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Vol. 16, No. 18, pp. 2350-2364, September 15, 2002
1 Laboratoire du Cytosquelette, INSERM U366; 2 Atelier de Transgenèse, Département Réponse et Dynamique Cellulaire, CEA-Grenoble, 38054 Grenoble, France; 3 Centre Européen des Sciences du Goût, CNRS, 21000 Dijon, France; 4 Department of Cell Biology, Institute of Biomembranes, University Medical Centre Utrecht, 3584 CX Utrecht, The Netherlands
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ABSTRACT |
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 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
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Neurons contain abundant subsets of highly stable microtubules that
resist depolymerizing conditions such as exposure to the cold. Stable
microtubules are thought to be essential for neuronal development,
maintenance, and function. Previous work has indicated an important
role of the microtubule-associated protein STOP in the induction of
microtubule cold stability. Here, we developed STOP null mice. These
mice were devoid of cold-stable microtubules. In contrast to our
expectations, STOP
/ mice had no detectable defects in brain anatomy
but showed synaptic defects, with depleted synaptic vesicle pools and
impaired synaptic plasticity, associated with severe behavioral
disorders. A survey of the effects of psychotropic drugs on STOP/
mice behavior showed a remarkable and specific effect of long-term
administration of neuroleptics in alleviating these disorders. This
study demonstrates that STOP is a major factor responsible for the
intriguing stability properties of neuronal microtubules and is
important for synaptic plasticity. Additionally, STOP/ mice may
yield a pertinent model for study of neuroleptics in illnesses such as
schizophrenia, currently thought to result from synaptic defects.
[Key Words: Microtubule; neuron; STOP; knock-out; synaptic plasticity; neuroleptic]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Neurons contain abundant subpopulations of stable microtubules that
resist depolymerizing conditions such as exposure to the cold and to
depolymerizing drugs. It is thought that stable
microtubules are important for the generation and maintenance of
neuronal morphology and function (Baas and Heidemann 1986
; Guillaud et
al. 1998). Neuronal microtubules are stabilized by
microtubule-associated proteins (MAPs). Among these MAPs, only a few
exhibit the capacity to reconstitute the microtubule cold and drug
stability observed in normal neurons (Houseweart and Cleveland 1999).
Cold stability actually represents an extreme state of microtubule
stabilization, and it has been argued that the functional importance of
the various MAPs found in neurons could be related to their ability to
induce microtubule cold stability (Houseweart and Cleveland 1999).
Thus, eliminating MAPs such as tau, which cannot induce microtubule cold stability, has little effect in mice (Harada et al. 1994), whereas
suppression of MAPs with cold-stabilizing activity such as BPAG1 (Yang
et al. 1999) or doublecortin (Francis et al. 1999) has major
detrimental effects on the maintenance or development of the nervous
system. Although several MAPs can induce microtubule cold stability,
previous works have indicated a major contribution of STOP proteins.
STOP proteins are calmodulin-binding and calmodulin-regulated MAPs.
STOPs are encoded by a single gene (Denarier et al. 1998a) but have
several tissue- or developmental-specific variants. Neurons contain the
major STOP variants, N-STOP and E-STOP (Bosc et al. 1996; Guillaud et
al. 1998). Other cell types such as fibroblasts contain less abundant
STOP variants such as F-STOP (Denarier et al. 1998b). Acute STOP
inhibition by injection of specific blocking antibodies suppresses
microtubule cold stability in both neuronal and nonneuronal cultured
cells (Denarier et al. 1998b; Guillaud et al. 1998). Furthermore, in
cultured neuronal cells, STOP inhibition impairs neuronal
differentiation (Guillaud et al. 1998).
To evaluate the role of STOP directly, we used gene targeting and
derived STOP null mice. The lack of STOP induced a dramatic loss of
microtubule cold stability in both neuronal and nonneuronal microtubules. Although stable microtubules are thought to provide a key
structural support for neuronal growth, adult STOP/ mice display
apparently normal brain histology. However, in the absence of STOP,
synaptic abnormalities were observed with depleted synaptic vesicle
pools and defects in both short- and long-term plasticity. These
anomalies were associated with severe behavioral disorders that were
specifically alleviated by long-term administration of neuroleptics.
These results demonstrate that STOP is a major factor responsible for
microtubule cold-stability and that it is important for synaptic
function. Additionally, STOP/ mice may provide novel opportunities
for study of synaptic plasticity and response to neuroleptic drugs, in
the context of recent models in which synaptic defects are a causal
mechanism for neuroleptic-sensitive diseases such as schizophrenia
(Mirnics et al. 2001).
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Results |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Generation of STOP-deficient mice
STOP-deficient mice (STOP/ mice) were derived by gene targeting.
Exon 1 was replaced with a cassette consisting of the neo gene
that conferred positive selection and a LacZ reporter gene (Fig. 1A,a). Mating between heterozygous
mice yielded the expected Mendelian ratio of mice bearing the STOP
targeted gene. STOP/ mice were viable, with no obvious anatomical
defects. To verify that STOP proteins were absent in homozygous
mutants, we compared STOP/ mice to wild-type mice for STOP content.
Immunoblot analysis of brain extracts from adult wild-type mice showed
the presence of STOP proteins (E-STOP and N-STOP), whereas no signal
was observed with corresponding extracts from STOP/ mice (Fig.
1A,b). Immunological detection of STOP in brain sections from wild-type
mice showed widespread labeling of neuronal tissues, whereas sections
from STOP/ mice remained unstained (Fig. 1A,c).
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Microtubule stability in STOP/ mice
To assess the consequence of STOP deficiency on microtubule
stability, we compared microtubule cold stability in cells from wild-type and STOP/ mouse embryos. In neuronal cells from wild-type mouse embryos, microtubules resisted exposure to the cold (45 min,
0°C). In contrast, in cells from STOP/ embryos, all neuronal microtubules depolymerized during cold exposure (Fig. 1B,a). Glial cells from wild-type mouse embryos also contained abundant
subpopulations of cold-stable cytoplasmic microtubules that were
completely absent in the corresponding cells from STOP/ mice (Fig.
1B,b). A similar loss of microtubule cold stability was observed in
fibroblasts from STOP/ mice (not shown). These results show a
widespread multisystem loss of microtubule cold stability in
STOP-deficient mice.
Brain anatomy of STOP/ mice
Since cold-stable microtubules are believed to be important for
neuronal growth (Baas and Heidemann 1986), we expected the brain
anatomy of STOP/ mice to be abnormal. However, systematic examination of the brain by light microscopy did not show obvious structural changes in STOP/ mice nor evidenced neuronal
degeneration (Fig. 1A,c). In wild-type mice, immunolabeling for STOP
was most intense in the olfactory bulb glomeruli, hippocampus, and
cerebellum (Fig. 2A). Examination of cell
layers in these brain regions showed apparently normal organization in
STOP/ mice as illustrated in Figure 2B. Taking advantage of the
reporter LacZ gene included in the targeting vector, we did
X-gal staining on brain sections. Similar patterns of staining were
observed in heterozygous and STOP/ mice, showing that the cells
with high STOP promoter activity were still present in STOP/ mice
(Fig. 2C). This argues against major developmental or degeneration
problems associated with the absence of STOP.
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Olfactory bulb patterning was normal in STOP-deficient mice, both
morphologically (Fig. 2B) and as assessed by quantification of the
number of glomeruli per histological section (wild-type mice: 55 ± 3
and STOP/ mice: 58 ± 4, mean ± s.e.m., n = 2 mice in each
group) and by measurements of the glomerulus diameter (wild-type mice:
106 ± 4 µm and STOP/ mice: 103 ± 2 µm, mean ± s.e.m.,
n = 2 mice in each group). Flattened sections of the somatosensory
cortex showed normal organization of the barrel field in STOP/ mice
as revealed by staining for cytochrome oxidase (Fig. 2D). The detailed
analysis of the posteromedial barrel subfield (corresponding to
the whisker array) indicates a normal number of barrels (five
rows and 32 barrels) in STOP/ mice.
Lastly, we examined axonal and dendritic organization of neurons
expressing a high level of STOP. Staining for zinc revealed a normal
organization of the hippocampal mossy fibers in the stratum lucidum of
CA3 region without sprouting in dentate gyrus (Fig. 2E).
Somatodendritic configuration of CA1 pyramidal cells was also normal as
revealed by intracellular labeling (Fig. 2G). Calbindin immunostaining
indicated that the typical dendritic arborization of Purkinje cells was
also preserved in STOP / mice (Fig. 2F). Together these results
indicate that there is no disruption of cellular layering, sensory
patterning, or axonal and dendritic organization in the brain of
STOP/ mice.
Electron microscopy analysis of synapses
To assess possible consequences of STOP deficiency at the
ultrastructural level, we carried out an electron microscopy (EM) examination of the CA1 region of the hippocampus. The hippocampal ultrastructure (not shown) and organization of synapses (Fig. 3A,B) in the wild-type and the STOP/
mice did not show any major difference. However, in the STOP/
Schaffer collateral terminals, the number of synaptic vesicles seemed
smaller than in the wild-type (Fig. 3A,B). This observation was
confirmed by quantifying the surface density of the synaptic vesicles
in the presynaptic nerve terminals, in the stratum radiatum region of
CA1. A striking twofold decrease in the synaptic vesicle density was
observed in STOP/ mice compared to wild-type mice (Fig. 3E). In the
wild-type hippocampus, cryoimmuno-EM studies of STOP showed a
widespread, cytoplasmic distribution.STOP labeling was
observed in both the dendritic spines in close proximity to
postsynaptic densities (Fig. 3C) and in axons, where it
seemed slightly more abundant (Fig. 3D).
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Biochemical analysis of synaptosomes
We then investigated whether the depletion of the vesicle pool
observed by EM in the active zone of synapses reflected a deficit in
the import of synaptic proteins. In this case, we expected to see a
perturbation of the protein composition of synaptosomes. However, an
analysis of a battery of known synaptic proteins in synaptosomal
fractions from wild-type and STOP/ mice did not show detectable
differences between the two groups (Fig.
4). Thus, with this method, there was no
sign of defects in protein import in STOP/ mice. In the same study,
our assay of STOP showed the presence of the protein in synaptosomes,
with a distinct enrichment in a fraction containing proteins associated
with insoluble synaptic structures (Fig. 4B).
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Synaptic transmission in STOP/ mice
The vesicle depletion observed in STOP/ mice prompted us to
examine synaptic function. We first assayed synaptic transmission in
the CA1 region of the hippocampus, where STOP expression is intense
(Fig. 2A). To assess basic synaptic transmission, we examined the
input-output relationship between the fiber excitability and the
synaptic strength. The amplitude of fiber volley from Schaffer collaterals was compared to the slope of field excitatory postsynaptic potentials (EPSPs) in the CA1 region of the hippocampus. The experiment was run at different electrical stimulus intensities. Input-output curves were qualitatively similar in STOP/ and wild-type mice (Fig.
5A,a,b). For quantitative
analysis we compared the slopes of these input-output curves between
six wild-type and six STOP/ mice. No significant difference was
observed (Fig. 5A,c).
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We then examined the quantal amplitude of the synaptic response in the
mutant mice by using whole-cell patch clamp techniques. The quantal
amplitude of AMPA-mediated synaptic response was estimated by measuring
strontium-induced asynchronous excitatory postsynaptic currents
(aEPSCs), in nine anatomically identified CA1 pyramidal cells from
STOP/ or wild-type mice. No difference in the amplitude of aEPSCs
was observed (Fig. 5F). These results indicate normal basic synaptic
transmission in STOP/ mice.
We then examined synaptic plasticity. In synaptic plasticity
experiments, the synaptic response to standard stimuli is measured by
the slope of EPSP. Baseline values of the slopes are determined using
repetitive stimulation at low frequencies (0.03-0.1 Hz). At time zero,
a conditioning stimulation protocol is applied. Synaptic adaptation is
evidenced by a stable deviation of EPSP slope values from the baseline
values. A high-frequency conditioning protocol (100 Hz,
duration 1 sec, tetanus) applied at the Schaffer collateral-CA1 pyramidal cell synapses produced a stable increase in
EPSP slopes in slices from wild-type mice (Fig. 5B,a), demonstrating synaptic potentiation in these mice. This potentiation persisted over
30 min, and such a persistence defines long-term potentiation (LTP).
The same protocol produced potentiation of synaptic transmission reduced by threefold in normalized experiments using slices from STOP/ mice (Fig. 5B,b,c).
We then examined long-term depression (LTD) at the same Schaffer
collateral-CA1 pyramidal cell synapses. We used the classical low-frequency conditioning protocol (LFS, 1 Hz, 900 pulses). Slices from STOP/ mice showed a significant decrease in the amplitude of
LTD compared to wild-type mice (Fig. 5C). Thus, both LTP and LTD were
impaired in STOP/ mice. LTP and LTD depend crucially on NMDA
receptor (for review, see Bear and Malenka 1994), and a possible basis
of this perturbation is an altered receptor activity in STOP/ mice.
However, the NMDA receptor activation during tetanus (Fig. 5D) and the
basal NMDA receptor activity, measured as the ratio of NMDA/AMPA-evoked
postsynaptic currents in whole-cell patch clamp recordings (Fig. 5E),
were similar in wild-type and STOP/ mice.
As shown in Figure 5B, synaptic potentiation in STOP/ mice was
altered during the first minutes following tetanus, as at later
timepoints. This prompted us to test whether short-term plasticity was
impaired at the Schaffer collateral-CA1 pyramidal cell synapses. We
investigated posttetanic potentiation (PTP) and paired pulse
facilitation (PPF). Like LTP, PTP is a form of synaptic potentiation
following a tetanus (100 Hz, duration 1 sec, tetanus). However, PTP
persists only for minutes following tetanus and, unlike LTP, is not
blocked by NMDA receptor antagonists. PPF is another form of short-term
synaptic plasticity observed when synapses are stimulated with paired
stimuli. PPF is defined as an increase in the synaptic response
following the second stimulus. PTP was reduced in STOP/ mice (Fig.
5G). In contrast, PPF was unaffected over a large range of decreasing
extracellular calcium concentrations in STOP/ mice (Fig. 5H; data
not shown).
We next examined synaptic plasticity at the mossy fiber-CA3 pyramidal
cell synapses. No significant differences in either LTP or PPF between
STOP/ and wild-type mice were observed (data not shown). To further
examine short-term plasticity, mossy fibers were stimulated using
increasing frequencies from 0.033 to 1 Hz. This protocol normally
induces a large, but transient, increase in the amplitude of mossy
fiber EPSP, a phenomenon called frequency facilitation. The amplitude
of frequency facilitation was significantly decreased in mutant mice
compared to wild-type mice (Fig. 5I).
Taken together these results show that several distinct forms of
long-term and short-term plasticity are affected in different regions of the hippocampus, in STOP/ mice.
Behavioral study of STOP-deficient mice
Although STOP/ mice were fully viable, they consistently
exhibited atypical behaviors. Phases of intense activity, apparently without goal orientation, accompanied by frequent random shifts between
activities were common. Occasionally, the mice displayed crisis,
lasting over 20 min, during which the animals continuously circled the
cage or demonstrated burrowing motion. The same mice also went through
periods of freezing behavior, during which they remained immobile and
were unreactive to their environment. Such crises did not mimic
epilepsy-like events and were never observed in wild-type mice. We used
video recording for quantitative assessment of mouse behavior. The time
spent by the mice feeding, sleeping, grooming, walking, or remaining
still while awake was measured over a 3-h period during the
night (Fig. 6A). Compared to wild-type mice, STOP/ mice spent more time walking or remaining still while
awake, with a decrease in the time spent feeding and sleeping (Fig.
6A,a). The mutant mice also displayed increased shifting between
behavioral states, mainly due to a significantly larger number of
periods of walking and stillness (Fig. 6A,b). State shifts in STOP/
mice often broke characteristic sequences of activity. For instance, in
wild-type mice, 71% of the sleeping phases were preceded by a phase of
grooming. The corresponding frequency was 47% in STOP/ mice (Fig.
6A,c), when the expected value for random activities was 35%. These
quantitative results substantiated the global impression of purposeless
and disorganized activity observed in STOP/ mice.
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To evaluate the major classes of behavioral tasks (Crawley 1999), we
applied a series of standard behavioral tests. Anxiety-like behavior
was evaluated using the light-dark crossing task (Misslin et al. 1990)
in which the animals are allowed to choose between a dark and an
adjacent bright compartment. Although mice prefer the dark compartment,
they normally exhibit brief incursions into the bright compartment.
Anxiety-like behavior is manifested by a decrease in the time spent in
the bright compartment and in the number of transitions between the two
boxes. Compared to wild-type mice, STOP/ mice showed dramatic signs
of anxiety-like behavior, spending virtually all of the time in the
dark compartment with very few transitions between the light and dark
compartments (Fig. 6B). To assess short-term memory and learning in
wild-type and STOP/ mice, we used an object recognition task
(Meziane et al. 1998). In this test, the animals were challenged to
discriminate between a familiar object and a novel object. Wild-type
mice (n = 6) performed the test successfully, whereas STOP/ mice
(n = 8) were unable to perform the test (data not shown). These mice showed no apparent interest in the objects, suggesting perturbed interactions with their physical environment. To assess social behavior, we monitored both social investigation and intermale aggression, using the resident-intruder test (Nelson et al. 1995; Mohn
et al. 1999). Both active social investigation and aggressive encounters were dramatically reduced in STOP/ mice compared to
controls (Fig. 6C), indicating severe social withdrawal in mutant mice.
Postnatal death in the progeny from STOP/ female mice
Progeny from heterozygous males and females are viable and develop
normally. However, pups born from heterozygous males crossed with
STOP/ females died in the perinatal period within 24-48 h after
birth, regardless of their genotype. Among 161 pups from 20 STOP/
females, none survived the perinatal period. The dead pups were never
cannibalized. When cross-fostered to wild-type mothers, pups from
STOP/ mothers were raised to weaning. Thus, pup death was directly
related to the mother's genotype. To determine the cause of death, we
first examined obvious explanations such as suckling defects due to a
lack of olfactory cues in the nipples of STOP/ females. When pups
from STOP/ mice were left with their mother but repetitively placed
in nursing position by human intervention, all of the pups showed
nipple searching and attachment behavior. The normal expression of this
guided behavior suggests that the STOP/ females emit the olfactory
cues necessary for suckling. In addition, in these conditions, milk
could be observed in the pups' stomachs. Thus, pup death was not due
to lactation defects in STOP/ females.
The most likely possibility then was that pup death was due to an
abnormal behavior of STOP/ mothers. Indeed, STOP/ females, whether from BALBc/129 SvPas or pure 129 SvPas backgrounds, showed severe defects in their nurturing behavior. Normal nurturing behavior in mice includes building a nest and retrieving pups. Such behavior was
uniformly observed in both wild-type (Fig. 6D,a) and heterozygous mice
(data not shown). Although STOP/ mothers were never aggressive toward the pups, they showed a consistent defect in maternal behavior (Fig. 6D,a). The nurturing behavior of STOP/ mothers did not improve with multiple pregnancies. Retrieving defects could be due to a
failure of the STOP/ mothers to recognize their progeny. The major
sensory cue in this behavior is thought to be olfaction (Gandelman et
al. 1971); however, STOP/ females showed normal behavior in the
hidden food olfactory test (data not shown) and, when placed close to
their progeny, obviously sniffed the pups. To test whether the
nurturing deficiency observed in STOP/ mice was related to the
hormonal status, nurturing behavior was examined in virgin females and
in males, using standard tests of pup retrieval (Brown et al. 1996).
Pup retrieval was significantly impaired in such female and male
STOP/ mice (Fig. 6D,b). Thus, the nurturing defect observed in
STOP/ mice was independent of obvious organic defects and of the
hormonal status.
Effects of anxiolytics and neuroleptics on STOP/ mice behavior
The synaptic defects and the multiple behavioral disorders observed
in STOP-deficient mice were reminiscent of current models of
schizophrenia (Mohn et al. 1999; Mirnics et al. 2001). This prompted us
to test the action of psychotropic drugs on STOP/ mice behavior.
Because maternal behavior is highly conserved among mammals (Fleming
1989), it provided a stringent criterion to evaluate drug influence in
a complex deficit. In pilot experiments (data not shown), STOP/
mice were treated with different dosages of either anxiolytic
(diazepam) or neuroleptics (chlorpromazine, haloperidol, or clozapine)
using a variety of schedules. Both types of drugs administered at the
time of or shortly following delivery were apparently ineffective in
improving nurturing behavior in STOP/ mothers. When mice were
treated with various drugs beginning at least 6 d prepartum there was a
positive effect, although incomplete, of neuroleptics on nest building,
maternal care, and pup retrieving in STOP/ mice, with best results
yielded by a combination of chlorpromazine and haloperidol. For
quantitative assessment of treatment effects, wild-type and STOP/
mice were subjected to the test of pup retrieval, following various
drug treatments. The severe impairment in pup retrieval observed in untreated STOP/ mothers was only slightly and not significantly improved by diazepam administration (Fig.
7A,a). In contrast, both STOP/ mothers
treated with neuroleptics and wild-type mothers succeeded in the pup
retrieval test (Fig. 7A,a,b). However, no pup survival was observed in
either treated or untreated groups of STOP/ females. These results
indicate a limited but specific beneficial effect of short-term
administration of neuroleptics on behavior in STOP/ mice.
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We then tested whether a longer neuroleptic treatment could induce pup
survival in STOP/ mothers. Seven STOP/ and seven wild-type
females were subjected to daily administration of a combination of
chlorpromazine and haloperidol, for 4 mo, starting at weaning. This was
maintained through subsequent pregnancy, delivery, and the postpartum
period. The seven wild-type mice showed normal maternal behavior and
all of their pups survived (Fig. 7B). Remarkably, in four of the seven
STOP/ females subjected to this long-term regime, nurturing
improvement was sufficient to permit pup survival with ratios of
surviving pups to newborns of 3/11, 4/8, 2/4, and 1/5 for the four
mice. The occurrence of pup survival in long-term neuroleptic-treated
STOP/ mice was striking because, in the absence of such treatment,
pup survival never occurs among the progeny from STOP/ mothers
(Fig. 7B). These results indicate a remarkable capacity of long-term
neuroleptic administration in reestablishing maternal abilities
compatible with pup survival in STOP/ mice.
We next tested whether neuroleptics' effects on behavior were
accompanied by modifications of synaptic response. On the input-output curves of field EPSPs, we found no change in basic synaptic
transmission in the CA1 region of the hippocampus following long-term
neuroleptic treatment whether in wild-type or STOP/ mice (data not
shown). In LTP experiments (Fig. 7C), there was still a significant
difference between wild-type and STOP/ mice at 30-40 min following
long-term neuroleptic administration, but potentiation was apparently
improved at early times following tetanus in STOP/ mice. Based on
these results, we tested the effect of long-term neuroleptic treatments on PTP. Indeed, PTP experiments showed an apparent increase in the
synaptic response in STOP/ mice following neuroleptic
administration (Fig. 7C).
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Microtubules assembled from pure tubulin in vitro are dynamic
structures that exhibit both treadmilling behavior and spontaneous length fluctuations (Margolis and Wilson 1978; Mitchison and Kirshner 1984). Such dynamic microtubules are strongly sensitive to
modifications in their physicochemical environment. For instance,
microtubule exposure to the cold or to depolymerizing drugs induces
rapid polymer disassembly. The intrinsic instability of microtubules seems to be intimately related to many of their basic functions in
cells (Desai and Mitchison 1997). However, stable polymers resistant to
exposure to the cold and to depolymerizing drugs are also encountered
among living cells (Baas et al. 1994; Lieuvin et al. 1994). For
instance, in mature neurons virtually all microtubules are cold-stable.
The origin of such microtubule stability has been a long-standing
enigma. The marked reduction of cold-stable microtubules observed in
STOP/ mice establishes a major role for STOP proteins in
microtubule cold stability.
It has long been thought that cold-stable, nondynamic microtubules in
neurons were required to sustain neurite extensions using a microtubule
cytoskeleton whose maintenance did not require continuous energy
consumption (Baas and Heidemann 1986; Guillaud et al. 1998).
Remarkably, in the present study, we demonstrate that mice completely
devoid of cold-stable microtubules fail to display any obvious
disturbance of neuronal cell morphology, differentiation, or survival.
The absence of cold-stable polymers seemed to principally affect
synaptic transmission.
Because synaptic plasticity involves morphogenetic events at synapses
and spines, and vesicle trafficking, it is believed that microtubules
and microtubule effectors are important for this aspect of synaptic
transmission (for review, see van Rossum and Hanisch 1999). In the
present study we found a twofold reduction of the synaptic vesicle pool
in CA1 hippocampal nerve terminals. Our biochemical data argue against
a major perturbation of synaptic proteins import from the
soma through microtubule-dependent vesicle transport (for review, see
Goldstein and Yang 2000). In the absence of major defects in protein
import, the size of the synaptic vesicle pool may depend on the
kinetics of vesicle recycling from the apical membrane and of vesicle
budding from the endoplasmic reticulum (for review, see Calakos and
Scheller 1996). STOP deficiency may perturb synaptic vesicle
turnover by affecting microtubule dynamics or microtubule interaction
with motors.
The observed reduction in the size of vesicle pools may account for the
perturbations of hippocampal CA1 PTP and mossy fiber frequency
facilitation in STOP-deficient mice, because both forms of synaptic
plasticity involve presynaptic vesicle trafficking (Salin et al. 1996;
Thomson 2000). In contrast, PPF, which is unaffected in STOP/ mice,
only involves ready-to-go vesicles (Thomson 2000). Both microscopy and
biochemical data indicate the presence of STOP in synapses. The
deleterious effect of STOP deficiency on LTP and LTD in the CA1
synapses of the hippocampus indicates that STOP has functions in the
postsynaptic compartment. Both CA1 LTP and CA1 LTD involve
Ca++- and calmodulin-dependent cascades in the postsynaptic
compartment (for review, see Soderling and Derkach 2000). STOP proteins
are unique Ca++-calmodulin-binding proteins (Bosc et al.
2001) and could be involved in these cascades. In contrast, mossy fiber
LTP, which is normal in STOP/ mice, is independent of postsynaptic
Ca++-dependent cascades (Mellor and Nicoll 2001).
The synaptic defects observed in STOP/ mice offer an obvious basis
for the behavioral disorders exhibited by these mice. STOP deficiency
affects a wide variety of basic behavioral tasks, including
disorganized activity, nurturing defects, anxiety-related behavior,
inability to perform object recognition tests, and social withdrawal,
and consequently cannot be attributed either to limited or defined
brain structures or to a single neurotransmitter pathway. For example,
the different aspects of maternal behavior that are perturbed in
STOP/ mice depend on adaptive neuronal responses in different brain
regions such as olfactory bulb and preoptic area of hypothalamus (Brown
et al. 1996; Numan and Sheehan 1997). The apparently nonfocal origin of
the behavioral deficiencies observed in STOP/ mice suggests that
the synaptic defects that we observed in the hippocampus are widespread
in the brain.
The pattern of synaptic defects in STOP-deficient mice is such that the
mice can live but exhibit multiple behavioral disorders that can be
modulated by long-term treatment with neuroleptics. These drugs are
major antipsychotic agents principally used in schizophrenia. There are
still important pending questions regarding the mechanism of action and
use of neuroleptics. For instance, there is an intriguing dissociation
between the rapid blocking effect of neuroleptics on dopaminergic
receptors and the apparent delayed effects of the drugs on symptoms
(Kandel et al. 2000). The behavioral disorders observed in
STOP-deficient mice are to our knowledge unique in showing a delayed
sensitivity to neuroleptic treatment mimicking that observed in
schizophrenia. Neuroleptics are principally known as dopamine receptor
blockers (Seeman et al. 1976) but apparently act on disorders affecting
other neurotransmitter pathways (Mohn et al. 1999; Leveque et al.
2000). Our data also indicate that some synaptic defects can be
alleviated by neuroleptic treatment. This result is of particular
interest in view of recent models in which schizophrenia arises from
synaptic disorders (Harrison 1997; Mirnics et al. 2001). STOP/ mice
thus appear to be an interesting model to investigate neuroleptic
effects in animals showing synaptic defects associated with severe
behavioral disorders. Interestingly, the human STOP gene,
located at position 11q14 (GenBank accession no. AP000588), lies within
a region which has been linked to major mental diseases including
schizoid disorders (Holland and Gosden 1990; St. Clair et al. 1990;
Brzustowicz et al. 2000).
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Materials and methods |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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STOP targeting construct and generation of STOP/ mice
We isolated genomic clone DNA containing the STOP locus from a
mouse strain 129 SvPas genomic DNA library (Denarier et al. 1998a). As
all characterized STOP cDNAs contain exon 1, we targeted exon 1 to
suppress STOP proteins. In a 7.2-kb genomic clone (Fig. 1A), the exon 1 was replaced by LacZ gene under the control of the endogenous
STOP promoter followed by a phosphoglycerate kinase-driven neomycin
resistance (pGK-neo) cassette [plasmid pWH9, a generous gift
from E. Hirsch (Torino, Italy)]. We used either R1 ES cells kindly
provided by Andras Nagy (Nagy et al. 1993) or AT1 ES cells raised in
the transgenic facility of our department. The AT1 ES line was
established from (129 SvPas × 129 SvPas) F1, 3.5-d blastocysts. The
targeting vector was electroporated into R1 or AT1 ES cells. Transfectants were plated on mitomycin-treated neomycin-resistant primary embryonic fibroblasts as feeder layer and cultured in DMEM high
glucose (Invitrogen) supplemented with 15% fetal calf serum and 1000 U/mL leukemia inhibitory factor (Esgro, Chemicon). Forty-eight hours
after transfection, 250 µg/mL G-418 (Invitrogen) was added to the
medium. Selection with 2 µg/mL gancyclovir (Syntex) was performed
from day 4 to day 8 postelectroporation. Resistant colonies were picked
at day 10 and expanded for freezing and DNA analysis. Recombinant ES
clones were identified by Eco RV digestion of genomic DNA and
hybridization with Eco RV-Eco RI probe located immediately in the 5'
position of the homologous region of the targeting construct (Fig. 1A).
Two recombinant ES clones (R1-7 and AT1-76) were aggregated with OF1
morula to generate chimaeric mice (Nagy and Rossant 1993). Germline
transmitting mice from both R1-7 and AT1-76 ES clones were mated to
either BALBc or 129 SvPas mice to produce heterozygous mutant mice on
either mixed BALBc/129 SvPas or pure 129 SvPas backgrounds. All
wild-type mice and mice homozygous for the mutant STOP gene (STOP/)
were obtained from the intercross of heterozygous animals. BALBc and
129 SvPas animals were purchased from Charles River Laboratories.
Analysis of brain anatomy
For histological analysis, we perfused mice aged 8-12 wk with 4%
paraformaldehyde (PFA). Brains were postfixed 2 h in 4% PFA at 4°C.
Brain slices (50 µm) were stained with cresyl violet or assayed for
cytochrome oxidase (Liu et al. 1993). For analysis of olfactory bulb
glomeruli and barrel field, littermate mice were used. The examination
was done blind to genotype. For the olfactory bulb, sections throughout
the full length were examined. The number and the size of glomeruli
were assessed on 20 coronal sections (50 µm) located after the point
where the lateral ventricle first appeared. Measurements were not
corrected for shrinkage. For barrel field, the examination was done on
sections (80 µm) tangential to the barrels. For STOP staining, brains
were prepared as above and then cryoprotected in 20% sucrose in PBS.
Brain slices (20 µm) were successively incubated with 1%
H2O2 (15 min), 3% BSA (30 min), affinity-purified
STOP antibody 23C (100 µg/mL overnight; Guillaud et al. 1998) and
with anti-rabbit peroxydase-conjugated antibody, and then revealed with
ethylcarbazole (DAKO AEC). For mossy fiber staining, mice were perfused
first with 50 mM sodium sulfide followed by 4% PFA. Hippocampi were
then dissected, sliced (50 µm), and revealed according to Danscher
(1981). For 
-galactosidase activity staining, animals were perfused
with 0.2% glutaraldehyde and 2% formaldehyde in PBS. Brain slices (50 µm) were incubated in PBS containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride and 1 mg/mL X-Gal as
substrate for 3-5 h at 30°C.
Floating sections of cerebellum (50 µm) were successively incubated with calbindin antibody (generous gift of J. Baudier, Grenoble, France), 10 µg/mL overnight) and with anti-rabbit Cy3 antibody.
Electron microscopy analysis
The hippocampus was dissected out from transcardially fixed mice.
Perfusion was performed with 2% PFA and 0.2% glutaraldehyde in 0.1 M
phosphate-buffer at pH 6.9. For morphometric analyses, hippocampal
slices were postfixed in 2.5% glutaraldehyde in 0.1% cacodylate
buffer for 2 h, fixed with 1 % OsO4 for 1 h at 4°C, and
embedded in Epon. Matching areas from the hippocampus were identified
in semithin sections (500 nm), and for the comparison of the wild-type
and the mutant mice ultrathin sections (70 nm) were cut, prepared, and
viewed under a transmission electron microscope (JEOL 1200EX). To
determine the surface density of synaptic vesicles, cross-sections of
50 synapses per animal made of CA1 region beneath the pyramidal cell
layer were photographed randomly, and the numbers of synaptic vesicles
in each nerve preterminal were counted on the electron micrographs. For
this analysis, three wild-type and three STOP/ hippocampi were used.
For localization of STOP, ultrathin frozen sections were prepared from
hippocampus slices fixed in 2% PFA and 0.2% glutaraldehyde in 0.1 M
phosphate-buffer at pH 6.9. Briefly, blocks of hippocampus were
embedded in gelatin, and infiltrated in sucrose before freezing in
liquid nitrogen. Ultrathin sections were cut at 120°C, and the
retrieval of sections from the knife was achieved with a 1:1 mixture of 2% methylcellulose and 2.3 M sucrose according to the method of Liou et al. (1996)
