Previous Article | Next Article 
The Journal of Neuroscience, September 1, 1998, 18(17):6952-6962
Essential Role of the fosB Gene in Molecular,
Cellular, and Behavioral Actions of Chronic Electroconvulsive
Seizures
Noboru
Hiroi1,
Gerard
J.
Marek1,
Jennifer R.
Brown2,
Hong
Ye2,
Frederic
Saudou2,
Vidita A.
Vaidya1,
Ronald S.
Duman1,
Michael E.
Greenberg2, and
Eric J.
Nestler1
1 Laboratory of Molecular Psychiatry, Departments of
Psychiatry and Neurobiology, Yale University School of Medicine, New
Haven, Connecticut 06508, and 2 Division of Neuroscience,
Children's Hospital and Department of Neurobiology, Harvard Medical
School, Boston, Massachusetts 02115
 |
ABSTRACT |
The role of Fos-like transcription factors in neuronal and
behavioral plasticity has remained elusive. Here we demonstrate that a
Fos family member protein plays physiological roles in the neuronal,
electrophysiological, and behavioral plasticity associated with
repeated seizures. Repeated electroconvulsive seizures (ECS) induced
isoforms of 
FosB in frontal cortex, an effect that was associated
with increased levels of the NMDA receptor 1 (NMDAR1) glutamate
receptor subunit. Induction of FosB and the upregulation of NMDAR1
occurred within the same neurons in superficial layers of neocortex.
Activator protein-1 (AP-1) complexes composed of FosB were
bound to a consensus AP-1 site in the 5'-promoter region of the NMDAR1
gene. The upregulation of NMDAR1 was absent in mice with a targeted
disruption of the fosB gene. In addition, repeated ECS
treatment caused progressively shorter motor seizures (tolerance) in
both rats and wild-type mice, as well as reduced NMDA-induced inward
currents in pyramidal neurons from superficial layers of the neocortex
of wild-type mice. These behavioral and electrophysiological effects
were also significantly attenuated in fosB mutant mice.
These findings identify fosB gene products as
transcription factors critical for molecular, electrophysiological, and
behavioral adaptations to motor seizures.
Key words:
transcription factor; seizure; ECS; cerebral cortex; c-Fos; FosB; FosB; FRA; JunD; AP-1; neural plasticity; depression; epilepsy; NMDA receptor
| |
INTRODUCTION |
The long-term responses of the brain
to chemical and electrical stimulation have served as useful models to
gain insight into the molecular mechanisms underlying neural plasticity
(Hyman and Nestler, 1993
). Increasing evidence suggests that
transcriptional regulation of genes is one pivotal mechanism of
plasticity. As a result, identification of transcription factors whose
induction is required for neuronal and behavioral adaptations to
chemical and electrical stimulation is a critical step in understanding complex forms of neural plasticity in the brain.
Different types of seizures are dynamic behavioral manifestations of
excessive activation of distinct brain regions. Electroconvulsive seizures (ECS) depend on activation of the motor cortex, whereas most
chemically induced seizures occur, at least initially, via activation
of limbic structures (McNamara, 1994). In addition to inducing changes
in behavior, repeated ECS are associated with a number of neurochemical
changes in relevant brain regions (e.g., Hiroi et al., 1996; Nibuya et
al., 1996), with little sign of neuronal cell loss (Devanand et al.,
1994). Thus, the ECS paradigm is a model to study the roles of
transcription factors in activity-dependent plasticity. Yet, in
contrast to limbic seizures (see McNamara, 1994; Watanabe et al.,
1996), the molecular factors involved in ECS are poorly understood.
Considerable attention has focused on the transcription factor products
of the fos and jun families of immediate-early
genes, which are induced rapidly but transiently in the brain in
response to acute seizure (Morgan and Curran, 1991a). Such
transcription factors are believed to then regulate the expression of
specific late-response genes, which would lead to some of the
functional and structural adaptations to the original stimulus. Indeed,
Fos and Jun proteins bind to AP-1 (activator protein-1) sites, which have been found in the 5'-promoter regions of genes that encode certain
peptide neurotransmitters, receptors, protein kinases, and other
intracellular signaling molecules (see Sheng and Greenberg, 1990;
Hughes and Dragunow, 1995). Although some of these potential target
genes can be regulated by AP-1 complexes in vitro, it has not yet been possible to demonstrate a physiological target gene for
these transcription factors in the brain in vivo. Moreover, whereas several Fos and Jun family member proteins are induced acutely,
there is evidence that their induction desensitizes after chronic
perturbation (Winston et al., 1990; Hope et al., 1992, 1994a,b;
Pennypacker et al., 1995).
We have demonstrated previously that repeated ECS induce a long-lasting
AP-1 complex in cerebral cortex that is composed of novel Fos-like
proteins of 35-37 kDa, termed "chronic FRAs" (Fos-related antigens) (Hope et al., 1994b). Persistent induction of the chronic FRAs and the associated chronic AP-1 complex is in marked contrast to
the very short-lived induction of several Fos-like proteins and the
acute AP-1 complex by an acute seizure (Sonnenberg et al., 1989).
The aims of the present study were to characterize further the chronic
FRAs and the chronic AP-1 complex in response to repeated ECS and then
to identify potential target genes for this transcription factor and
its role in electrophysiological and behavioral adaptations to ECS.
| |
MATERIALS AND METHODS |
Animal treatments. We used male Sprague Dawley rats
(200-350 gm; Camm Research Institute, Wayne, NJ) and fosB
mutant mice and their age-matched wild-type littermates (2-3 months
old) (Brown et al., 1996). Animals were divided into five groups. One
group received sham treatments for 6-7 d and was killed 18-20 hr
after the last sham treatment. A second group received sham treatments for 6-7 d and was killed 2 or 6 hr after acute ECS treatment (45 mA
and 0.3 sec for the rat; 9 mA and 0.3 sec for the mouse) on the last
day. A third group received daily ECS treatments for 6-7 d and was
killed 18-20 hr after the last treatment. In a separate time-course
experiment, a fourth group was given sham treatment for 6 d and
killed 18-20 hr later or given sham treatment for 5 d and a
single ECS on the 6th day and killed 18-20 hr later. A fifth group
received sham or ECS treatments for 2 d and was killed 24 hr after
the last treatment. This group was used for electrophysiology.
Tissue preparation. Animals were decapitated, and the
frontal cortex was isolated by gross dissection. The sample was the entire frontal cortex, including the medial prefrontal and motor cortex. Brain samples were Dounce-homogenized in electrophoretic mobility shift assay buffer ("EMSA buffer") (Korner et al.,
1989; Hope et al., 1994a): 20 mM HEPES, pH 7.9, 0.4 M NaCl, 20% glycerol, 5 mM
MgCl2, 0.5 mM EDTA, 0.1 mM
EGTA, 1% Nonidet P-40, 10 µg/ml leupeptin, 0.1 mM
p-aminobenzamide, 1 µg/ml pepstatin, 0.5 mM PMSF, and 5 mM DTT. Homogenates were incubated on ice for
30 min and were centrifuged at 12,000 × g for 20 min
at 4°C. Supernatants were used for Western blotting and gel shift
assays.
Immunoblotting. Aliquots of tissue extracts (containing 50 µg of protein) were diluted in EMSA buffer containing 2% SDS and 
-mercaptoethanol and were subjected to SDS-PAGE (6.5 or 10%
acrylamide/0.4% bis-acrylamide in resolving gels) at 75 V overnight.
Proteins were transferred electrophoretically to polyvinylidene
fluoride membranes (Immobilon-P; Millipore, Bedford, MA).
Membranes were then incubated with 1 or 2% nonfat dry milk in 10 mM sodium phosphate buffer (PBS) for 60 min at room
temperature and in primary antiserum overnight at 4°C. Membranes were
washed and incubated with goat anti-rabbit or horse anti-mouse IgG
peroxidase conjugate (1:4000 or 1:2000, respectively;
Vector Laboratories, Burlingame, CA) for 2 hr. After washing in PBS,
membranes were developed by chemiluminescence (Renaissance; DuPont NEN,
Boston, MA). We used an anti-FRA antiserum (1:4000; Dr. M. J. Iadarola, National Institutes of Health) as described previously (Young
et al., 1991; Hope et al., 1994a). This antiserum was raised against a
sequence highly conserved in all known Fos family member proteins (DNA
binding domain). An excess amount of the immunogen eliminated specific
FRA bands. The other antibodies we used were a monoclonal anti-NMDA
receptor 1 (anti-NMDAR1) antibody (1:4000; PharMingen, San Diego, CA), a polyclonal anti-NMDAR2A affinity-purified antibody (1:1000; Chemicon,
Temecula, CA), and a polyclonal anti-NMDAR2B affinity-purified antibody (1:1000; Chemicon). The specificity of these
antibodies has been established (Brose et al., 1994; Siegel et al.,
1994; Snell et al., 1996), and these antibodies recognized specific bands at their predicted molecular weights. Levels of subunit immunoreactivity were quantified by use of a Macintosh-based image analysis system with National Institutes of Health software.
Immunoblots were routinely stained by amido black to confirm equal
loading and transfer of proteins.
Gel shift assay. Gel shift assays were performed according
to published procedures (Hope et al., 1994b). We used double-stranded oligonucleotides, which contained the AP-1 site from the
human metallothioneine II gene
(5'-TCGACGTGACTCAGCGCGC-3') (Sonnenberg et al.,
1989) or the AP-1 site from the rat NMDAR1 gene
(5'-GATCAAGCCTGAGTCACAG-3') (Bai
and Kusiak, 1993). (The bold nucleotides indicate double-stranded sequences; the underlined nucleotides indicate the consensus AP-1 site.) The probes were radioactively labeled with
[
-32P]dGTP and [-32P]dTTP using
Klenow DNA polymerase fill-in reaction. Protein (20 or 40 µg) was
mixed with the probes and incubated for 20 min at room temperature.
Samples were then electrophoresed in a nondenaturing gel system that
contained 6% acrylamide/0.24%
N,N'-methylene-bis-acrylamide, 25 mM
Tris-borate, pH 8.3, 1 mM EDTA, and 1.6% glycerol as
described previously (Hope et al., 1994a). Specificity of the resulting AP-1 binding activity was demonstrated by competition with
nonradioactive probe and mutant probe (data not shown) (see also Hope
et al., 1994b) as well as by supershift assays.
Supershift assays were conducted in the same manner as gel shift assays
except for the addition of antiserum as described (Hope et al., 1994b).
Briefly, 1 µl of antiserum against c-Fos, FosB/FosB, FRA-1, FRA-2,
c-Jun, JunB, or JunD (1:30 or 1:3) was added to samples in reaction
buffer (29 µl). These antibodies were provided by M. Gruda and R. Bravo of Bristol-Myers Squibb (Princeton, NJ). Samples were incubated
with antiserum for 2 hr at room temperature. One microliter of
radioactively labeled double-stranded AP-1 probe (Sonnenberg et al.,
1989) was then added, and the mixture was incubated for 20 min at room
temperature. The specificity of these antisera has been well
characterized, and each has been shown to disrupt successfully AP-1
binding activity of complexes containing the specific proteins at the
concentrations used here (Gruda et al., 1996).
Anatomical procedures. For in situ hybridization,
rat brains were removed rapidly from decapitated animals, immediately
frozen on dry ice, and cut at 10 µm. Sections were fixed for 5 min in 4% paraformaldehyde, were washed for 10 min in PBS, and were immersed for 30 min in 2× SSC (0.3 M NaCl and 30 mM
sodium citrate). Buffer (500 µl) containing 2 × 106 cpm of an oligoprobe directed against NMDAR1
mRNA (antisense, 5'-TTCCTCCTCCTCCTCACTGTTCACCTTGAATCGGCCAAAGGGACT-3';
sense, 5'-AGTCCCTTTGGCCGATTCAAGGTGAACAGTGAGGAGGAGGAGGAA-3') or an
oligoprobe directed against NMDAR2B (antisense,
5'-GGGCCTCCTGGCTCTCTGCCATCGGCTAGGCACCTGTTGTAACCC-3'; sense,
5'-GGGTTACAACAGGTGCCTAGCCGATGGCAGAGAGCCAGGAGGCCC-3') was placed onto
each slide. Sections were incubated overnight at 37°C. Sections were
rinsed for 2 hr in 1× SSC and for 1 hr in 0.5× SSC at room
temperature. After the last wash for 1 hr in 1× SSC at 37°C,
sections were dried and exposed to Hyperfilm--max. The optical
density of mRNA signals was measured from film using a Macintosh-based
image analysis system with National Institutes of Health software. For
each section, rectangular areas (1 × 0.3 mm) were chosen from the
superficial (II and III) and deep (V and VI) layers of the rat motor
cortex. Measurements were taken from both hemispheres of the entire
extent of the motor cortex, using standards provided by National
Institutes of Health software. For each rat, the entire extent of motor
cortex, represented by two to three levels in the anterior-posterior
axis of sections, was examined on both hemispheres. These in
situ hybridization experiments were repeated on two to three sets
of tissue sections. As a result, quantification of in situ
hybridization data was based on analysis of ~50 determinations of
sham- and of ECS-treated tissue.
For standard immunohistochemistry, mice and rats were anesthetized with
sodium pentobarbital at 120 mg/kg and were perfused with 0.9% saline
followed by 4% paraformaldehyde. Brains were post-fixed for 1-2 hr in
4% paraformaldehyde and were cryoprotected in 20% glycerol overnight.
Mouse sections were cut at 20 µm and were incubated with a monoclonal
antibody against calbindinD28K (1:1000; Sigma, St. Louis,
MO) or a monoclonal antibody against NMDAR1 (1:1000; PharMingen).
Sections were incubated with a biotinylated horse anti-mouse IgG
(1:500) and stained with ABC-DAB (Hiroi, 1995; Hiroi and Graybiel,
1996). Similarly, rat brain sections were stained with the polyclonal
anti-FRA antibody (1:5000) and biotinylated rabbit IgG (1:500). For
each section, rectangular areas (1 × 0.3 mm) were chosen from the
superficial (II and III) and deep (V and VI) layers of the motor
cortex. The number of immunoreactive nuclei was counted inside the
rectangular areas as described (Hiroi and Graybiel, 1996). The
specificity of these various antibodies has been well characterized
(Brose et al., 1994; Huntley et al., 1994; Siegel et al., 1994; Hiroi
and Graybiel, 1996).
For double-labeling immunofluorescence, rat and mouse brain sections
were incubated for 10 min with 1% Triton X-100, for 30 min with 5%
normal goat serum, and overnight with a mixture of a monoclonal
antibody against NMDAR1 (1:1000; PharMingen) and the polyclonal
anti-FRA antibody (1:5000) or polyclonal anti-FosB/FosB antibody
(1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Sections were then
incubated with a mixture of fluorescein-conjugated goat anti-mouse IgG
(1:100; Jackson Laboratories, Burlingame, CA) and Texas red-conjugated
goat anti-rabbit IgG (1:500; Jackson Laboratories). Resulting sections
were examined by confocal microscopy. Adsorption of the anti-FRA
antibody with the immunogen eliminated staining. The selectivity of the
anti-NMDAR1 antibody and the anti-FosB/FosB antibody has been
established previously (Huntley et al., 1994; Siegel et al., 1994;
Hiroi and Graybiel, 1996); in addition, omission of the primary
antibodies eliminated staining. The selectivity of the secondary
antibodies and their lack of cross-reactivity have been well
characterized (Berhow et al., 1996).
Some brain sections of wild-type and fosB mutant mice were
analyzed by use of a standard Nissl-staining procedure described elsewhere (Hiroi, 1995).
Behavioral analysis. The duration of motor seizures elicited
by ECS treatments was assessed in rats and in fosB mutant
and wild-type mice. Assessments were performed by an observer blinded to the genotypes of the mice. Motor seizure was defined as full-body extension and repetitive, jerky paw-leg movements. Sham-treated rats
and mice received ear clips without any current passed.
Electrophysiology. Brain slices were prepared from male
fosB mutant mice (n = 15) and their
age-matched wild-type littermates (n = 13) (2-3 months
old) (Brown et al., 1996). Briefly, a cervical dislocation was
performed, in adherence to protocols approved by the Yale University
Animal Care and Use Committee. After decapitation, the brain was
removed rapidly and placed in an ice-cold modified artificial
CSF (ACSF) in which sucrose (252 mM) was substituted for NaCl (Aghajanian and Rasmussen, 1989). A block of the brain containing the motor cortex was dissected free, and coronal slices (500 µm) were cut with an oscillating-blade tissue slicer (FHC, Brunswick, ME). Slices were then transferred to the stage of a fluid-gas interface chamber that had a constant flow of humidified 95%O2/5%CO2. The chamber was heated
slowly from room temperature to 34°C. The slices were perfused with
normal ACSF that consisted of (in mM): NaCl 126; KCl 3;
CaCl2 2; MgSO4 2; NaHCO3 26;
NaH2PO4 1.25; and D-glucose 10. The
ACSF flow rate was 1.5 ml/min. There was a 2 hr recovery period before
experiments.
Intracellular recording and single-electrode voltage clamping were
conducted in neocortical layer II/III pyramidal cells using an
Axoclamp 2-A (Axon Instruments, Foster City, CA). Stubby
electrodes (~8 mm, shank to tip) with relatively low capacitance and
resistance (40-60 M
) were pulled from filament-containing capillary
tubing (1.5 µm) with a Brown-Flaming electrode puller (Sutter
Instruments, San Rafael, CA) and were filled with 1 M
potassium acetate. Under voltage clamp, electrodes prepared in this
manner had rapid settling times (50-75 µsec), allowing switching
frequencies of 4-6 Hz and a loop gain of 10 nA/mV (30% duty cycle).
Phase lag was used to prevent oscillations; false clamping was avoided
by using optimal capacitance neutralization and by allowing settling to
a horizontal baseline, verified by monitoring input voltage
continuously. NMDA (6.25-50 µM) was bath-applied for 1.5 min, and the steady-state inward current was monitored. Cells were
clamped at 
55 mV to decrease Mg2+ block of the
NMDA responses. Pyramidal cells were identified according to criteria
described previously (McCormick et al., 1985; Connors and Gutnick,
1990). Regularly spiking layer II/III pyramidal cells were recorded in
the motor cortex. Data were collected by means of pClamp software via a
Digidata 1200 interface (Axon Instruments) and a Gould chart recorder.
Drugs and other agents were obtained from the following sources:
tetrodotoxin (TTX) from Sigma and NMDA from Research Biochemicals
(Natick, MA).
Statistical analyses. Data obtained in this study, whether
from Western blots, gel shift assays, immunohistochemistry, in situ hybridization, behavioral assays, or electrophysiological recordings, were first examined for their statistical significance by
use of the ANOVA. Differences that were significant with this measure
were further evaluated by use of Fisher LSD protected t tests.
| |
RESULTS |
Repeated ECS administration induces FosB/FosB in
frontal cortex
We attempted to identify Fos and Jun family member transcription
factors that are selectively induced in response to repeated administration of ECS. Although the chronic FRAs have been shown to be
immunochemically related to FosB, a truncated splice variant of FosB
(Dobrazanski et al., 1991; Mumberg et al., 1991; Nakabeppu and Nathans,
1991; Yen et al., 1991), the identity of the proteins has remained
controversial (Hope et al., 1994a; Chen et al., 1995; Bing et al.,
1996, 1997) (see Discussion). This is because their apparent molecular
weight on SDS-PAGE can be distinguished from FosB induced in brain
by acute stimuli (Hope et al., 1994a; Chen et al., 1995) and because
fosB mRNA is not detectable at the time the chronic FRAs
are induced (Chen et al., 1995; Bing et al., 1996, 1997). Indeed, these
findings have led to speculation that the chronic FRAs are products of
a novel fos-related gene.
As a first step in identifying the constituents of the AP-1 complex
induced by chronic ECS administration, we used antibodies selective for
each of the known Fos and Jun family member proteins in supershift
assays. The specificity and lack of cross-reactivity of these
antibodies have been well characterized (Gruda et al., 1996). As shown
in Figure 1A, chronic
ECS resulted in an ~70% increase in AP-1 binding activity in rat
frontal cortex, as observed previously (Hope et al., 1994b). The
specificity of the AP-1 complex has been well characterized using
mutant AP-1 probe and AP-1 probe for competition (see Hope et al.,
1994b). This chronic AP-1 complex was disrupted by an anti-FosB/FosB
antibody, whereas antibodies directed against other Fos family members,
c-Fos, FRA-1, or FRA-2, were without effect (Fig.
1B). The chronic AP-1 complex was also disrupted by
an anti-JunD antibody, whereas an anti-c-Jun antibody was without
effect. An anti-JunB antibody produced a small but consistent decrement
in levels of AP-1 binding. These results indicate that the chronic AP-1
complex is composed predominantly of a FosB/FosB-like protein(s)
coupled to JunD and, to a lesser extent, JunB.
View larger version (97K):
[in this window]
[in a new window]
|
Figure 1.
A, Induction of AP-1 binding by
acute (2 or 6 hr) and chronic (Ch) ECS treatments in rat
frontal cortex. Arrows on the left
indicate specific AP-1 activity, determined by competition with
nonradioactive probe (see also Hope et al., 1994b).
B, Disruption of chronic AP-1 binding activity in rat
frontal cortex by anti-Fos or anti-Jun family member antibodies
(Ab). 0, No antibody added. Two dilutions
(left, 1:30; right, 1:3) of primary
antiserum were used (see Materials and Methods). The figure is
representative of results obtained from at least six samples in each
group in at least three separate experiments. C,
Induction of AP-1 binding by acute (2 hr) and chronic ECS treatments in
the frontal cortex of wild-type littermates (fosB
+/+) and fosB mutant mice (fosB
/). For treatments, see Materials and Methods.
C, Sham-treated mice; ns, nonspecific
bands. The four right lanes show competition of the
specific AP-1 complex with nonradioactive AP-1 probe. The figure is
representative of results obtained from analysis of at least nine
samples in each group in at least three separate experiments.
D, Induction of Fos-like proteins by acute and chronic
ECS treatments in the frontal cortex of wild-type littermates
(fosB +/+) and fosB mutant mice
(fosB /). The figure is representative of
results obtained from analysis of at least nine samples in each group
in at least three separate experiments. The peptide block experiment
verifies the specificity of the immunoreactive bands (see Materials and
Methods).
|
|
To obtain further evidence that the chronic AP-1 complex required
fosB gene products, we examined the ability of chronic ECS to induce AP-1 binding activity in fosB mutant mice. It was
found that chronic ECS increased AP-1 binding activity in the frontal cortex of wild-type mice (Fig. 1C), similar to the increase
seen in rat. In contrast, the ability of chronic ECS to induce AP-1 binding activity in this brain region was completely absent in fosB mutant mice.
To identify the specific FosB/FosB-like proteins that comprise the
chronic AP-1 complex, we next analyzed extracts of frontal cortex from
fosB mutant and wild-type mice by Western blotting, using an
"anti-FRA" antibody that recognizes all known Fos-like proteins. As
shown in Figure 1D, chronic ECS resulted in the
induction of 35-37 kDa proteins (chronic FRAs) and a 45 kDa protein,
equivalent to the effects seen in the rat (Hope et al., 1994b). In
contrast, induction of the 35-37 kDa and 45 kDa FRAs was completely
absent in the fosB mutant mice. There persisted in the
mutant a low intensity band below 45 kDa, also seen in wild-type, which
was not regulated by seizure treatment (Fig. 1D).
Although the identity of this band remains unknown, it would seem to be
a specific FRA given its ability to be competed by blocking peptide as
shown in Figure 1D.
Also shown in Figure 1D is the induction of Fos-like
proteins in response to a single acute ECS. In wild-type mice, four
major Fos-like proteins were induced: a 55 kDa protein (presumably
c-Fos), a 45 kDa protein (presumably FosB), a 41 kDa protein
(presumably FRA-1 or FRA-2), and 35-37 kDa proteins (presumably
FosB). In the mutant mice, induction of the 45 kDa and 35-37 kDa
proteins was completely absent, consistent with the identification of
these proteins as FosB and FosB, respectively, whereas induction of the 55 and 41 kDa proteins was normal, consistent with their
identification as products of distinct genes. Indeed, these findings
confirm that the targeted disruption of the fosB gene
selectively blocked the production of fosB gene products
without altering other Fos family member proteins.
The ability of a single ECS to acutely (2 hr later) induce the various
Fos-like proteins in wild-type and fosB mutant mice is
consistent with results obtained with AP-1 binding (Fig.
1C). Thus, acute ECS increased AP-1 binding in frontal
cortex of mutant mice (in contrast to the lack of induction by chronic
ECS), although a lower level of induction was seen in mutant mice
compared with wild-type littermates. This is consistent with the loss
of induction of the 45 and 35-37 kDa proteins but normal induction of
the 55 and 41 kDa proteins. Also evident in Figure 1, A and
C, is the different migration pattern of the AP-1 complex
induced acutely versus chronically; the acute complex (in both rats and
mice) migrates slightly higher on the gels compared with the migration of the chronic complex. This difference in migration is consistent with
the different protein compositions of these two complexes.
The fosB gene produces two protein products through
alternative splicing: full-length FosB (45 kDa) plus FosB (~35
kDa) that lacks the C terminal of the full-length form. By the use of
antibodies directed against the N and C terminals of FosB, we found
that the chronic FRAs are recognized by the N-terminal antibody only (data not shown). These results, along with the finding that all of the
chronic FRA bands are absent in fosB mutant mice, indicate that the chronic FRAs and the 45 kDa FRA are isoforms of FosB and
FosB, respectively.
Repeated motor seizure regulates the expression of NMDA
receptor subunits
ECS treatment consistently induces a motor seizure, which is
characterized by clonic jerking of major muscle groups of the body
(McNamara, 1994). In contrast to limbic seizures, which occur via
activation of excitatory pathways in limbic brain structures (e.g.,
hippocampus and amygdala), motor seizures depend on activation of NMDA
glutamate receptors in the motor cortex (Clineschmidt et al., 1982;
Nomikos et al., 1992; McNamara, 1994). NMDA receptors are multimeric
proteins composed of several subunits. The NMDAR1 subunit is an
obligatory constituent of functional NMDA receptors; this subunit can
form complexes with any of several NMDAR2 subunits. NMDA receptors
composed of different combinations of subunits exhibit distinct
electrophysiological properties (Monyer et al., 1992; Nakanishi, 1992)
and distinct developmental courses (Sheng et al., 1994).
Given the obligatory role of the NMDA receptor in the production of
motor seizures, we asked whether repeated ECS might alter NMDA receptor
subunit expression. The rodent cerebral cortex contains NMDAR1 and two
forms of NMDAR2, NMDAR2A and NMDAR2B; two other known forms of NMDAR2,
NMDAR2C or NMDAR2D, are not abundantly expressed in this brain region
(Monyer et al., 1992; Nakanishi, 1992; Ishii et al., 1993). Thus, we
examined regulation of NMDAR1, NMDAR2A, and NMDAR2B by ECS. As shown in
Figure 2A, chronic ECS (with animals examined 18-20 hr after the last ECS) significantly increased levels of immunoreactivity of NMDAR1 [t(14) = 2.03; p < 0.05] and NMDAR2B [t(9) = 2.53;
p < 0.05] in rat frontal cortex as determined by
Western blotting. No change was seen in levels of NMDAR2A
immunoreactivity [t(14) = 0.83; p > 0.05]. In contrast to chronic ECS, a single ECS treatment (with
animals examined 18-20 hr later) decreased levels of NMDAR1 [29%;
t(14) = 2.43; p < 0.05], increased levels
of NMDAR2B [70%; t(15) = 2.57; p < 0.05], and had no effect on levels of NMDAR2A [t(15) = 0.91; p > 0.05].
View larger version (49K):
[in this window]
[in a new window]
|
Figure 2.
A, Effect of chronic ECS treatment
on the levels of NMDA receptor subunit immunoreactivity in rat frontal
cortex as determined by Western blotting. Data are expressed as
mean ± SEM. S, Sham (n = 7);
E, chronic ECS (n = 9);
R1, NMDAR1; R2A, NMDAR2A;
R2B, NMDAR2B; *, statistically significant difference
from controls; NS, no statistically significant
difference from controls (Fisher LSD protected t tests).
B, Effect of chronic ECS treatment on levels of
R1 and R2B mRNA in the rat frontal cortex
as determined by in situ hybridization
(n = 4 for S; n = 4 for E). The right column (sense)
shows the lack of signals with sense probes (see Materials and
Methods).
|
|
To gain greater insight into the anatomical localization of ECS-induced
upregulation of NMDA receptor subunit expression, we studied levels of
NMDAR1 and NMDAR2B mRNAs in the rat frontal cortex by in
situ hybridization. This analysis revealed that upregulation of
NMDAR1 mRNA levels occurs most strikingly in superficial layers of the
frontal cortex [t(100) = 2.62; p < 0.05]
but not in deep layers [t(100) = 0.57; p > 0.05] (Fig. 2B). Similarly, upregulation of NMDAR2B
mRNA levels was significant in superficial layers [t(93) = 2.21; p < 0.05] but not in deep layers
[t(74) = 0.52; p > 0.05]. For both R1 and
R2B, upregulated mRNAs were most apparent in the superficial (II and
III) layers of the dorsal and lateral cortices (see a band-like
pattern).
NMDAR1 is a putative physiological target of the chronic
AP-1 complex
Given that the observed induction of NMDAR1 and of the chronic
AP-1 complex required chronic (as opposed to acute) ECS administration, we considered the possibility that the gene encoding this receptor subunit may be a physiological target for the chronic AP-1 complex composed predominantly of FosB and JunD. Support for this
possibility is the finding that the 5'-promoter of the NMDAR1 gene
contains a consensus AP-1 site (Bai and Kusiak, 1993).
As a first step in assessing the role of FosB in regulating NMDAR1
gene expression, we determined whether induction of the proteins occurs
in the same cells by use of double-labeling immunofluorescence. This
experiment revealed that chronic ECS produced a dramatic (fourfold)
increase in Fos-like immunoreactivity in layers II and III throughout
the rat frontal cortex (sham, 30 ± 3 FRA-positive nuclei per 0.3 mm2 area ± SEM; ECS, 127 ± 6;
p < 0.01; based on analysis of 21 samples derived from
four rats in each group), with no increase observed in deep cortical
layers (sham, 10 ± 2; ECS, 12 ± 2; p > 0.05). A similar pattern was seen for NMDAR1-like immunoreactivity,
which showed the most dramatic increase in superficial cortical layers (data not shown). This is similar to the pattern of elevated levels of
NMDAR1 and NMDAR2B mRNAs seen by in situ hybridization (see Fig. 2B). Moreover, increased levels of Fos-like
proteins were found in neurons with upregulated NMDAR1-like
immunoreactivity (Fig. 3). NMDAR1
immunoreactivity showed clear cytoplasmic and membrane staining,
whereas Fos-like immunoreactivity showed nuclear localization,
consistent with the known subcellular distribution of these proteins
(Morgan and Curran, 1991a; Ehlers et al., 1995). Although this
experiment was performed with the anti-FRA antibody, the large majority
of the Fos-like immunoreactivity detected after chronic ECS probably
represents FosB isoforms (and to a lesser extent FosB) based on the
preponderance of these proteins as revealed by Western blotting (Fig.
1D). Indeed, equivalent results were obtained by use
of a selective anti-FosB/FosB antibody (data not shown).
Nevertheless, some caution is needed when ascribing immunohistochemical
staining to a particular protein.
View larger version (126K):
[in this window]
[in a new window]
|
Figure 3.
Colocalization of FRA and NMDAR1 immunoreactivity
in rat motor cortex (A, B) and medial
prefrontal cortex (C, D).
A, C, Sham treatment; B,
D, chronic ECS treatment. Rats received six daily ECS
treatments and were used on day 7. Red staining
represents FRA immunoreactivity; green staining
represents NMDAR1 immunoreactivity. The left sides in
A and B are medial to the right
sides. The right sides in C and
D are layer I. All the images were centered in layer II.
Note the generally higher basal levels of FRA immunoreactivity in motor
cortex (A) than in prefrontal cortex
(C). The observation that some FRA-negative cells
express NMDAR1 in both brain regions under basal conditions suggests
that, although the AP-1 transcription factor seems to be important for
ECS induction of NMDAR1 expression, it may not be required for basal
expression of the subunit. Scale bar, 25 µm. The figure is
representative of results obtained from analyses of four sham- and four
ECS-treated rats.
|
|
We next determined whether the consensus AP-1 site present within the
NMDAR1 gene promoter can bind the AP-1 complex induced by chronic ECS.
Using a double-stranded oligonucleotide that corresponds to this region
of the NMDAR1 promoter, we found that repeated ECS administration
resulted in a clear increase in binding activity to this AP-1 site in
extracts of rat frontal cortex [t(16) = 5.94; p < 0.01] (Fig.
4A,B).
The specificity of this binding is demonstrated by its competition by
unlabeled probe, shown in Figure 4A.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 4.
A, Induction of AP-1 binding by
chronic ECS treatment in rat frontal cortex using a radioactively
labeled probe corresponding to the AP-1 site in the NMDAR1 gene
promoter. Specificity of the AP-1 binding was established by
competition with nonradioactive probe (30 ng). S, Sham;
E, chronic ECS. B, Quantification of AP-1
binding activity shown in A. The entire AP-1 complex
indicated by arrows in A was measured.
Data are expressed as mean ± SEM (n = 8 for
S; n = 10 for E). **,
Statistically significant difference from control
(p < 0.01) (t test).
C, Effect of chronic ECS treatment on levels of NMDAR1
and NMDAR2B immunoreactivity in frontal cortex of wild-type littermates
(+/+; n = 6 for S;
n = 13 for E) and
fosB mutant mice (/; n = 5 for
S; n = 8 for E). *,
Statistically significant difference between S +/+ and
E +/+ (p < 0.05); #,
statistically significant difference between S +/+ and
S / (Fisher LSD protected t
tests).
|
|
To study directly a role for FosB in mediating the ECS-induced
increase in NMDAR1 expression, we compared the ability of chronic ECS
to upregulate levels of NMDAR1 immunoreactivity in frontal cortex of
wild-type and fosB mutant mice. As shown in Figure
4C, chronic ECS increased levels of NMDAR1 immunoreactivity in the frontal cortex of wild-type mice [t(29) = 2.02;
p < 0.05], similar to the effect seen in rat (see
Fig. 2A). In contrast, chronic ECS failed to alter
levels of the receptor subunit in this brain region of mutant mice. The
basal levels of NMDAR1 did not differ between the mutant and wild-type
mice [t(24) = 0.72; p > 0.05]. Moreover,
induction of NMDAR1 and of FosB/FosB-like immunoreactivity showed
similar colocalization in the cortex of wild-type mice (data not shown)
as seen in the rat (Fig. 3). As would be expected, there was no
detectable FosB/FosB-like immunoreactivity in fosB mutant
mice (data not shown). Together, these results provide direct evidence
that the NMDAR1 gene is indeed a potential physiological target for
fosB gene products in the brain in vivo.
Because the 5'-region of the NMDAR2B gene cloned to date also contains
an AP-1-like site (Sasner and Buonanno, 1996), we were interested in
determining whether ECS regulation of this subunit was similarly
aberrant in the mutant mice. Indeed, chronic ECS did not alter levels
of NMDAR2B immunoreactivity in the fosB mutant mice
[t(24) = 0.72; p > 0.05] in contrast to
the increase seen in wild-type littermates [t(43) = 2.01;
p < 0.05] (Fig. 4C). However, basal levels
of NMDAR2B immunoreactivity were significantly higher in the mutant
mice than in wild-type littermates [t(24) = 2.87; p < 0.01]. It remains unclear, therefore, how FosB
and FosB serve to regulate NMDAR2B gene expression under both basal and
stimulated conditions.
fosB gene products are required for behavioral
tolerance to repeated ECS
We next studied whether induction of FosB/FosB could be
correlated with some form of behavioral plasticity to ECS treatment. As
shown in Figure 5,
left, we found that repeated administration of ECS
induced a progressively shorter motor seizure in rats, an effect that
was near maximal by day 3. This form of tolerance has not been
recognized previously but is consistent with the clear tolerance that
develops in humans undergoing chronic treatment with ECS (Fink,
1990).
View larger version (18K):
[in this window]
[in a new window]
|
Figure 5.
Left, Effect of chronic ECS
treatment on the duration of motor seizures in rats
(n = 9). *, Statistically significant difference
from day 1 (Fisher LSD protected t tests).
Right, Effect of chronic ECS treatment on the duration
of motor seizures in fosB mutant mice
(n = 21) and their wild-type littermates
(n = 13). Data are expressed as mean ± SEM;
*, statistically significant difference from day 1; #, statistically
significant difference between fosB mutant mice and
their wild-type littermates (Fisher LSD protected t
tests).
|
|
A similar reduction in the duration of motor seizures was observed in
wild-type mice (Fig. 5, right). The reduction occurred with
a time course equivalent to that seen in rats but was smaller in
magnitude (~17%). In contrast to wild-type mice, fosB
mutant mice showed a significant delay in the development of tolerance to motor seizures, even though the motor seizure induced in the mutant
mice by the first ECS was indistinguishable from that exhibited by
wild-type littermates [t(32) = 0.59; p > 0.05]. Continued treatment with ECS, however, did eventually result in
a significant reduction in seizure duration, with equivalent motor
seizures observed in mutant and wild-type mice by day 6.
fosB gene products are required for
electrophysiological adaptation to repeated ECS
Given alterations in NMDA receptor subunit expression and in
duration of motor seizures after repeated ECS, we next studied electrophysiological responses of layer II/III pyramidal cells to NMDA
in the motor cortex of wild-type and fosB mutant mice. Recordings were made from cells that showed characteristics of regularly spiking pyramidal cells (Fig.
6A) (McCormick et al., 1985; Connors and Gutnick, 1990). In sham-treated wild-type mice, we
found that increasing concentrations of NMDA (from 6.25 to 25 µM) induced increasing inward currents that reached ~90
pA at 25 µM NMDA (Fig.
6B,C). Consistent with previous
findings in rat cerebral cortex, NMDA concentrations of 50 µM or greater, bath applied for 1-2 min periods, induced
very large (4-5 nA) inward currents in cells from wild-type mice (data
not shown), currents that far exceed the physiological range and are
associated with excitotoxicity. Thus, we did not regularly test 50 µM NMDA in wild-type mice and typically used a 25 µM NMDA concentration.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 6.
NMDA-induced inward currents in pyramidal cells in
the motor cortex. A, Responses of a layer II/III
pyramidal cell of the motor cortex to an incremental series of current
pulses (0.2 nA steps). An intracellular recording with a K acetate
electrode is shown. B, In voltage-clamp mode at a
holding potential of 55 mV, current traces from a
layer II/III pyramidal cell of a sham-treated, wild-type (+/+) mouse in
response to a 1.5 min bath application of 6.25, 12.5, and 25 µM NMDA separated by at least 10 min. This same cell
responded to 50 µM NMDA with a 4.5 nA inward current
(data not shown). C, NMDA-induced inward currents (at
NMDA concentrations of 6.25, 12.5, and 25 µM) in
sham-treated, wild-type mice (n = 3).
D, Current traces at a holding potential
of 55 mV from layer II/III pyramidal cells from wild-type (+/+) or
fosB mutant (/) mice in response to a 1.5 min bath
application of 25 µM NMDA. S, Sham;
E, two daily ECS treatments (animals were killed 24 hr
later). E, A summary of the results of experiments
measuring the NMDA-induced inward current (+/+; n = 7 for S; n = 6 for E;
/, n = 8 for S;
n = 6 for E). *, Statistically
significant difference between sham- and ECS-treated groups (Fisher LSD
protected t tests). F, A summary of
experiments in six separate slices (+/+, n = 4 for
S; n = 2 for E) in
which the NMDA (25 µM)-induced inward currents were
measured both before and after bath application of tetrodotoxin
(TTX; 2 µM), which completely blocked the
spiking activity of each cell tested.
|
|
In these experiments, mice were studied 24 hr after the second of two
daily ECS, because it was at this time point that maximal differences
in seizure duration were observed between wild-type and mutant mice
(see Fig. 5, right). Such repeated treatment with ECS
decreased the NMDA-induced inward current 24 hr after the last ECS
treatment in wild-type mice [69%; t(11) = 3.36;
p < 0.01] (Fig.
6D,E). In contrast, repeated ECS
treatment did not alter the 25 µM NMDA-induced inward
currents in cells from mutant mice [t(13) = 1.13;
p > 0.05], although the baseline currents were lower
in sham-treated mutant mice than in sham-treated wild-type mice [78%
decrease; t(13) = 4.53; p < 0.001].
However, the failure of cells from mutant mice to show an adaptation to
repeated ECS did not represent a "floor" effect, because a higher
concentration of NMDA (50 µM), which elicited larger
currents-but still ones in the physiological range-in the mutants,
also revealed a lack of adaptation to repeated ECS in the mutant mice
[t(10) = 0.53; p < 0.05] (Fig.
6E). The NMDA-induced inward currents seemed to be
caused by the postsynaptic action of NMDA on the pyramidal cells,
because the fast sodium channel blocker TTX did not alter the
NMDA responses in six cells from both ECS- and sham-treated mice
[t(5) = 0.34; p > 0.05] (Fig.
6F).
fosB mutant mice have normal cytoarchitecture in the
frontal cortex
Targeted disruption of a gene can cause severe anatomical
abnormalities attributable to developmental effects of the disruption, which could lead to significant phenotypic abnormalities in the adult
animals (e.g., Xu et al., 1994). This is a particular concern in the
present study, because FosB/FosB-like proteins are expressed during
cortical development (Kaminska et al., 1995). Thus, we examined the
cytoarchitecture of superficial layers of the frontal cortex of
fosB mutant mice to assess the anatomical integrity of this
brain region. We first examined the calcium-binding protein calbindin
that is expressed in GABAergic, nonpyramidal neurons and in pyramidal
neurons within cerebral cortex (Kubota et al., 1994; Gabbott et al.,
1997; Kawaguchi and Kubota, 1997). Calbindin is particularly enriched
in superficial cortical layers, where it can be used
immunohistochemically to stain both cell bodies and a dense fiber
network (Celio, 1990; van Brederode et al., 1991). As shown in Figure
7, calbindin immunoreactivity showed a
normal distribution in the motor and medial prefrontal cortex of mutant
mice. The dorsomedial corner of the frontal cortex showed lower levels
of calbindin-positive cell bodies and fibers in wild-type and mutant
mice, although there was considerable individual variation in the size
of this calbindin-poor area in both types of mice. We also examined
NMDAR1, which is highly expressed in cortical pyramidal and other
neurons (see Monyer et al., 1992; Nakanishi, 1992). Again, superficial
layers showed apparently normal patterns of NMDAR1-immunoreactive
neurons in the motor area of the prefrontal cortex of mutant mice (Fig.
7). This is consistent with equivalent levels of NMDAR1 in the frontal
cortex of sham-treated wild-type and mutant mice as determined by
Western blotting (see Fig. 4C). Finally, Nissl staining
revealed apparently normal organization of cells in these brain regions
(Fig. 7). Although these analyses do not eliminate the possibility of a
subtle abnormality, they demonstrate that the various biochemical,
behavioral, and electrophysiological phenotypes in the fosB
mutant mice are not associated with a gross abnormality in the
cytoarchitecture of the superficial layers of cortex in these
animals.
View larger version (94K):
[in this window]
[in a new window]
|
Figure 7.
Cytoarchitecture of the frontal cortex.
Immunoreactivity of calbindin (CALB)
(top), immunoreactivity of NMDAR1 (R1)
(middle; showing the motor cortex), and Nissl staining
(bottom; showing the dorsolateral quadrant of the
frontal cortex) are shown. Scale bars: top,
bottom, 500 µm; middle, 250 µm. The
figure is representative of results obtained from analysis of four
wild-type littermates (+/+) and four fosB mutant mice
(/).
|
|
| |
DISCUSSION |
We showed in the present study, by use of a mutant mouse that
lacks the fosB gene, that the 45 kDa FRA and the chronic
FRAs induced by repeated ECS were FosB and novel isoforms of FosB, respectively, and that they complexed predominantly with JunD/JunB to
form the chronic AP-1 complex. We provided several lines of evidence
that a specific NMDA receptor subunit, which was upregulated by chronic
ECS, was a physiological target gene for this chronic AP-1 complex.
Altered expression of NMDA receptor subunits was associated with
altered electrophysiological responses of cortical pyramidal neurons to
NMDA, an effect that was lost in fosB mutant mice. Moreover,
tolerance developed to the production of motor seizures in rats and
mice, and the development of this tolerance was delayed in
fosB mutant mice.
Chronic FRAs are isoforms of FosB
The present study provides strong evidence that all of the chronic
FRAs induced by repeated motor seizures are encoded in the
fosB gene. Together with our previous finding that FosB
overexpressed in cell lines migrates at 35-37 kDa (J. S. Chen et
al., 1997), the present result establishes the 35-37 kDa FRAs induced
in the cortex by repeated motor seizures as isoforms of FosB.
FosB mutant mice also exhibit lack of induction of the
chronic FRAs in striatum in response to chronic cocaine administration
(Hiroi et al., 1997) and in hippocampus after kainate lesions
(Mandelzys et al., 1997). Although it has remained unclear whether
chronic FRAs induced by various chemical and electrical treatments are
identical, these observations indicate that diverse types of chemical
and electrical stimulation induce common transcription factors, FosB
isoforms, and, to a lesser extent, FosB in a region-specific manner in
brain.
fosB gene products are required for seizure-associated
reorganization of NMDA receptors
The present study provides several lines of evidence that the
NMDAR1 gene is a physiological target for fosB gene
products, presumably FosB. First, repeated ECS administration
upregulated levels of NMDAR1, an obligatory subunit of NMDA receptors,
in the frontal cortex of rats and wild-type mice, an effect that was
completely absent in fosB mutant mice. Second, upregulation of NMDAR1 and induction of FosB/FosB-like proteins were colocalized to the same subpopulations of neurons in the frontal cortex. Third, repeated ECS treatment increased AP-1 binding activity at the AP-1 site
encoded in the promoter of the NMDAR1 gene. Fourth, the NMDAR1 subunit
was upregulated at the mRNA level, consistent with the notion that
regulation of gene transcription is involved. Fifth, the absence of
fosB gene products alone was sufficient for the defective
upregulation of NMDAR1; other Fos family proteins were induced by a
single ECS equally in both the mutant and wild-type mice. These
findings suggest that FosB isoforms (and perhaps FosB), rather than
other Fos family member proteins, are responsible for the upregulation
of NMDAR1 by repeated ECS treatment. It should be pointed out, however,
that we do not eliminate the possibility that fosB gene
products act on other genes to bring about the upregulation of the
NMDAR1 gene.
fosB gene products are required for the normal
development of tolerance to motor seizures
The present study also demonstrates that tolerance develops with
respect to the duration of the motor seizure elicited by repeated ECS
administration in rodents. Such tolerance to ECS has been widely noted
in clinical practice during the use of repeated ECS for the treatment
of affective disorders such as depression (Fink, 1990). Although this
tolerance can complicate the clinical use of ECS (Fink, 1994), it also
could reflect neural adaptations related to its therapeutic efficacy.
Our results establish that this tolerance in part depends on
transcriptional regulation by fosB gene products. The
results also indicate that the acute induction of motor seizure by a
single ECS does not require the protein products of the fosB
gene but that its behavioral adaptation does.
It should be noted that tolerance to the duration of motor seizure was
not completely abolished in fosB mutant mice, because tolerance, equivalent to that seen in wild-type mice, eventually developed after a larger number of ECS treatments. This observation raises the possibility that, in the absence of fosB gene
products, other factors eventually substitute (see Hummler et al.,
1994). It is unlikely, however, that other Fos family member proteins played a compensatory role, because no upregulation was seen in these
proteins (see Fig. 1D). Alternatively, only an
initial phase of tolerance might require fosB gene products.
With respect to this possibility, it is interesting to note that such a
phase-specific involvement of a transcription factor (e.g., cAMP
response element binding protein) has been demonstrated in
Drosophila (DeZazzo and Tully, 1995), Aplysia
(Bailey et al., 1996), and mice (Bailey et al., 1996). If this were the
case here, tolerance to motor seizure would involve more than one
neuroadaptive process with distinct temporal properties. According to
this scheme, transcriptional regulation by fosB gene
products would be the basis of the rapid, initial reduction in seizure
duration.
The tolerance elicited by repeated administration of ECS is
qualitatively different from the consequences of repeated limbic seizures. Repeated subthreshold stimulation of limbic structures (e.g.,
amygdala and perforant path) leads to a gradual reduction in seizure
threshold and eventually to intense limbic seizures (i.e., kindling)
(McNamara, 1994). Given that kainate-induced seizures are associated
with induction of the 35-37 kDa FRAs (Sonnenberg et al., 1989; Morgan
and Curran, 1991b; Pennypacker et al., 1994), which are FosB
isoforms (Mandelzys et al., 1997), it would be interesting to study
NMDA receptor subunits and other potential target genes for FosB
within the context of limbic seizures (see Friedman et al., 1994).
fosB gene products are required for chronic ECS-induced
desensitization of NMDA responses
One major finding of the electrophysiological studies is that
repeated ECS result in desensitization of NMDA-induced inward currents
in neocortical layer II/III pyramidal cells of wild-type mice at the
time when behavioral tolerance developed. This reduction in
NMDA-induced inward currents is attenuated in fosB mutant
mice. This suggests that fosB gene products are essential
for the electrophysiological adaptation to reduce excessive
glutamatergic transmission during motor seizures.
Paradoxically, the higher basal levels of the NMDAR2B subunit in
fosB mutant mice were associated with electrophysiologically less effective NMDA receptors. By itself, a change in the ratio of
NMDAR2A versus NMDAR2B subunits combined with the NMDAR1 subunits would not be expected to alter NMDA responses, because the conductance and mean open time for heteromeric expression of NMDAR1/NMDAR2A and
NMDAR1/NMDAR2B combinations is similar (Stern et al., 1992). Rather, the NMDAR2B subunit, coexpressed with the NMDAR1 subunit, is
more sensitive to NMDA, glycine, and serine than is the NMDA2A subunit
(Hess et al., 1996) and is less sensitive to Mg2+
block and inhibition by Zn2+ (Stern et al., 1992; N. Chen et al., 1997). Nevertheless, upregulation of NMDAR2B in the
absence of a change in NMDAR2A could cause reduced inward currents to
NMDA by producing greater basal desensitization in the presence of a
given ambient glutamate concentration. This could explain the reduced
basal responsiveness to NMDA, as well as the lack of adaptation in NMDA
responsiveness after repeated ECS treatment, in fosB mutant
mice. Such a paradox between an increased number of receptors and
development of tolerance has been noted for another ionotropic
receptor, the nicotinic cholinergic receptor, after chronic nicotine
treatment (Marks et al., 1993).
Another important dissociation we observed is that constitutively
higher levels of NMDAR2B (see Fig. 4C) and initially smaller NMDA-evoked inward currents (Fig.
6B,C) in mutant mice were not paralleled by a change in the duration of motor seizure by the initial
ECS: a single, acute ECS induced the same duration of motor seizure in
wild-type and mutant mice (see Fig. 5, right, day 1). One
possibility is that NMDAR2B alone may not be sufficient for setting the
duration of motor seizure but may be sufficient for reducing
NMDA-induced inward currents.
It should be pointed out that the present study was not designed to
establish causal links among the neuronal (NMDAR1 upregulation), electrophysiological (inward current change), and behavioral (tolerance to motor seizures) phenotypes.
Role for FosB as a mediator of neural and
behavioral plasticity
The chronic FRAs have been shown to accumulate in a
region-specific manner in brain in response to several treatments,
including chronic seizures, chronic psychotropic drug treatments, and
kainate and other lesions (Hope et al., 1994a,b; Nye et al., 1995;
Pennypacker et al., 1995; Doucet et al., 1996; Hiroi and Graybiel,
1996; Moratalla et al., 1996; Nye and Nestler, 1996; Mandelzys et al.,
1997; Pich et al., 1997). Thus, accumulation of the chronic FRAs may be
a common response of the brain to chronic perturbation (Hiroi et al.,
1996). By establishing the identity of these proteins as novel FosB
isoforms, the results of the present study contribute to understanding
the general role these transcription factors play in mediating
long-term adaptations in the brain. By identifying a particular NMDA
receptor subunit as a putative target gene for these transcription
factors, the present study provides a model system to study the roles
of fosB gene products and NMDA receptor regulation in many
forms of neural and behavioral plasticity known to involve NMDA
receptor activation.
| |
FOOTNOTES |
Received Feb. 5, 1998; revised June 19, 1998; accepted June 23, 1998.
This work was supported by United States Public Health Service
Grants MH51399 to E.J.N. and HD18655 and NS28829 to M.E.G. and
by grants from the National Alliance for Research on Schizophrenia and
Depression and the Abraham Ribicoff Research Facilities, State of
Connecticut Department of Mental Health and Addiction Services. We
thank Drs. M. Iadarola, M. Gruda, and R. Bravo for their generous gifts
of antibodies and Dr. M. Picciotto for her valuable comments on this
manuscript.
Correspondence should be addressed to Dr. Eric J. Nestler, Department
of Psychiatry, Yale University School of Medicine, 34 Park Street, New
Haven, CT 06508.
Dr. Hiroi's present address: Laboratory of Molecular Psychobiology,
Departments of Psychiatry and Neuroscience, Albert Einstein College of
Medicine, Bronx, NY 10461.
| |
REFERENCES |
-
Aghajanian GK,
Rasmussen K
(1989)
Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices.
Synapse
3:331-338[ISI][Medline].
-
Bai G,
Kusiak JW
(1993)
Cloning and analysis of the 5' flanking sequence of the rat N-methyl-D-aspartate receptor 1 (NMDAR1) gene.
Biochim Biophys Acta
1152:197-200[Medline].
-
Bailey CH,
Bartsch D,
Kandel ER
(1996)
Toward a molecular definition of long-term memory storage.
Proc Natl Acad Sci USA
93:13445-13452[Abstract/Free Full Text].
-
Berhow MT,
Hiroi N,
Kobierski LA,
Hyman SE,
Nestler EJ
(1996)
Influence of cocaine on the JAK-STAT pathway in the mesolimbic dopamine system.
J Neurosci
16:8019-8026[Abstract/Free Full Text].
-
Bing G,
McMillian M,
Kim H,
Pennypacker K,
Feng Z,
Qi Q,
Kong L-Y,
Iadarola M,
Hong JS
(1996)
Long-term expression of the 35,000 mol. wt Fos-related antigen in rat brain after kainic acid treatment.
Neuroscience
73:1159-1174[ISI][Medline].
-
Bing G,
Wang W,
Qi Q,
Feng Z,
Hudson P,
Jin L,
Zhang W,
Bing R,
Hong J-S
(1997)
Long-term expression of Fos-related antigen and transient expression of FosB associated with seizures in the rat hippocampus and striatum.
J Neurochem
68:272-279[Medline].
-
Brose N,
Huntley GW,
Stern-Bach Y,
Sharma G,
Morrison JH,
Heinemann SF
(1994)
Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex.
J Biol Chem
269:16780-16784[Abstract/Free Full Text].
-
Brown JR,
Ye H,
Bronson RT,
Dikkes P,
Greenberg ME
(1996)
A defect in nurturing in mice lacking the immediate early gene fosB.
Cell
86:297-309[ISI][Medline].
-
Celio MR
(1990)
Calbindin D-28k and parvalbumin in the rat nervous system.
Neuroscience
35:375-475[ISI][Medline].
-
Chen JS,
Nye HE,
Kelz MB,
Hiroi N,
Nakabeppu Y,
Hope BT,
Nestler EJ
(1995)
Regulation of FosB and FosB-like proteins by electroconvulsive seizure and cocaine treatment.
Mol Pharmacol
48:880-889[Abstract].
-
Chen JS,
Kelz M,
Hope BT,
Nakabeppu Y,
Nestler EJ
(1997)
Chronic Fos-related antigens: stable variants of FosB induced in brain by chronic treatments.
J Neurosci
17:4933-4941[Abstract/Free Full Text].
-
Chen N,
Moshaver A,
Raymond LA
(1997)
Differential sensitivity of recombinant N-methyl-D-aspartate receptor subtypes to zinc inhibition.
Mol Pharmacol
51:1015-1023[Abstract/Free Full Text].
-
Clineschmidt B,
Martin GE,
Bunting PR
(1982)
Anticonvulsant activity of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cylcohepten-5,10-imine (MK-801). A substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties.
Drug Dev Res
2:123-134.
-
Connors BW,
Gutnick MJ
(1990)
Intrinsic firing patterns of diverse neocortical neurons.
Trends Neurosci
13:99-104[ISI][Medline].
-
Devanand DP,
Dwork AJ,
Hutchison ER,
Bowlig TG,
Sackeim HA
(1994)
Does ECT alter brain structure?
Am J Psychiatry
151:957-970[Abstract/Free Full Text].
-
DeZazzo J,
Tully T
(1995)
Dissociation of memory formation: from behavioral pharmacology to molecular genetics.
Trends Neurosci
18:212-218[ISI][Medline].
-
Dobrazanski P,
Noguchi T,
Kovary K,
Rizzo CA,
Lazo PS,
Bravo R
(1991)
Both products of the fosB gene, FosB and its short form, FosB/SF, are transcriptional activators in fibroblasts.
Mol Cell Biol
11:5470-5478[Abstract/Free Full Text].
-
Doucet JP,
Nakabeppu Y,
Bedard PJ,
Hope BT,
Nestler EJ,
Jasmin BJ,
Chen JS,
Iadarola MJ,
St-Jean M,
Wigle N,
Blanchet P,
Grondin R,
Robertson GS
(1996)
Chronic alterations in dopaminergic neurotransmission produces a persistent elevation of deltaFosB-like protein(s) in both the rodent and primate striatum.
Eur J Neurosci
8:365-385[ISI][Medline].
-
Ehlers MD,
Tingley WG,
Huganir RL
(1995)
Regulated subcellular distribution of the NR1 subunit of the NMDA receptor.
Science
2
Top
Abstract
Introduction
