriko Takahashi4, Toshikazu Shirai1,{dagger}, Hiroyuki Nishimura4 and Sachiko Hirose1,*

1Second Department of Pathology and 2Department of Otorhinolaryngology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan, 3Central Laboratory of First Clinical College, China Medical University, Shenyang, People's Republic of China and 4Department of Biomedical Engineering, Toin Human Science and Technology Center, Toin University of Yokohama, Yokohama 225-8502, Japan

Received November 19, 2002; Revised January 22, 2003; Accepted March 14, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Systemic lupus erythematosus (SLE), a complex multigenic disease, is characterized by hypergammaglobulinemia, autoantibody production and immune complex-type lupus nephritis. In addition to these signs and symptoms in SLE, there can be symptoms of neurological disorders, including anxiety. To clarify mechanisms governing the anxiety seen in lupus, we carried out genome-wide scans, and found that the region including interferon-{alpha} (IFN-{alpha}) on NZB chromosome 4 is significantly linked to the anxiety-like behavior seen in SLE-prone New Zealand Black (NZB)xNew Zealand White (NZW) F1 (B/W F1) mice. This finding was confirmed by anxiety-like performances of mice with heterozygous NZB/NZW alleles in the susceptibility region onto the NZW background. In B/W F1 mice, neuronal IFN-{alpha} levels were elevated, and blockade of the µ1 opioid receptor or corticotropin-releasing hormone receptor 1, possible downstream effectors for IFN-{alpha} in the brain partially overcame the anxiety-like behavior seen in the B/W F1 mice. Consistently, neuronal corticotropin-releasing hormone levels were higher in B/W F1 than NZW mice. Furthermore, pretreatment of µ1 opioid receptor antagonist abolished anxiety-like behaviour seen in IFN-{alpha}-treated NZW mice. Anxiety is shown to be mediated by multiple mediators. Our data suggest that a genetically determined endogenous excess amount of IFN-{alpha} in the brain may form one aspect of anxiety-like behavior seen in SLE-prone mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Anxiety, a complex and higher brain function, is defined as unwarranted or inappropriate fear (1). Different components of anxiety are mediated in multiple brain regions by several neurotransmitters, including glutamate, GABA and monoaminergic neurotransmitters (2). The involvement of multiple molecules/genes in anxiety was noted using pharmacological and gene manipulating approaches (3).

In addition to primary psychiatric disorders with accompanying anxiety-like symptoms, an increasing body of evidence revealed that systemic lupus erythematosus (SLE) also can include symptoms of neurological disorders such as anxiety (4). Since, SLE is a complex multigenic disease (5), the genetic complexity of the pathogenesis of anxiety in lupus has to be considered. Like human SLE, spontaneous SLE-prone MRL/lpr, BXSB, New Zealand Black (NZB) and NZBxNew Zealand White (NZW) F1 (B/W F1) strains of mice show alterations in behavior (6). In particular, B/W F1 mice show increased anxiety as well as learning disturbances, impaired motor coordination, altered sensitivity to pain stimuli and reduced exploration (6–8). In contrast to MRL/lpr, BXSB and NZB mice with anatomical abnormalities in the brain, organization of the central nervous system (CNS) is essentially normal in B/W F1 mice (9,10). The B/W F1 strain is generally considered to be the best model available for human SLE, because of the high occurrence in females and the severe fatal lupus nephritis. Therefore, this strain may be a suitable model to determine functional mechanisms involved in genetically determined anxiety in lupus.

Neurons express an unusual number of molecules that were originally thought to be specific for immune functions (11). One such molecule is interferon (IFN)-{alpha}, the type I IFN family (12,13), and there are at least 18 IFN-{alpha} genes in humans (14). In the periphery, IFN-{alpha} produced mainly in activated leukocytes exerts diverse effects such as antiviral properties, antitumor action, inhibition of cell growth and modulatory effects on immune responses (15). In addition, IFN-{alpha} was suggested to act on the CNS both in vitro and in vivo in case of exogenous administration. In humans, immunotherapy with IFN-{alpha} often induces anxiety as well as irritability, sadness, loss of interest, depression, anorexia and fever (16–18). Since electrophysiological experiments revealed its function as a neuronal modulator (17,19–21), the basis for these behavioral changes can probably be ascribed to alterations in neuronal activities. The neuromodulatory actions of IFN-{alpha} were suggested to be mediated through opioid and corticotropin-releasing hormone (CRH) systems (21).

To determine what molecules mediate the anxiety-like behavior seen in lupus, we did genome-wide screening in B/W F1xNZW back-cross mice, and mapped one major susceptibility allele for the anxiety-like behavior seen in SLE-prone B/W F1 mice to the close vicinity of the IFN-{alpha} gene. The possible involvement of the IFN-{alpha} gene and its downstream effector molecules in the anxiety-like behaviour seen in B/W F1 mice was examined using flow cytometry (FCM) analysis and pharmacological approaches.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple genes are involved in the increased anxiety seenin B/W F1 mice
Previous studies showed the anxiety-like behavior of SLE-prone B/W F1 mice, compared with NZW mice, determined using a reliable measure for anxiety (22), the elevated plus maze test (7,8). We first examined the performance of NZB as well as B/W F1 and NZW mice in the elevated plus maze test to determine if only NZB-derived genes are sufficient or whether NZW-derived genes also contribute to the anxiety-like behaviour seen in B/W F1 mice. Three-month-old B/W F1 mice showed evidence of increased anxiety based on significantly fewer entries in open arms than did the age-matched NZW mice (Fig. 1A), as reported (7). NZB mice also showed fewer entries, as compared with NZW mice. When observation of B/W F1 mice was compared with that of NZB mice, the number of entries was significantly fewer. Thus, multiple genes derived from both NZB and NZW strains of mice seem to contribute to the anxiety-like behavior seen in B/W F1 mice.



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  		Figure 1. The susceptibility region for anxiety-like behavior on chromosome 4. (A) Numbers of entries in open arms of NZB (4.3±1.3, mean±SE, n=8), NZW (8.0±1.2, n=13), and B/W F1 (1.1±0.3, n=11) mice in the elevated plus maze test. (B) MAPMANAGER/QTL scans on chromosome 4 for the elevated plus maze test in B/W F1xNZW backcross mice. (C) Relation of genotypes for particular loci to the performance in the elevated plus maze test. The backcross mice were separated into four groups according to combination of genotypes for D4MIT119 and D2MIT254 loci. B/W and W/W indicate NZB/NZW heterozygotes and NZW/NZW homozygotes respectively. *P<0.05, **P<0.01.

 
The major susceptibility allele for anxiety in B/W F1 miceis located on chromosome 4
To map susceptibility alleles for the anxiety seen in B/W F1 mice, we assessed the performance of the 215 B/W F1xNZW back-cross progeny at 2 months, using the elevated plus maze test. Genome-wide scans for quantitative trait loci (QTL) revealed that the decreased number of entries in the open arms was significantly linked to the D4MIT119 locus on NZB chromosome 4 (provisionally designated Anx, LOD 3.2, significant by permutation test; Fig. 1B). Indeed, the back-cross progeny with the heterozygous NZB/NZW (B/W) type for the D4MIT119 locus showed significantly fewer entries in open arms (2.3±0.3) than noted in the homozygous NZW/NZW (W/W) type (4.0±0.3; P=0.0001).

QTL scans also showed that the D2MIT254 locus on NZW chromosome 2 was potentially linked to decreased entries into open arms (P=0.016 by {chi}2 test, LOD 1.3). The back-cross progeny with the W/W type for the D2MIT254 locus showed significantly fewer entries (2.6±0.3) than seen with the B/W type (3.7±0.3) (P=0.013).

We then separated the backcross progeny into four groups according to genotypes of D4MIT119 and D2MIT254 loci. As shown in Figure 1C, progeny with the B/W type D4MIT119 and the W/W type D2MIT254 loci had the least number of entries. The entry numbers increased over that seen with the B/W type both loci, that with the W/W type both loci, and that with the W/W type D4MIT119 and B/W type D2MIT254 loci, respectively, in that order. This finding indicates that a NZB-derived gene linked to D4MIT119 and a NZW-derived gene linked to D2MIT254 additively contribute to the increased anxiety in B/W F1 mice.

QTL for the number of entries in closed arms, an indicator of locomotor activity (23), showed suggestive linkage to the centromeric region on chromosome 19 (LOD 2.0), but no linkage to the D4MIT119 locus. The number of entries in closed arms of the backcross progeny with the B/W type for the D4MIT119 locus (12.2±0.6) was comparable to that of W/W type (13.2±0.5; P=0.22).

To confirm the effect of Anx on chromosome 4, we generated mice with the heterozygous B/W-type Anx allele onto NZW background (NZW.Anx-B/W) by selective backcrossing. Detailed genotyping with microsatellite markers on chromosome 4 revealed that the region ~20 cM in length, including Anx, was replaced by the heterozygous B/W type allele (Fig. 2A). As shown in Figure 2B, NZW.Anx-B/W mice showed significantly fewer entries in the open arms of the elevated plus maze test than did the control NZW mice. Similarly, when these mice were subjected to the light–dark compartment test (24), another behavioral test to evaluate anxiety, NZW.Anx-B/W mice spent longer in the dark compartment than NZW mice (Fig. 2C). However, the ambulation count in the open field was not different between NZW and NZW.Anx-B/W mice (Fig. 2D). Thus, Anx proved to be closely related to anxiety, but not to general motor activity.



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  		Figure 2. Anxiety-like behavior in mice with the heterozygous NZB/NZW-Anx allele onto NZW background (NZW.Anx-B/W). (A) Genotypes for loci on chromosome 4 in NZW.Anx-B/W mice. Loci with NZB/NZW heterozygous genotype (B/W) and NZW/NZW homozygous genotype (W/W) are shown as solid and open boxes respectively. Marker map locations are expressed in cM according to MGI. (B) Numbers of entries in open arms of NZW (6.8±1.8, n=8) and the NZW.Anx-B/W mice (3.0±1.4, n=5) in the elevated plus maze test. (C) Time in the dark compartment of NZW (223.6±14.4, n=5) and the NZW.Anx-B/W mice (275.8±6.2, n=8) in the light–dark compartment test. (D) Ambulation count of NZW (91.0±14.2, n=7) and the NZW.Anx-B/W mice (91.9±12.3, n=7) in the open field.

 
Higher neuronal IFN-{alpha} levels in B/W F1 mice
Among several known genes mapped near Anx on chromosome 4, we focused on IFN-{alpha} because both humans and mice administered with IFN-{alpha} show an increase in anxiety (4,6,8,16). To assess IFN-{alpha} expression in the brain, we carried out FCM analysis using the entire brain of NZB, NZW and B/W F1 mice, and evaluated quantitatively the expression level of IFN-{alpha} protein in the neurons. As markers for neurons, we used an anti-Thy-1 mAb (25,26). We also used a T cell marker, CD3, to eliminate any contaminating Thy-1 positive T lymphocytes. IFN-{alpha} protein was below levels of detection in neurons from NZW mice. However, a slight but significant expression was recognized in neurons from NZB and B/W F1 mice (Fig. 3A).



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  		Figure 3. The µ1 opioid receptor mediates anxiety-like behavior induced by IFN-{alpha}. (A) Comparison of IFN-{alpha} expression among NZB (green line), NZW (blue line), and B/W F1 (red line) mice. Entire brain cells were stained with antibodies to IFN-{alpha}, Thy-1.2 and CD3. Thy-1+, CD3- neurons were gated as depicted in box in left panel, and the intensities of IFN-{alpha} expression were examined, using flow cytometry analysis. The solid histogram indicates the control fluorescence. (B and C) Numbers of entries in open arms of vehicle (8.1±1.6, n=10), naloxone (6.7±1.8, n=7) (B), or naloxonazine (10.7±1.4, n=7) (C) treated NZW, and those of B/W F1 (0.9±0.3, n=9, 2.7±1.0, n=9, and 4.0±1.3, n=8, respectively) mice in the elevated plus maze test. (D) Effect of naloxonazine on the elevated plus maze performance of IFN-{alpha}-treated NZW mice. Vehicle or naloxonazine was administered 24 h before measurement of the elevated plus maze test. Then, vehicle or IFN-{alpha} was administered 2 h before measurement. Numbers of entries in open arms were measured in vehicle-treated (8.3±1.6, n=6), IFN-{alpha}-treated (4.2±1.3, n=6), or IFN-{alpha} and naloxonazine-treated (9.1±1.4, n=8) NZW mice.

 
Involvement of the µ1 opioid receptor in IFN-{alpha}-induced anxiety
To determine if the increased expression of IFN-{alpha} contributes to the anxiety-like behavior, and to search for possible effector molecules, we screened effects of receptor systems suggested to be linked to the function of IFN-{alpha} in the brain and to be related to anxiety. We first examined the effect of an opioid receptor system, which was shown to be a possible receptor for IFN-{alpha}-elicited responses (27). When treated with naloxone, an opioid receptor antagonist, B/W F1 mice easily entered into the open arms in the elevated plus maze test compared with findings using vehicle-treated B/W F1 mice. However, the same dose of naloxone could not alter the behavior of the NZW mice (Fig. 3B). Treatment with naloxonazine, a µ1-specific opioid receptor antagonist also led to the significant increase in the number of entries by B/W F1 mice, while the effect on NZW mice was not significant (Fig. 3C). Difference between the two strains was diminished but remained significant after administration of both these antagonists, hence recovery from increased anxiety was partial.

To demonstrate further the direct functional link between IFN-{alpha} and µ1 opioid receptor in anxiety-like behavior, we evaluated the effect of naloxonazine on IFN-{alpha}-treated NZW mice. IFN-{alpha}-treated NZW mice exhibited reduced entries in open arms, and pretreatment of naloxonazine abolished the effect of IFN-{alpha} (Fig. 3D). Thus, µ1 opioid receptor is likely to be a mediator for IFN-{alpha}-elicited anxiety.

CRH-CRHR1 system is functionally related to anxiety-likebehavior in B/W F1 mice
Electrophysiological data suggested that stimulation of central opioid receptors by IFN-{alpha} led to activation of CRH-producing neurons in vitro (17,19–21). Thus, we next examined CRH protein levels using FCM. As shown in Figure 4A, the CRH level in neurons from the entire brain was higher in B/W F1 than that in NZW mice. In addition, administration of human recombinant IFN-{alpha} to NZW mice led to a subtle increase in neuronal CRH levels compared with findings in vehicle-treated NZW mice (Fig. 4B). This indicates the definitive link between IFN-{alpha} and the CRH system in vivo.



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  		Figure 4. Involvement of activated CRH-CRHR1 system in anxiety-like behavior of B/W F1 mice. (A–C) Neuronal CRH (A and B) or phospho-CREB (C) protein levels were compared between naive (A) or elevated plus maze experienced (C) NZW (blue line) and B/W F1 (red line) mice, or between naive (blue line) and IFN-{alpha}-administered (red line) NZW mice (B). Entire brain cells were stained with antibodies to CRH, Thy-1.2 and CD3 (A and B) or phospho-CREB, Thy-1.2 and CD3 (C). Then, Thy-1+, CD3- neurons were gated, and intensities of CRH (A and B) or phospho-CREB (C) expression were examined using flow cytometry analysis. Solid histograms indicate the control fluorescence. (D) Numbers of entries in open arms of vehicle (6.8±1.8, n=10) or CP-154,526 (7.5±1.0, n=6) treated NZW, and those of B/W F1 (1.0±0.3, n=8 and 4.2±1.3, n=9, respectively) mice in the elevated plus maze test.

 
To determine if higher CRH levels would lead to an increased neuronal activity via CRH receptor (CRHR) system, we examined CREB phosphorylation, since this phosphorylation is induced after the activation of CRHR1 (28) and CRHR2 (29). Two hours after placement of the mouse on an open arm in the elevated plus maze, the entire brain was removed, and the level of phosphorylated CREB (pCREB) was assessed, using FCM with phospho CREB antibody. The amount of pCREB was below levels of detection in neurons from NZW mice, but was detectable in neurons from B/W F1 mice (Fig. 4C).

To elucidate the functional significance of the activated neuronal activity induced by the CRH–CRHR system, we determined if blockade of the CRHR would alter the anxiety-like behavior in B/W F1 mice. Treatment with CP-154,526, a specific CRHR1 antagonist, significantly raised the number of entries in open arms in B/W F1, but not in NZW mice (Fig. 4D). The anxiety-like behavior was partially but significantly relieved with this treatment, as was observed in treatments with specific antagonists for opioid receptor (Fig. 3B and C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
With the recent advance in gene manipulation technology, transgenic or gene knockout mice provide a valuable tool for understanding the neural substrates of anxiety, and several genes have been shown to be involved (3). In contrast, a QTL approach will also be useful for uncovering further genetic variants that contribute to the observed anxiety-like behavioral phenotype. In the present studies using the elevated plus maze test, we did a QTL analysis to search for susceptible alleles to the anxiety-like behavior seen in SLE-prone B/W F1 mice and found that genes derived from both NZB and NZW parents contribute to the anxiety-like behavior seen in B/W F1 mice. One NZB-derived allele, Anx, was mapped to the vicinity of the IFN-{alpha} gene on chromosome 4. The increased anxiety-like behavior in mice with the heterozygous B/W-type Anx allele onto NZW background, compared with findings in NZW mice, was confirmed using both the elevated plus maze test and light–dark compartment test, indicating that the effect of Anx is dominant. The same QTL scan showed the potential linkage with the NZW-derived allele on chromosome 2, suggesting that multiple susceptibility alleles are involved.

Turri et al. (30) mapped QTL susceptible to anxiety-like behavior, using F2 intercrosses of mouse lines originally derived from a cross between two inbred strains, BALB/c and C57BL/6, and established by replicated bidirectional selection for open-field activity. Such models by selection for extremes of animal behavior have long been used to derive strains that can be potentially useful for genetic studies for behavioral disorders. Extensive studies by the above authors proposed several susceptibility regions with significant linkage. Importantly, a region overlapping with Anx allele was included, suggesting that the same genetic pathway is possibly involved in anxiety-like behavior seen in both bidirectional selection models and SLE-prone mice. Further studies are needed to clarify the relationship of causative genes in both models.

In the present study, we focused on IFN-{alpha} gene as a possible candidate for Anx, since the administration of IFN-{alpha} can often cause anxiety-like behavior (4,6,8,16). The amount of IFN-{alpha} protein was below the level of detection in neurons from the NZW entire brain, in accordance with the previous observations that IFN-{alpha} mRNA was below the level of detection in the BALB/cByJ mouse brain, determined using northern blot hybridization (31). In contrast, a slight but significant expression was detected in neurons from NZB and B/W F1 mice; both showed increased anxiety-like behavior compared with NZW mice. These findings suggest that genetically determined up-regulated IFN-{alpha} may be involved in SLE-associated anxiety-like behavior.

Electrophysiological data suggest a link between IFN-{alpha}, µ opioid receptor and the CRH–CRHR system in vitro, in which IFN-{alpha} was found to bind opioid receptors in the mouse brain (32). In addition, a single injection of IFN-{alpha} can cause anxiety-like behavior in mice (8). However, it still remains to be determined that these systems are indeed involved in vivo in IFN-{alpha}-elicited anxiety. Using pharmacological approaches, we found that the µ1 opioid receptor is a possible downstream molecule for anxiety-like behavior seen in B/W F1 and in IFN-{alpha}-treated NZW mice. Our in vivo study also revealed a definitive link between IFN-{alpha} and CRH when recombinant human IFN-{alpha} was given to NZW mice. Involvement of the CRH–CRHR1 system in anxiety-like behavior was verified using CRH transgenic (33) and CRHR1 knockout mice (34). Our data showed that the CRH–CRHR1 system is also involved in anxiety-like behavior seen in B/W F1 mice, resulting in the phosphorylation of CREB in neurons.

Previous reports proposed an idea that neurological abnormalities in SLE-prone mice are the consequence of autoimmune inflammatory process (6,7). Our QTL analysis was done using young back-cross mice before the onset of SLE, indicating that the anxiety-like behavior observed in the present studies is not simply the result of SLE. The linkage analysis in the same back-cross mice revealed that the B/W-type D4MIT119 allele was significantly associated with high serum levels of IgM class autoantibodies (data not shown), but not with lupus nephritis accompanied by the class switch from IgM to IgG autoantibodies (35). Thus, it is likely that Anx is involved in both anxiety-like behavior and early lupus phenotype, and that additional genetic factors are needed in the florid SLE in B/W F1 mice (36).

The IFN-ß gene is also mapped close to Anx; however, it is unlikely to be a candidate gene for the anxiety seen in B/W F1 mice. CNS side effects occur less frequently in cases with IFN-ß treatment than those with IFN-{alpha} treatment (37–39). Furthermore, human IFN-{alpha} but not human IFN-ß increases immobility time in mice and rats forced to swim (40,41). Human IFN-{alpha} but not IFN-ß exerts opioid-like effects in the CNS (27).

Interestingly, an up-regulated serum IFN-{alpha} level was evident in patients with SLE (42,43). In addition, levels of IFN-{alpha} were specifically increased in cerebrospinal fluid of patients with lupus psychosis. Immunohistochemical studies at autopsy also revealed that IFN-{alpha} protein was evident in neurons and in the microglia in one such patient, but not in subjects who died of other diseases (43). Thus, it would be worth examining if the up-regulated IFN-{alpha} plays a role in anxiety via opioid receptor and/or CRH systems in CNS lupus patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
NZB, NZW and B/W F1 mice obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan) were maintained in our laboratory according to guidelines of Juntendo University. Back-cross mice were produced by crossing female B/W F1 mice with male NZW mice. Mice carrying the heterozygous NZB/NZW-Anx allele on the NZW background (NZW.Anx-B/W) were obtained by selective back-crossing for seven generations of B/W F1 mice to NZW, using marker-assisted selection.

Behavioral tests
The elevated plus maze test was done according to the method of Pellow et al. (22). The apparatus consisted of two open and two closed arms of 30x5 cm, and the 5x5 cm center platform elevated 50 cm from floor level. The closed arms had walls 15 cm high. The mouse was placed in the center facing an open arm, and then allowed to explore the maze for 10 min. The numbers of entries into open or closed arms were recorded. Entering an arm was defined as the four legs of the mouse crossing from the center.

The apparatus used in the light–dark compartment test (24) was a box consisting of dark (1 lx, 25x10x30 cm) and light (500 lx, 25x30x30 cm) compartments. A partition containing a 3x3 cm hole separated the two compartments. Mice were placed in the light compartment, and time spent in the dark compartment was measured for 5 min after first entry in the dark compartment.

Ambulation was measured by counting the number of times mice crossed from one square (10x10 cm) to another in the open field (50x50 cm), essentially as described (44).

Drugs and treatments
Recombinant human IFN-{alpha} (1x107 U i.p.; Pepro Tech EC Ltd, London, UK) was injected 5 or 2 h prior to flow cytometry or behavioral analyses, respectively. Naloxone hydrochloride (1 mg/kg s.c.; Sigma, St Louis, MO, USA) was administered 30 min prior to the behavioral test. CP-154,526 (3 mg/kg s.c.; Pfizer Inc., Groton, NY, USA) and naloxonazine (35 mg/kg s.c.; Sigma-RBI, St Louis, MO, USA) were given 45 min and 24 h prior to the test, respectively. The drugs were dissolved in 0.9% physiological saline except for CP-154,526 which was dissolved in a minimum amount of 0.1 N HCl and diluted in 0.9% physiological saline.

FCM analysis
To obtain a single cell suspension, brain cells were forced through nylon mesh 108 µm in diameter after incubation with 4 mg/ml collagenase D (Wako, Tokyo, Japan) and 360 U DNase I (Invitrogen, Carlsbad, CA, USA) for 1 h. Approximately 2x106 cells were incubated with biotinylated anti-Thy1.2 mAb (prepared from hybridoma; ATCC, Rockville, MD, USA) and phycoerythrin-conjugated anti-CD3 mAb (PharMingen, San Diego, CA, USA) followed by incubation with streptavidin-conjugated allophycocyanin to detect neurons. After three washes with PBS containing 0.2% BSA and 0.1% sodium azide, the cells were treated with PBS containing 4% paraformaldehyde, and stained with rabbit antibodies directed against mouse IFN-{alpha} (PBL Biomedical Laboratories, New Brunswick, NJ, USA), human CRH (Biogenesis, Poole, UK) or phospho CREB (Cell Signaling Technology, Beverly, MA, USA) in PBS containing 0.3% saponin and 1% BSA followed by Alexa 488-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). After washing in PBS containing 0.2% BSA and 0.1% sodium azide, cells were fixed in PBS containing 1% paraformaldehyde.

Samples were run on a FACStar flow cytometer and measured using Cell/Quest software (Becton Dickinson, San Jose, CA, USA).

Genotyping
DNA was extracted from the mouse tail. PCR reactions were done for genotyping with microsatellite markers distributed approximately every 10 cM in the entire genome, except for the sex chromosome. Microsatellite primers were purchased from Research Genetics (Hunteville, AL, USA). A three-temperature PCR protocol (94, 58 and 72°C) was conducted for 45 cycles in a Geneamp 9600 Thermal Cycler (Perkin-Elmer-Cetus). PCR products were diluted 2-fold with loading buffer consisting of xylene cyanol and bromophenol blue dyes in 50% glycerin and were run on 18% polyacrylamide gels. After electrophoresis, gels were visualized after ethidium bromide staining.

Statistics
The linkage of a particular locus with anxiety-like behavior was examined by estimating the numbers of entries in the open or closed arms in the elevated plus maze test in 215 B/W F1xNZW back-cross mice, using a computer package program of MAPMANAGER/QTL. Statistical significance was considered with a P-value of less than 0.05, using Student's t-test unless otherwise specified, {chi}2-test or ANOVA.


   ACKNOWLEDGEMENTS
 
We thank Pfizer Inc. (Groton, NY, USA) for the generous gift of CP-154,526, and M. Ohara for language assistance. This work was supported in parts by research grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Second Department of Pathology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel: +81 358021039; Fax: +81 338133164; Email: sacchi{at}med.juntendo.ac.jp Back

{dagger} Emeritus Professor. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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