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The Journal of Neuroscience, August 15, 2002, 22(16):7177-7194
Impairment of L-type Ca2+ Channel-Dependent Forms of
Hippocampal Synaptic Plasticity in Mice Deficient in the Extracellular
Matrix Glycoprotein Tenascin-C
Matthias R.
Evers,
Benedikt
Salmen,
Olena
Bukalo,
Astrid
Rollenhagen,
Michael R.
Bösl,
Fabio
Morellini,
Udo
Bartsch,
Alexander
Dityatev, and
Melitta
Schachner
Zentrum für Molekulare Neurobiologie, Universität
Hamburg, D-20246 Hamburg, Germany
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ABSTRACT |
The extracellular matrix glycoprotein tenascin-C (TN-C) has been
suggested to play important functional roles during neural development,
axonal regeneration, and synaptic plasticity. We generated a
constitutively TN-C-deficient mouse mutant from embryonic stem cells
with a floxed tn-C allele, representing a standard for
future analysis of conditionally targeted mice. The gross morphology of
the CNS was not detectably affected, including no evidence for
perturbed nerve cell migration, abnormal oligodendrocyte distribution,
or defective myelination. Despite the apparent normal histology of the
hippocampus and normal performance in the water maze, theta-burst
stimulation (TBS) of Schaffer collaterals elicited reduced long-term
potentiation (LTP) in the CA1 region of TN-C-deficient mutants, as
compared with wild-type littermates. However, high-frequency stimulation evoked normal LTP not only in CA1, but also at mossy fiber-CA3 and medial and lateral perforant path-granule cell synapses in the dentate gyrus. Low-frequency stimulation failed to induce long-term depression in the CA1 region of TN-C-deficient animals. Recordings of TBS-induced LTP in the presence of nifedipine, an antagonist of L-type voltage-dependent Ca2+ channels
(VDCCs), did not affect LTP in TN-C-deficient mice, but reduced LTP in
wild-type mice to the levels seen in mutants. Furthermore, chemical
induction of a L-type VDCC-dependent LTP in the CA1 region by
application of the K+ channel blocker
tetraethylammonium resulted in impaired LTP in TN-C mutants. Thus,
reduction in L-type VDCC-mediated signaling appears to mediate the
deficits in certain forms of synaptic plasticity in constitutively
TN-C-deficient mice.
Key words:
tenascin-C; knock-out mutation; extracellular matrix
glycoprotein; gene targeting; hippocampus; long-term potentiation; long-term depression; CA1; CA3; dentate gyrus; water maze; TEA; L-type
voltage-dependent Ca2+ channels; VDCC; nifedipine
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INTRODUCTION |
Tenascin-C (TN-C) is a member of a
family of closely related extracellular matrix (ECM) glycoproteins that
includes TN-R, TN-X, TN-Y, and TN-W (Bristow et al., 1993 ;
Chiquet-Ehrismann et al., 1994; Erickson, 1994; Hagios et al., 1996;
Weber et al., 1998). Of the three tenascins that are detectable in the
nervous system, TN-C has been studied most extensively (Faissner and
Schachner, 1995; for review, see Bartsch, 1996). It is highly conserved
during evolution and most prominently expressed during development of the nervous system and several non-neuronal tissues (Jones and Jones,
2000). In the nervous system, it is downregulated after maturation but
persists in restricted areas that exhibit neuronal plasticity, as for
instance the hippocampus (Ferhat et al., 1996).
High levels of TN-C expression at critical stages of neuronal
development and regeneration and synaptic plasticity in the adult have
prompted several laboratories to investigate functional properties of
this ECM constituent in vitro. These studies have implicated
TN-C in diverse functions, including cell proliferation, migration,
axon guidance, and tissue development and repair. In vitro
studies with neuronal cells have revealed that diverse functions of the
molecule are localized to distinct domains and presumably also related
to different cellular receptors linked to varying intracellular signal
transduction pathways (Jones and Jones, 2000).
To investigate the functions of TN-C in vivo, two
TN-C-deficient mouse mutants have been generated independently, and
both have been shown to develop normally (Saga et al., 1992; Forsberg et al., 1996). However, more detailed studies on one of these mutants
(Saga et al., 1992) have revealed subtle abnormalities (Fukamauchi et
al., 1996; Kiernan et al., 1999; Mackie and Tucker, 1999). The
interpretation of whether these abnormalities are caused by the
complete lack of TN-C or a reduced expression of abnormal fragments
thereof has been a matter of debate (Mitrovic and Schachner, 1995;
Steindler et al., 1995; Settles et al., 1997).
Expression of TN-C is upregulated in the hippocampus after potentiation
of synaptic activity in vivo (Nakic et al., 1996, 1998). These observations indicate a function of TN-C in
synaptic plasticity, an aspect not yet investigated in TN-C-deficient
mice. With the long-term aim of elucidating such a role and relating it
to behavior, we have made a first step toward generating conditionally targeted mutants. To ascertain that future observations could be
compared with a constitutively deficient standard, we generated a
TN-C-deficient mouse starting from embryonic stem (ES) cells harboring
a floxed tn-C allele by excision of targeted sequences in vitro. In this study, we report on the use of these
constitutively TN-C-deficient mice to elucidate the contribution of
this ECM constituent to synaptic plasticity along the hippocampal
trisynaptic circuit. We demonstrate that TN-C plays an important role
in modulating L-type voltage-dependent
Ca2+ channel (VDCC)-mediated plasticity at
Schaffer collateral-CA1 synapses.
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MATERIALS AND METHODS |
Antibodies
The polyclonal antibodies pK7 and KAF 9-2 to TN-C and the
monoclonal antibodies 619 to TN-R and 513 to myelin-associated
glycoprotein (MAG) have been described (Morganti et al., 1990; Bartsch
et al., 1994). Polyclonal antibodies to glutamic acid decarboxylase
(GAD) were purchased from Chemicon International Inc. (Temecula, CA). A
monoclonal antibody to synaptophysin was purchased from Calbiochem (Schwalbach, Germany), and monoclonal antibodies PARV-19 to parvalbumin and G-A-5 to glial fibrillary acidic protein (GFAP) were purchased from
Sigma-Aldrich (Deisenhofen, Germany). For indirect immunofluorescence and Western blot analysis, Cy 3-conjugated antibodies and horseradish peroxidase-conjugated goat antibodies to rabbit or mouse IgG (all from
Dianova, Hamburg, Germany) were used, respectively.
Targeting vector construction
A 129/SvJ mouse genomic library in  Fix (Stratagene,
Amsterdam, The Netherlands) was screened with a TN-C cDNA probe derived from parts of exons 4 and 5 (corresponding to nucleotides 2064-2395 of
GenBank accession number D90343). A clone with an insert of
~18 kb covering exons 2-5 of the mouse tn-C gene was
isolated. The replacement-type targeting vector was prepared by
inserting a 1.78 kb cassette containing the phosphoglycerate kinase
promotor-driven neomycin resistance cassette amplified from the pKO
Scrambler V901 vector (Lexicon Genetics Inc., The Woodlands, TX)
flanked by a pair of PCR-generated loxP (Hoess et al., 1982) and FRT
(Andrews et al., 1985) sites into an EcoRV site located 117 bp upstream of exon 2, which contains the start codon (see Fig.
1a). A second PCR-generated loxP site adjacent to a
BamHI site was inserted into the DraI site
located 91 bp downstream of exon 2. The final targeting vector
contained 1.6 kb of homologous DNA upstream
(BglII-EcoRV fragment) of the above described
cassette and 5.9 kb downstream of the single loxP site
(DraI-BamHI fragment).
Gene targeting and generation of TN-C-deficient mouse mutants
Eighty micrograms of the NotI-linearized targeting
vector were electroporated into 107 R1 ES
cells (Nagy et al., 1993) using a double-pulse protocol (3 µF, 800 V
prepulse; 500 µF, 240 V pulse) with the Gene Pulser system (Bio-Rad,
München, Germany). G418-resistant ES cell clones harboring
homologous recombination events were identified by BamHI digestion of genomic DNA and Southern blot analysis using the external
probe 5'A (see Fig. 1a). Correctly targeted ES cell clones were electroporated under the same conditions as described above with
20 µg of the Cre expression plasmid pIC-CRE (Gu et al., 1994) allowing transient expression of Cre recombinase. G418-sensitive ES
cell colonies were screened for Cre-mediated excision events again by
Southern blot analysis using the probe 5'A on BamHI-digested DNA. Cre-recombined ES cell clones lacking the entire exon 2 were injected into C57BL/6J blastocysts. Male chimeras derived from two
independently targeted ES cell clones were mated with C57BL/6J females
to obtain germ line transmission. Heterozygous (tn-C +/ ) mice were intercrossed to yield homozygous TN-C-deficient
(tn-C /) and wild-type (tn-C +/+) littermates
with a C57BL/6J-129SvJ genetic background.
Southern blot and PCR analyses
Genomic DNA (10 µg each) obtained from tail tips was digested
with BamHI, separated on a 0.8% agarose gel, and
transferred onto Hybond-N membranes (Amersham Biosciences, Freiburg,
Germany) under alkaline conditions. The PCR-generated probe 5'A (see
Fig. 1a) was  -32P-labeled
using the Megaprime DNA labeling kit (Amersham Biosciences). Genotyping
was performed routinely by multiplex PCR analysis using primers derived
from the intron between exons 1 and 2 (5'-AGC CCC TGC CTA CCT TTT CCT
AAT G-3'; see Fig. 1a, 1A), from the single loxP-BamHI site (5'-CCA GCT TTA TCG GAT CCA TAA CTT CG-3';
see Fig. 1a, 1C), and from exon 2 (5'-CTT CGG GAG
TGA GGG CAA ACA-3'; see Fig. 1a, 1B). PCR was
performed in 25 µl reaction mixtures containing standard buffer plus
1.5 mM MgCl2 and 0.4 µM of each primer. The cycling conditions
consisted of an initial 150 sec denaturing step at 94°C, followed by
30 cycles of 30 sec at 94°C, 45 sec at 65°C, and 50 sec at 72°C.
A 461 bp fragment was indicative of the wild-type allele, and a 225 bp
fragment was indicative of the targeted allele.
RNA preparation, Northern blot, and reverse
transcription analyses
Mice were killed by cervical dislocation (n = 6 for each genotype), and various organs (i.e., thymus, lung) and brain
regions (i.e., hippocampus, cerebellum) were quickly dissected and
frozen in liquid nitrogen. Total RNA was isolated using the RNeasy
system (Qiagen, Hilden, Germany). Electrophoresis and capillary
blotting onto a Hybond-N membrane (Amersham Biosciences) were performed following standard procedures (Sambrook et al., 1989). Northern blots
were hybridized with 5-15 × 106 cpm
of a -32P-labeled TN-C cDNA probe
derived from the plasmid pJT1 (Bartsch et al., 1992). For reverse
transcription analysis, 250 ng of total RNA were transcribed using
Omniscript Reverse Transcriptase (Qiagen) with
Oligo-(dT)23 primers. One-tenth of each reaction
was amplified with the forward primer 5'-AGA GAC TTT GCT TTT CCC GAC
CTG-3' located in exon 1 and the reverse primer 5'-CAC CGC CCA CGA TTG TAG CA-3' located in exon 3 of the tn-C gene (see Fig.
1e). A 1036 bp fragment was indicative of the wild-type
mRNA, and a 454 bp fragment was indicative of an aberrant transcript
lacking exon 2, namely containing exon 1 spliced directly to exon 3.
Western blot analysis
Tissue samples (cerebra and cerebella) of 7-d-old TN-C-deficient
mice and wild-type littermates (n = 6 for each
genotype) were Dounce homogenized on ice in lysis buffer [20
mM Tris, pH 7.4, 0.15 M NaCl, 0.5% (w/v) Nonidet
P-40] complemented with the Complete Protease Inhibitor Mix (Roche
Diagnostics, Mannheim, Germany). The homogenates were centrifuged at
20,000 × g at 4°C for 30 min to remove insoluble
material, and the supernatants were collected. Protein concentrations
were determined with the Micro BCA protein assay (Pierce Chemical Co.,
Rockford, IL). Samples were subjected to 10% SDS-PAGE under reducing
conditions and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) following standard protocols (Towbin et al.,
1979). TN-C-immunoreactive bands were detected using the polyclonal
TN-C antibody pK7 (1:10,000), horseradish peroxidase-conjugated goat
antibodies to rabbit IgG (1:10,000), and a chemiluminescence system
(Amersham Biosciences).
Light and electron microscopy
Two-month-old TN-C-deficient mice and wild-type littermates
(n = 7 for each genotype) were deeply anesthetized and
perfused through the left ventricle with 4% paraformaldehyde and 2%
glutaraldehyde in PBS, pH 7.4. Brains and eyes with attached optic
nerves were removed and postfixed in the same fixative. Light and
electron microscopic analysis were performed as described (Weber et
al., 1999). Briefly, parasagittal sections of cerebella and cerebra were prepared with a Vibratome (Leica, Bensheim, Germany). Sections of
retinas were prepared from central regions (i.e., close to the
optic disk). Cerebellar, cerebral, and retinal Vibratome sections and
optic nerves were incubated in 2% osmium tetroxide for 2 hr, dehydrated in an ascending series of methanol, and embedded in Epon 812 (Sigma-Aldrich). For light microscopic analysis, 3-µm-thick sections
were stained with Toluidine blue and analyzed with an Axiophot (Carl
Zeiss, Göttingen, Germany). Ultrathin sections were
counterstained with lead citrate and examined with an EM 10C electron
microscope (Zeiss).
Indirect immunofluorescence and detection of perineuronal nets
TN-C-deficient mice, 4-6 weeks old, and age-matched wild-type
littermates (n = 10 for each genotype) were deeply
anesthetized and perfused through the left ventricle with 4%
paraformaldehyde in PBS, pH 7.4. Brains were removed and postfixed in
the same fixative overnight at 4°C. Indirect immunofluorescence was
performed as described (Weber et al., 1999). Briefly, sections (30 µm) were blocked in PBS containing 2% BSA for 2 hr followed by
incubation with antibodies against parvalbumin, GAD (both 1:100 in
PBS/0.1% BSA), or synaptophysin (1:100 in PBS/0.1% BSA) overnight at
4°C. After washing, sections were incubated with Cy3-conjugated
antibodies to mouse IgG or rat IgG (1:300) for 2 hr at room temperature
and, after washing, mounted with Aqua-Poly/Mount (Polysciences,
Warrington, PA).
Indirect immunofluorescence for the detection of TN-C (KAF 9-2;
1:100), MAG (513; 1:100), and TN-R (619; undiluted cell culture supernatant) was performed on cryostat sections. Longitudinal sections
of fresh frozen optic nerves with attached retinas and parasagittal
sections of fresh frozen cerebella were processed as described (Bartsch
et al., 1992, 1994). Primary antibodies were detected with Cy
3-conjugated antibodies to rabbit or mouse IgG.
Visualization of perineuronal nets with the plant lectin Wisteria
floribunda (WFA) (Sigma-Aldrich) was performed as described (Weber
et al., 1999). Briefly, fixed Vibratome sections (see above) were
incubated with the lectin at a final concentration of 20 µg/ml. After
washing, the lectin was detected with Cy 3-conjugated streptavidin
(1:600; Dianova).
Nissl and Timm's staining
For Nissl staining, fixed Vibratome sections (see above;
n = 3 for each genotype) were washed with water
followed by 70% ethanol, incubated with a 10% thionin (Sigma-Aldrich)
solution (in ethanol) for 15 min at room temperature, dehydrated in
ascending series of ethanol, followed by isopropanol and xylol, and
mounted with EUKITT (Merck, Darmstadt, Germany). Timm's staining was
performed to visualize mossy fibers in the inner molecular layer of the hippocampus. Animals (n = 3 for each genotype) were
deeply anesthetized and transcardially perfused with 1% sodium sulfide
(Sigma-Aldrich) followed by 3% glutaraldehyde and finally with 1%
sodium sulfide. Brains were postfixed in the same fixative overnight at
4°C. Parasagittal brain sections (30 µm) were cut with a Vibratome
(Leica), mounted onto gelatin-coated slides, and air dried overnight.
Sections were washed three times in PBS (10 min each), incubated for 60 min at 30°C in Timm's solution (2.5% citric acid, 2.4% sodium citrate, 1.7% hydroquinone, 5% silver nitrate, and 25% gum
arabicum; all chemicals from Sigma-Aldrich). Finally, sections
were washed with tap water for 10 min, washed twice with distilled
water for 5 min each, and mounted with glycerol.
Golgi impregnation
Animals (n = 6 for each genotype) were deeply
anesthetized and transcardially perfused with 0.9% sodium chloride
followed by 10% formaldehyde. Brains were postfixed in the same
fixative for 1 week at room temperature and processed following a
modified Golgi-Kopsch protocol. Chromation in 3.6% potassium
dichromate (Sigma-Aldrich) for 5 d was followed by impregnation in
0.75% silver nitrate (Sigma-Aldrich) for 5 d. Both chromation and
impregnation were repeated twice for 4 d each. Thereafter, 100 µm sagittal sections were cut with a Vibratome (Leica). Free-floating
sections were dehydrated in an ascending series of ethanol, followed by methylsalicylate, isopropanol, and xylene, and mounted with DePeX (Serva).
Water maze test
Twelve- to 15-week-old males (19 TN-C-deficient mice and 19 wild-type littermates) were maintained in groups of two to three mice
(at least one mouse of each genotype per group) under standard housing
conditions (20 ± 1°C, 50% humidity; food and water ad libitum). After being subjected to several behavioral
paradigms (open field, light/dark avoidance, and elevated plus maze
tests), animals were housed singly for 1 week before being tested in
the water maze.
Mice were trained during the dark period in a 155 cm diameter water
maze (water at 20 ± 1°C, made opaque by a nontoxic white paint;
14-cm-diameter platform placed 1 cm below the water surface, white
walls 20 cm above the water surface; maximal trial duration 90 sec, 15 sec on top of the platform at the end of each trial). The maze was
placed at the center of the experimental room (4 × 4 m)
provided with several cues and illuminated by white bulbs (light
intensity was set to be 100 lux at the surface level of the maze).
During the experiment, the mice were kept in a room adjacent to the
experimental room illuminated by dim red light. They were transported
to the water maze in a plastic cup handled by a long stick. The opening
of the cup was placed toward the wall of the maze to let the mice glide
into the water. Mice were started from six symmetrical starting
positions in a pseudo-randomized order. After staying on the platform
for 15 sec, the mice were given the opportunity to climb on a wire-mesh
grid attached to a long stick and then returned to their home cage and
placed under red light. We started the training with a visible platform
to train all animals to associate the platform with the escape from the
pool. This was done to avoid the possibility that learning of this
particular feature of the task overlapped or interfered with learning
of the spatial components. For the "visible platform" protocol
(days 1-2, four trials per day, intertrial interval of 2 hr), the pool
was surrounded by black curtains to occlude the sight of extra maze
cues. The platform was cued by a 15-cm-high dark cylinder placed onto
it and located pseudo-randomly in different locations across trials.
For the "spatial task" acquisition, all animals were trained over
3 d (days 3-5, six trials per day, intertrial interval of 1 hr).
The platform was hidden, and the curtain was removed to reveal extra
maze cues. At the end of the acquisition phase, the platform was
removed, and the animals were kept swimming for 60 sec (probe trial).
Time spent in different areas with a surface equal to 10% of the total
maze was used to test the preference of the animals for the former
platform location. The next day (day 6), the hidden platform was placed
at a new position, and after five acquisition trials another 60 sec
probe trial (platform removed) was performed. From day 7 on, a protocol
for three consecutive fast relearning trials was started
("trial-to-criterion" task) (Chen et al., 2000). Each animal was
trained, for up to nine trials per day (intertrial interval of 5-8
min), for a platform location until it reached the criterion of three
consecutive trials with an average escape latency of  15 sec before
being trained for a new location on the next day. In this way, an
animal was trained for one platform location over a minimum of four
trials (the escape latency of the first trial was not determined) up to
an open number of trials. All mice were tested until they reached the
criterion for three different platform locations. The number of trials
required to reach the criterion was evaluated to analyze the
performance of the two genotypes. All trials were video recorded and
analyzed with the video tracking system EthoVision (Noldus, Wageningen, The Netherlands).
All the data were analyzed with nonparametric statistics. Differences
between the two genotypes were tested with the Mann-Whitney U test. Time spent in the different areas was tested against
the chance level of 10% with the Wilcoxon signed rank test. To test dependent data within a genotype, Wilcoxon matched pair and Friedman tests were used. Because there is no nonparametric procedure for multifactorial analysis, a parametric ANOVA for repeated measurements having the genotype as between factor and trials as within factor was used.
Electrophysiological recordings
TN-C-deficient mice (4-6 weeks old) and their wild-type
littermates were used in all electrophysiological experiments except for CA1 long-term potentiation (LTP) recordings, for which 12- to
13-week-old mice were used in addition. Hippocampal slice preparation and recordings of CA1 LTP, CA1 long-term depression (LTD), and CA3 LTP
were performed as described (Eckhardt et al., 2000). All recordings and
analyses were done without knowing the genotype of mice.
LTP and LTD in the CA1 region of the hippocampus. Briefly,
recordings of focal field EPSP (fEPSP) were performed in the
stratum radiatum with glass pipettes filled with artificial CSF
(ACSF) and having a resistance of 1-2 M . Schaffer collaterals were
stimulated by a bipolar electrode. Basal synaptic transmission was
monitored at 0.05 Hz. The inter-theta burst stimulation (TBS) interval
was 20 sec, and four TBSs were applied to induce LTP. TBS consisted of
10 bursts delivered at 5 Hz. Each burst consisted of four pulses delivered at 100 Hz. Duration of pulses was 0.2 msec, and stimulation strength was set to provide fEPSPs with an amplitude of ~50% from the subthreshold maximum. For comparison of paired-pulse facilitation at different interpulse intervals, the stimulation strength was set to
25-30% of the subthreshold maximum. A voltage-dependent Ca2+ channel (VDCC)-dependent form of
potentiation was induced by application of 25 mM
tetraethylammonium (TEA), a K+ channel
blocker (Aniksztejn and Ben Ari, 1991; Huang and Malenka, 1993), for 7 min. To block the L-type VDCC-dependent component of TBS-induced LTP,
nifedipine (20 µM; Sigma-Aldrich) was bath applied in the dark.
Homosynaptic LTD was induced by two trains applied at 1 Hz for 10 min
with a 10 min interval between them. Stimulation strength during
baseline recordings and after induction of LTD was set to 30-40% of
maximal fEPSPs. Stimulation strength was set to 60-70% when 1 Hz
trains were delivered. Both TBS and the protocol to induce LTD reliably
produced homosynaptic NMDA receptor-dependent LTP and LTD, respectively
(Eckhardt et al., 2000).
LTP in the CA3 region of the hippocampus. To record mossy
fiber responses in CA3 pyramidal cells, the stimulating electrode was
placed close to the inner part of the granule cell layer, and the
recording electrode was placed in the stratum lucidum. Recordings and
stimulations were both performed with glass pipettes filled with ACSF
and having a resistance of 2 M. The LTP-inducing high-frequency
stimulation (HFS) consisted of trains of stimuli applied at 100 Hz for
1 sec, which were repeated four times with an interval of 20 sec. To
evoke LTP exclusively in mossy fiber synapses, which are known to
undergo LTP in a NMDA receptor-independent manner, the NMDA receptor
antagonist AP-5 (50 µM; Tocris Cookson Ltd.,
Bristol, UK) was applied 15 min before and during HFS. To confirm that
the fEPSPs recorded were evoked by the stimulation of mossy fibers and
not by the associational-commissural pathway, an agonist of
metabotropic glutamate receptors (L-CCG1, 10 µM; Tocris Cookson Ltd.) was applied at the end
of each experiment. Slices in which responses were reduced by at least
70% were selected for analysis. To demonstrate dependency of the
recorded LTP on protein kinase A (PKA), slices were incubated for 2 hr
in a 4 ml chamber in the presence of Rp-cAMPS (100 µM; Biolog, Bremen, Germany), a
membrane-permeable competitive inhibitor for PKA that was also included
in the ACSF used for perfusion of slices at a concentration of 15-20
µM.
LTP in the dentate gyrus. The negative-going responses in
the dentate gyrus were identified as fEPSPs evoked by stimulation of
medial or lateral perforant path if they exhibited paired-pulse depression or facilitation, respectively, and changed their direction when the stimulation electrode was moved more laterally or medially. Because disinhibition is known to be an important precondition for
successful induction of LTP in the dentate gyrus in vitro (Hanse and Gustafsson, 1992), five trains of short HFS (SHFS) were
delivered in the presence of the GABAA receptor
antagonist picrotoxin (100 µM; Tocris Cookson
Ltd.) at 100% of supramaximal strength to elicit LTP. The intertrain
interval was 20 sec, the number of pulses per train was 10, and the
interpulse interval was 10 msec. Only slices showing potentiation
>20% during the first 10 min after HFS were selected for further analysis.
To facilitate visual analysis of sweeps, stimulus artifacts were erased
in the figures. Effects produced by stimulation or pharmacological
treatments are given as mean ± SEM percentage of the baseline
value. Differences between groups were tested for significance using
the nonparametric Mann-Whitney U test and considered
significant at p < 0.05.
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RESULTS |
Generation of TN-C-deficient mice
We flanked exon 2 of the murine tn-C gene with a
loxP-FRT-pgk/neo-loxP-FRT cassette and a single loxP site by homologous
recombination in R1 ES cells (Nagy et al., 1993) (Fig.
1a). G418-sensitive ES cell
clones harboring the desired recombination event were identified by
Southern blot analysis (data not shown). The neo selection marker and the entire exon 2 with adjacent intronic sequences were
excised in vitro by transient expression of Cre recombinase. A sequence coding for the signal peptide and tenascin assembly (TA)
domain was thus deleted, leading to a predicted frameshift in the TN-C
transcript [see also Forsberg et al. (1996)]. Cre-recombined, G418-sensitive ES cell clones were identified by Southern blot analysis
with the probe 5'A flanking the sequence included in the targeting
vector. The presence of the artificially introduced BamHI
site (Fig. 1a) and the lack of exon 2 were indicated by the
appearance of a 4.6 kb band in addition to the wild-type signal at 11.5 kb. The expected band pattern is identical to the one obtained from DNA
of heterozygous offspring (Fig. 1b, lane labeled tn-C +/). Two independent ES cell clones were used to
generate chimeric mice. Germ line transmission was achieved with
chimeras from both ES cell clones as evident from Southern blots (Fig. 1b, lane labeled tn-C +/).
Homozygous TN-C-deficient (tn-C/) and wild-type
littermates (tn-C +/+) used during this study were obtained
from intercrossings of heterozygous (tn-C +/) animals at
Mendelian frequencies. Genomic DNA samples from wild-type, heterozygous, and homozygous mice were digested with BamHI
and subjected to Southern blot analysis. Analysis with the probe 5'A confirmed the pattern expected from the successful homologous recombination and the subsequent Cre recombinase-mediated excision event in vitro (Fig. 1b). Multiplex PCR was
performed to determine routinely the genotype of animals (Fig.
1c). TN-C-deficient mice developed normally by gross
inspection and had a normal life span and fertility.
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Figure 1.
Targeted disruption of the murine
tn-C gene. a, Diagram of a part of the
murine tn-C gene (denoted as tn-C +)
covering exons 2-5, the targeting vector, the targeted
tn-C gene, and the resulting mutant (denoted as
tn-C -) allele after Cre recombinase-mediated excision
in vitro. Artificial introduction of a
BamHI site combined with the excision of the floxed exon
2 converted a 11.5 kb BamHI genomic fragment indicative
of the wild-type allele (tn-C +) to a 4.6 kb
BamHI genomic fragment indicative of the mutant allele
(tn-C -). Selected restriction enzyme recognition sites
are indicated as follows: B, BamHI;
Bg, BglII; D,
DraI; V, EcoRV. Probe
5'A and primers 1A,
1B, and 1C are indicated.
b, Southern blot analysis of representative tail DNA
samples from wild-type (tn-C +/+), heterozygous
(tn-C +/), and homozygous (tn-C
/) mice. DNA was digested with BamHI
and subjected to hybridization using probe 5'A
(a). c, Determination of genotypes
by PCR. Multiplex PCR using primers 1A,
1B, and 1C (a) as
performed routinely from representative tail DNA samples.
d, Northern blot analysis of total RNA from wild-type
(lane 1, 3) and TN-C-deficient mice
(lanes 2, 4). RNA was isolated
from cerebrum (lanes 1, 2) and cerebellum
(lanes 3, 4) of 7-d-old mice.
Wild-type samples gave rise to a broad band with a size of ~6-8 kb.
Note that the weak band in lane 4 is shifted to lower
molecular mass with respect to the intensive wild-type band in
lane 3. Probing blots with a GAPDH-specific probe
revealed no differences in RNA amounts loaded. e, RT-PCR
analysis of TN-C-specific cDNAs derived from wild-type (lanes
1, 3, 5, 7)
and TN-C-deficient mice (lanes 2, 4,
6, 8). Reverse transcription was
performed on total RNA from cerebrum (lanes 1,
2), cerebellum (lanes 3,
4), lung (lanes 5,
6), and thymus (lanes 7,
8) of 7-d-old mice. In the subsequent PCR, primers were
used as depicted. Note that the mutant TN-C message lacking exon 2 could be amplified from all TN-C-deficient tissues tested.
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To test whether the mutated tn-C allele is transcribed,
Northern blot and RT-PCR analyses were performed. After hybridizing with a mouse cDNA probe derived from plasmid pJT1 (Bartsch et al.,
1992), Northern blot analysis of total RNA from cerebella of 7-d-old
TN-C-deficient mice revealed very weak expression levels of a mutated
TN-C transcript (Fig. 1d, lane 4). In
contrast, a strong signal for the wild-type TN-C message of a size of
~6-8 kb was easily detectable in total RNA from cerebella and to a lesser extent in total RNA from cerebra of 7-d-old wild-type
littermates (Fig. 1d, lanes 1, 3). As
expected, the mutant transcript appeared to be shorter than the message
expressed in wild-type tissues (Fig. 1d, compare lanes
3, 4). Expression levels of this aberrant message were almost undetectable in total RNA from TN-C-deficient cerebra of the same developmental stage (Fig. 1d, lane
2), but RT-PCR analysis confirmed transcription in all tissues
from 7-d-old TN-C-deficient mice that were tested (cerebrum,
cerebellum, lung, and thymus) (Fig. 1e). Furthermore, by
using primers derived from sequences of exon 1 and 3, this approach
indicated that the mutated mRNA contained exon 1 directly spliced to
exon 3 (Fig. 1e). The nucleotide sequence of this aberrant
message was determined and revealed that the lack of exon 2 led to a frame shift, as it was described by Forsberg et al. (1996) for
an independently generated TN-C-deficient mutant. By replacing the
coding region of exon 2 with a neomycin cassette, these authors kept
both splice sites intact but also detected mainly the same unexpected
splicing event. In the other published TN-C-deficient mutant, Saga et
al. (1992) replaced parts of exon 2 and the 5' region of intron 2 with
a  -galactosidase gene and a neomycin cassette. Only when these authors used a -galactosidase gene-specific probe could
transcription of the mutated tn-C gene be detected.
To analyze whether the mutant mRNA was translated into a truncated
protein, we analyzed in detail the amount of TN-C proteins in the
mutant. The TN-C protein content was evaluated in thymus, lung, and
cerebellum of 7-d-old TN-C-deficient and age-matched wild-type mice by
Western blot analysis (Fig.
2a). All of these tissues are
known to express high levels of TN-C at this developmental stage
(Bartsch et al., 1992). As expected, TN-C protein was easily detected
with the polyclonal antibody pK7 in protein extracts of all three
tissues tested from wild-type mice (Fig. 2a,
lanes labeled tn-C +/+). A significant reduction
of TN-C immunoreactivity was observed in protein extracts from
heterozygous tissues (Fig. 2a, lanes labeled
tn-C +/). Samples from homozygous TN-C-deficient littermates did not give rise to TN-C immunoreactive signals (Fig. 2a, lanes labeled tn-C /). To
study the possibility of a residual expression of TN-C or truncated
forms thereof, we performed quantitative immunoblot analysis using the
polyclonal TN-C antibody pk7 (Fig. 2b). In crude lysates
from cerebra of 7-d-old wild type mice, TN-C immunoreactivity was
detectable in as little as 0.1 µg of total protein (Fig.
2b). In contrast, no immunoreactive bands were visible in
200 µg of total protein from TN-C-deficient tissue (Fig.
2b). Thus, if residual TN-C protein should be expressed in
the mutant, it comprises <0.05% of the wild-type level.
Immunohistological analysis of sections from fresh frozen cerebella
with the polyclonal antibodies KAF 9-2 (Fig.
2c,d) and pK7 (data not shown) confirmed the
result obtained by immunoblot analysis. Although intense TN-C immunoreactivity was visible on wild-type sections (Fig.
2c), no specific signal was detectable in mutant cerebella
(Fig. 2d). Thus, we consider our TN-C-deficient mouse to be
a true null mutant. Forsberg et al. (1996) proved TN-C deficiency in
their mutant using the same antibodies for Western blot analysis. TN-C
protein levels in the mutant described by Saga et al. (1992) were
indicated to be below 1% of wild-type levels on the basis of
experiments with variable exposure times of Western blots using
monoclonal TN-C antibodies (Settles et al., 1997). However, the latter
mutant has been controversial. Mitrovic and Schachner (1995) reported the detection of residual amounts of a truncated TN-C by Western blot
analysis and immunocytochemistry, whereas Steindler et al. (1995) and
Settles et al. (1997) could not detect any residual TN-C proteins in
the same mutant using the same antibodies.
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Figure 2.
Western blot and immunohistological analysis of
TN-C-deficient mice. a, Western blot analysis of crude
protein extracts from brain, thymus, and lung tissue of 7-d-old mice
from the indicated genotypes (tn-C +/+, tn-C
+/, and tn-C /) using polyclonal TN-C
antibody pK7. In each lane, 10 µg of total protein was loaded.
Intense immunoreactive bands specific for TN-C were easily detected in
tissues from wild-type mice, but no signals were detectable in samples
from TN-C-deficient animals. Heterozygous mice showed a reduced signal
intensity of TN-C-immunoreactive bands. b, Quantitation
of the TN-C protein content in different genotypes. The indicated
amounts of total protein from cerebra of 7-d-old wild-type
(tn-C +/+) and homozygously TN-C-deficient littermates
(tn-C /) were applied and detected after blotting
with the polyclonal TN-C antibody pK7. In wild-type animals, TN-C was
detectable in as little as 0.1 µg total protein, whereas in mutant
littermates no band was detectable even in 200 µg total protein.
Molecular weight markers are indicated at the left margins in
kilodaltons. c, d, Immunohistological
localization of TN-C by indirect immunofluorescence on fresh frozen
sections of cerebella of 7-d-old wild type mice
(c) and TN-C-deficient littermates
(d) using polyclonal antibody KAF 9-2. Intense
TN-C immunoreactivity is visible on sections from wild-type mice,
whereas no immunoreactivity is detectable in age-matched TN-C deficient
littermates. Sections incubated only with secondary antibody showed no
immunoreactivity (data not shown). egl, External
granular layer; igl, internal granular layer;
pcl, Purkinje cell layer. Scale bar (shown in
d): c, d, 150 µm.
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Morphological analysis
Cell culture experiments have implicated TN-C in diverse functions
during neural development, including neurite elongation, nerve cell
migration, or inhibition of axonal regeneration (Faissner and
Schachner, 1995; for review, see Bartsch, 1996). TN-C is strongly expressed in the developing cerebellar cortex by astrocytes and Golgi
epithelial cells and remains expressed at high levels in the adult
(Bartsch et al., 1992). Moreover, TN-C has been demonstrated to promote
neurite extension from cerebellar granule cells and migration of
granule cells from the external to the internal granular layer in
vitro (Husmann et al., 1992). However, light microscopic inspection of the cerebellar cortex of TN-C-deficient mutants revealed
an apparently normal histoarchitecture (Fig.
3b). All cortical layers
formed normally with a thickness indistinguishable from that of
wild-type mice (Fig. 3, compare a, b). There was also no evidence for ectopically positioned granule cells or other neural cell types in mutant cerebella (Fig. 3b). Moreover,
electron microscopic analysis revealed no obvious defects of Golgi
epithelial cells. For instance, end feet of Golgi epithelial cells
formed an ultrastructurally intact glial-limiting membrane (data not shown). Ultrastructural abnormalities of cerebellar nerve cell types,
and in particular of parallel fibers, were also not detectable (data
not shown).
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Figure 3.
Morphological and immunohistochemical analysis of
TN-C-deficient mice. Light microscopic analysis of the cerebellar
cortex (a, b) and retina
(c, d) of 2-month-old wild-type
(a, c) and TN-C-deficient
(b, d) mice demonstrates an apparently
normal histoarchitecture of both CNS structures in the mutant. All
cerebellar (compare a, b) and retinal
(compare c, d) layers of TN-C-deficient
animals display a normal thickness, and there is no evidence for
ectopically positioned cell types in TN-C-deficient tissues. TN-R
immunoreactivity in the cerebellar cortex of wild-type mice
(e) is homogeneously distributed in the molecular
layer (mol) and internal granule cell layer
(igl) and is accumulated in the white matter
(wm). A similar distribution and intensity of TN-R
positivity is detectable in the cerebellar cortex of TN-C-deficient
mice (f). The distribution of
oligodendrocytes and myelin in the optic nerve (on) of
adult wild-type (g) and TN-C-deficient
(i) mice was visualized with MAG antibodies
(h and j are the phase-contrast images of
g and i, respectively). MAG
immunoreactivity is absent from the retinal end of the optic nerve and
from the retina of both genotypes. Arrows in
g and i indicate the transition zone from
myelinated to nonmyelinated segments of retinal ganglion cell axons.
Electron microscopic analysis of optic nerves from TN-C-deficient mice
(k) revealed a normal ultrastructure of the
meninges and glia limitans (k). Virtually all
ganglion cell axons of mutant mice (some labeled with ax
in k and l) are surrounded by a
myelin sheath (some labeled with m in k
and l). Analysis at a higher magnification
demonstrates the presence of ultrastructurally intact CNS myelin
sheaths in TN-C-deficient optic nerves. as, Astrocyte;
ax, axons; igl, internal granule cell
layer; inl, inner nuclear layer; ipl,
inner plexiform layer; m, myelin sheath, mol, molecular
layer; on, optic nerve; onl, outer
nuclear layer; pcl, Purkinje cell layer;
wm, white matter. Scale bars: (shown in
b) a, b, 100 µm; (shown
in d) c, d, 100 µm;
(shown in f) e, f,
200 µm; (shown in j) g-j, 200 µm;
k, 2 µm; l, 1 µm.
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The retina is another CNS structure displaying high levels of TN-C
expression during development and significant levels of immunoreactivity in the adult (Bartsch et al., 1994). Again, obvious morphological alterations of TN-C-deficient retinas were not observed (Fig. 3d), and all retinal cell types of TN-C-deficient mice
were positioned appropriately.
To evaluate the possibility that the lack of TN-C is compensated by
increased levels of TN-R expression, we performed TN-R immunohistochemistry on brain sections from 2-month-old TN-C mutants. In the cerebellar cortex of wild-type mice, TN-R immunoreactivity was
homogeneously distributed in the molecular layer and internal granule
cell layer and particularly intense in the white matter. A similar
distribution and intensity of TN-R positivity was observed in the
cerebellar cortex of TN-C-deficient littermates (Fig. 3, compare
e, f).
Examination of TN-C-deficient optic nerves
TN-C is strongly expressed in the developing optic nerve and
remains expressed at high levels in the unmyelinated retinal end of the
adult nerve (Bartsch et al., 1994). Substrate-bound TN-C is a
nonadhesive substrate for cells of the oligodendrocyte cell lineage
when offered as a patterned substrate (Bartsch et al., 1994). In
addition, migratory activity of oligodendrocyte progenitor cells is
reduced on homogenous TN-C substrates (Kiernan et al., 1996). We
therefore hypothesized that elevated levels of TN-C at the retinal end
of the optic nerve prevent migration of oligodendrocyte progenitor
cells into the retina and as a consequence intraretinal formation of
myelin (Bartsch et al., 1994). Thus, we analyzed in detail the primary
visual pathway of TN-C-deficient mice and, in particular, the
distribution of oligodendrocytes and myelin along retinal ganglion cell
axons. The distribution of oligodendrocytes and myelin in 2-month-old
wild-type (Fig. 3g) and TN-C-deficient mice (Fig.
3i) was visualized in longitudinally sectioned optic nerves
using MAG antibodies. Strong and homogenously distributed MAG
immunoreactivity was observed in distal regions of optic nerves of both
genotypes. However, a sharp transition from a MAG-immunoreactive to a
MAG-immunonegative region was observed at the retinal end of the optic
nerve of both genotypes (Fig. 3g,i), reflecting
the characteristic differential distribution of oligodendrocytes and
myelin in the primary visual pathway of mice. As a next step, we
studied optic nerves of 2-month-old TN-C mutants and age-matched
wild-type mice at the ultrastructural level. Astrocytes are the
cellular source of TN-C in developing optic nerves. Electron
microscopic analysis revealed a normal ultrastructure of astrocytes and
an apparently normal structure of the glial-limiting membrane of
TN-C-deficient nerves (Fig. 3k). The ultrastructure of
retinal ganglion cell axons was also not altered detectably (Fig.
3k,l) when compared with wild-type nerves
(data not shown). There was also no evidence for a disturbed myelination in TN-C-deficient optic nerves. Virtually all ganglion cell
axons of mutant mice were surrounded by a myelin sheath (Fig. 3k). Inspection of TN-C-deficient optic nerves at higher
magnification revealed the presence of myelin sheaths with a normal
ultrastructure (Fig. 3l).
Normal histoarchitecture of the TN-C-deficient hippocampus
Levels of TN-C immunoreactivity are high in the developing
hippocampus and decrease during maturation (Ferhat et al., 1996). In
the adult rat, residual TN-C immunoreactivity is detectable in the
strata oriens and radiatum of the CA1, stratum oriens of the CA3
region, alveus, and the molecular layer of the dentate gyrus, where it
is most prominent in the hilar region (Nakic et al., 1998). We obtained
similar results on fresh frozen sections from 4- to 6-week-old
wild-type mice by indirect immunofluorescence using the polyclonal TN-C
antibody KAF 9-2. As in rats, TN-C expression was weak and diffusely
distributed (Fig. 4a, CA1).
TN-C-deficient littermates showed no detectable immunoreactivity (Fig.
4b).
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Figure 4.
Hippocampal morphology of TN-C-deficient mice.
a, b, TN-C immunoreactivity was
detectable in the hippocampal CA1 region of 5-week-old wild-type mice
(a) by indirect immunofluorescence using the
polyclonal antibody KAF 9-2. Specificity of weak signals is
demonstrated by comparison with TN-C-deficient littermates
(b). c, d, Nissl
staining revealed an apparently normal histology of CA1 through CA3
regions and of the dentate gyrus of TN-C-deficient mutants
(c) when compared with wild-type mice
(d). e-h, Immunohistochemical
localization of the presynaptic marker synaptophysin (e,
f) and Timm's staining (g,
h) revealed a similar laminated organization of the CA3
subfield in wild-type (e, g) and
TN-C-deficient mice (f, h).
i-l, GAD (i, j; only CA1
subfield shown) and parvalbumin immunoreactivity (k,
l) showed normal distribution and appearance of GAD- and
parvalbumin-positive interneurons in TN-C-deficient mice
(j, l) when compared with wild-type
littermates (i, k). m, n,
Immunohistochemistry of WFA lectin-binding sites showing the
interneuron-enwrapping perineuronal nets in the CA1 region of wild-type
(m) and TN-C-deficient (n)
mice. They were not detectably altered in the mutant. o,
p, The morphology of dendrites and spines of hippocampal
pyramidal cells of wild-type (o) and
TN-C-deficient animals (p), visualized with the
Golgi method, is indistinguishable between genotypes. Scale bars:
(shown in b) a, b, 25 µm; (shown in d) c, d,
50 µm; (shown in j) i,
j, 25 µm; (shown in l)
e-h, k, l, 50 µm; (shown in
n) m, n, 25 µm; (shown
in p) o, p, 5 µm.
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To further investigate the functional role(s) of TN-C in the adult
hippocampus, we studied the histoarchitecture of the hippocampal formation of TN-C-deficient mutants. CA1 through CA3 regions and dentate gyrus of the hippocampus appeared histologically normal as
judged from Nissl-stained sections (Fig. 4, compare c,
d) and neurofilament immunohistochemistry (data not shown).
No significant difference in the distribution of astrocytes was
detectable between genotypes, as suggested from immunostainings with
antibodies reactive to GFAP (data not shown). Finally, neither
synaptophysin immunoreactivity nor Timm's staining revealed
abnormalities in the laminated organization of the CA3 region or mossy
fiber projection (Fig. 4, compare e and f,
g and h). Weber et al. (1999) demonstrated
abnormal perineuronal nets and Saghatelyan et al. (2001) detected
reduced perisomatic inhibition of CA1 pyramidal cells by GABAergic
interneurons in mutant mice deficient in the closely related ECM
glycoprotein TN-R. To elucidate the distribution and
appearance of interneurons and their enwrapping perineuronal nets in
TN-C-deficient mutants, we analyzed GAD (Fig. 4, compare i,
j), parvalbumin (Fig. 4, compare k,
l), and WFA (Fig. 4, compare m,
n for CA1 region) immunoreactivity in comparison to
wild-type littermates. No significant differences between
TN-C-deficient mice and wild-type littermates were detectable. Additionally, examination of Golgi preparations revealed a normal appearance of dendrites and spines of hippocampal pyramidal cells (Fig.
4, compare o, p). Thus, the lack of TN-C did not
detectably affect the histoarchitecture of the murine hippocampus.
Unaltered performance in the water maze task
The protocol of the water maze used to evaluate our mutant was
designed such that TN-C-deficient mice and wild-type littermates were
tested under conditions (visible platform, hidden platform acquisition,
and relearning) that either do or do not implicate the hippocampal
formation in solving the task. In particular, the
"trial-to-criterion" protocol was designed in a way that the memory
of earlier platform locations would interfere with the learning of new locations. Therefore, the most recently encoded location should be selectively retrieved time by time, a characteristic feature of "episodic-like memory," which has been shown to be hippocampus dependent (Aggleton and Brown, 1999; Wood et al., 2000).
The performance of TN-C-deficient mice and wild-type littermates in the
water maze indicated that both genotypes quickly and reliably learned to locate the platform under all conditions. No difference between genotypes was observed for any of the parameters analyzed during the "visible platform," "acquisition," "relearning,"
and "training-to-criterion" phases. Thus, TN-C-deficient mice
showed apparently normal hippocampus-dependent and
hippocampus-independent learning and memory as far as assessed in the
water maze with the protocol conducted.
Wild-type and TN-C-deficient littermate mice both swam normally and
climbed successfully onto the escape platform in the pool. Performance
in the initial visible platform phase revealed no sensorimotor or
motivational abnormalities. Mice from both genotypes quickly reached
average escape latencies of <10 sec (Fig.
5a). The ANOVA analysis for
repeated measure of escape latency, distance moved, velocity, and
minimal distance to the wall (thigmotaxis) as analyzed for the visible
platform, acquisition, and relearning did not show any effect of
genotype and interaction between genotype and trials. As an index for
the use of a spatial strategy during the acquisition and relearning
phases, the percentage of time spent in five different areas in the
arena was analyzed (see Fig. 5e for a description of the
arena). Both genotypes showed a clear preference for the area
surrounding the platform as compared with the chance levels of 10% as
well as compared with the percentage of time spent in the other four
equivalent areas (data not shown). It is interesting to note that
during the relearning phase, mice from both genotypes continued to show
a preference for the area where the platform was located during the
acquisition phase. During a 60 sec probe trial at the end of the
acquisition phase, mice from genotypes spent a significantly higher
percentage of time in the area surrounding the northeast (NE) platform
location (see Fig. 10b). Mutants and wild-type mice spent
more time than the chance level also in the northwest (NW) area, which
may be attributable to the fact that the starting position during this
trial was in the west side of the arena. Indeed, no preference for this
area was observed during the acquisition phase. As shown in Figure 5c, during the probe trial after the relearning [starting
position from the south (S)], both TN-C-deficient and control mice
showed a preference for the area surrounding the platform position (NW) as well as for the position of the platform in the former acquisition phase (NE).
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Figure 5.
Unaltered learning, spatial memory, and relearning
of TN-C-deficient mice. a, Latencies to climb the
platform during the visible platform task, the spatial learning
acquisition, and the relearning. Each value represents one trial
(mean ± SEM). Mice from both genotypes quickly learned to locate
the platform in all three phases. b, During the probe
trial at the end of the acquisition phase, both genotypes showed a
preference for the area surrounding the platform in NE.
The preference for the area in NW might be attributable
to the fact that animals were
started from the west (mean ± SEM).
c, After five trials with the platform located at a new
position (NW), both genotypes showed a preference
for the area around the platform location (NW) as
well as for the area where the platform was located during the former
acquisition phase (NE). Asterisks
indicate a difference to the chance level of 10% at a significance of
p < 0.005 (Wilcoxon signed rank test; mean ± SEM). d, The analysis of the number of trials needed to
reach criterion for three successive platform locations during the
trial-to-criterion task (days 7-12) revealed no difference between
genotypes (mean ± SEM). e, Scheme of the circular
water maze (diameter, 155 cm) with different platform locations
(platform diameter, 14 cm) surrounded by a circular area equal to 10%
of the total area of the maze.
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It is known that searching strategies and swimming paths are of
paramount importance when evaluating the performance of mice in the
water maze (Lipp and Wolfer, 1998). Because during the second probe
trial TN-C-deficient mice spent more time in the five analyzed areas
(NW, NE, E, S, SW) as compared with wild-type littermates
(p = 0.007), we tested whether there was a
different searching strategy used by TN-C-deficient mice and wild-type
littermates. Therefore, we analyzed the time spent in an imaginary ring
including the area around the platform position during relearning (NW). No difference was found in the time spent in this ring between genotypes (p > 0.1). We also analyzed total
distance moved, mean velocity, mean distance to the wall, annulus
crossing, absolute turning angle, and absolute turning velocity during
the probe trials performed after learning and relearning. No difference was found between TN-C-deficient mice and wild-type littermates (data
not shown), indicating that TN-C mutants and wild-type mice used
indistinguishable searching strategies.
In the training-to-criterion task, both genotypes reached the criterion
on an average of seven trials for the first and second platform
locations and decreased to five trials for the third one (three being
the possible minimum number of trials to reach the criterion) (Fig.
5d). The percentage of time spent in different areas during
the first trial with a new platform location was used to check whether
the animals showed a preference for the area surrounding the platform
location of the previous day. Both TN-C-deficient and wild-type mice
showed a clear preference for the last platform location and, to a
lesser extent, for the second to last platform location (data
not shown), showing not only that they used navigation to search for
the platform, but that they could also retain the memory trace over a
long period of old spatial information.
Impaired TBS-induced LTP in the CA1 region
Stimulus-response curves for fEPSPs evoked by stimulation of
Schaffer collaterals and paired-pulse facilitation measured at interpulse intervals between 10 and 200 msec were not different between
TN-C-deficient mutants and wild-type littermates, demonstrating normal
basal levels of excitatory transmission and its presynaptic modulation
in the case of constitutive TN-C deficiency (Fig.
6a,b). Furthermore,
we investigated hippocampal synaptic plasticity in TN-C-deficient mice,
starting with the most widely studied form of plasticity, LTP in the
CA1 region. TBS of Schaffer collaterals reliably produced short-term
potentiation (STP) and LTP in all slices measured from 1-month-old
wild-type animals (Fig. 6c). The mean level of STP measured
as maximal potentiation during 1 min after TBS was 191.1 ± 14.2%, and the level of LTP seen 50-60 min after TBS was 148 ± 4.0%. The levels of STP in 1-month-old TN-C-deficient mice (167.3 ± 8.0%) were not significantly different, whereas TBS-induced LTP was
significantly reduced (119.3 ± 3.0%) (Fig.
6c,e), as compared with wild-type mice. To
analyze whether the deficit in LTP was age dependent, we recorded LTP
in 3-month-old TN-C-deficient mice and their wild-type control
littermates. Again, no significant difference in STP was revealed,
whereas LTP was significantly impaired in TN-C mutants (Fig.
6c,e).
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Figure 6.
LTP and LTD in the CA1 region are impaired
in TN-C-deficient mice. a, Input-output curves for
slopes of fEPSPs evoked by stimulation of Schaffer collaterals at
different stimulation strengths. No significant difference between
genotypes was found. b, Paired-pulse facilitation
(PPF) was measured as the ratio between the
slopes of fEPSPs evoked by the second and first pulses and plotted for
several interpulse intervals. Field EPSPs were recorded at 30% from
the subthreshold strength. To measure slopes of overlapping fEPSPs
evoked by paired-pulse stimulation (for interpulse intervals <50
msec), fEPSPs evoked by a single-pulse stimulation were subtracted from
fEPSPs evoked by paired-pulse stimulation. Examples of fEPSPs evoked by
paired-pulse stimulation are shown in c, right
panels. No significant difference between genotypes was found.
c, TBS of Schaffer collaterals (applied at time point 0)
evoked a high increase in the slopes of fEPSPs recorded in the CA1
region of slices from wild-type mice. In slices from TN-C-deficient
mice (tn-C /), the potentiation appeared lower than
in wild-type mice (tn-C +/+). The mean slope of fEPSPs
recorded 0-10 min before TBS was taken as 100%. Data represent mean + SEM; n indicates the number of tested slices;
N indicates the number of tested mice. Right
panels show fEPSPs recorded before and 60 min after TBS. Scale
bars, 20 msec and 500 µV. d, Two trains of
low-frequency stimulation (1 Hz, indicated by horizontal
bars) of Schaffer collaterals reliably decreased the slopes of
fEPSPs in slices from wild-type mice (tn-C +/+). In
slices from TN-C-deficient mice (tn-C /), the slope
returned to the baseline. The mean slope of fEPSPs recorded 10 min
before the first train was taken as 100%. Data represent mean + SEM;
n indicates the number of tested slices;
N indicates the number of tested mice. Right
panels show fEPSPs in TN-C-deficient (tn-C
/) and wild-type (tn-C +/+) mice before and
60 min after induction of LTD. Calibration: 10 msec, 500 µV.
e, Cumulative plots representing levels of STP and LTP
from all experiments. Each symbol represents a single
experiment. Cumulative probability at any given value X
is the probability to observe potentiation less than or equal to
X. Lower values of LTP (there is no overlap of values
measured in young tn-C / and tn-C +/+
mice) are evident for TN-C-deficient mutants when compared with
wild-type littermates. f, Cumulative plots representing
levels of STD and LTD from all experiments. Each symbol
represents a single experiment. Cumulative probability at any given
value X is the probability to observe depression less
than or equal to X. Lower values of both STD and
particularly LTD are evident for TN-C mutants when compared with
wild-type littermates.
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Abolished low-frequency stimulation-induced LTD in the
CA1 region
Another NMDA receptor-dependent form of long-term plasticity in
the CA1 region is LTD that can be evoked by two trains of low-frequency
stimulation (LFS) delivered within a 10 min interval. During LFS, a
transient facilitation was observed with similar levels of facilitation
and time course for both wild-type and TN-C-deficient mice (Fig.
6d, indicated by 1 Hz). Transient STD followed
the facilitation. The mean level of STD (measured as the maximal
depression during 1 min after the second LFS) was significantly lower
in mutants (reduction to 68.8 ± 3.7% from 100%) than in
wild-type animals (reduction to 51.1 ± 3.5% from 100%) (Fig.
6f). Long-term reduction of the fEPSP slope by >15% was seen in seven of eight slices prepared from wild-type mice. On
average, fEPSP slopes were reduced 50-60 min after induction of LTD by
28.0 ± 5.5% (Fig. 6d). In TN-C-deficient mice, the
reduction was not larger than by 12% (Fig. 6f). The
mean slope of fEPSPs measured 50-60 min after the second LFS was close
to the baseline level (100.6 ± 3.5%) (Fig. 6d). Thus,
STD is reduced and LTD is abolished in TN-C-deficient mice.
Normal HFS-induced LTP in the CA3 region
To investigate the regional specificity of abnormalities in
synaptic plasticity of the TN-C-deficient hippocampus, LTP at mossy
fiber synapses in the CA3 region was particularly interesting to study,
because it shows features clearly distinct from CA1 LTP and LTD, being
independent of postsynaptic NMDA receptors, mediated by cAMP, and
activated by adenylate cyclase and PKA (Weisskopf et al., 1994). Field
EPSPs evoked in CA3 pyramidal cells by mossy fiber stimulation are
known to be fast and to exhibit paired-pulse facilitation and
potentiation during 0.33 Hz stimulation. These criteria were taken to
search for responses that were further characterized pharmacologically
using L-CCG1, an agonist of type II metabotropic glutamate receptors,
which is known to reduce synaptic transmission in CA3 mossy fiber
synapses (Maccaferri et al., 1998; Eckhardt et al., 2000).
Low-frequency stimulation (0.33 Hz) potentiated fEPSPs to ~250% in
wild-type and TN-C mutant mice (Fig.
7a). L-CCG1 similarly diminished the amplitude of fEPSPs in both genotypes (Fig.
7b). The NMDA receptor antagonist AP-5 did not affect the
amplitude of selected fEPSPs in either wild-type or TN-C mutant mice
(Fig. 7c). HFS performed in the presence of AP-5 induced a
strong increase in fEPSP amplitudes (Fig. 7c). Post-tetanic
potentiation (PTP) during the first 1 min after HFS was ~900%, and
mean potentiation measured 50-60 min after induction of LTP was
~200%, resembling reported profiles of LTP in CA3 in mice
(Maccaferri et al., 1998; Eckhardt et al., 2000). There was no
difference between wild-type littermate and TN-C mutant mice in PTP or
LTP (Fig. 7c-e).
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Figure 7.
Normal LTP in the CA3 region of TN-C-deficient
mice. a, Stimulation of mossy fibers with a frequency of
0.33 Hz similarly increased the amplitudes of fEPSPs in acute slices
from both TN-C-deficient (tn-C /) and wild-type
(tn-C+/+) mice. b, Application of the
type II metabotropic glutamate receptor agonist L-CCGI (10 µM) reduced the amplitude of fEPSPs in slices from both
TN-C-deficient (tn-C /) and wild-type
(tn-C +/+) mice to the same level. c,
High-frequency stimulation (HFS) of mossy fibers
(applied at time point 0) evoked a similar increase in slopes of fEPSPs
in slices from TN-C-deficient (tn-C /) and wild-type
(tn-C +/+) mice, respectively. The
potentiation was impaired in wild-type slices treated with a
competitive inhibitor for PKA, Rp-cAMPS. The time interval of the
application of the NMDA receptor antagonist AP-5 is shown by a
horizontal bar. Mean slope of fEPSPs recorded 0-10 min
before HFS was taken as 100%. Right panels show
averaged fEPSPs recorded before and 60 min after induction of LTP in
TN-C-deficient (tn-C /) and wild-type
(tn-C +/+) mice. Calibration: 10 msec, 100 µV.
d, e, Cumulative plots representing
levels of PTP (d) and LTP
(e) from all experiments. Each
symbol represents a single experiment. Cumulative
probability at any given value X is the probability to
observe a potentiation less than or equal to X. Note
similar values of PTP and LTP in TN-C-deficient (tn-C
/) and wild-type (tn-C +/+) mice and
that Rp-cAMPS completely blocked LTP in five of seven experiments.
100% corresponds to the baseline level.
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To verify that we analyzed PKA-dependent mossy fiber LTP, HFS was
applied after incubation and perfusion of slices from wild-type mice
with the PKA antagonist Rp-cAMPS. This treatment strongly diminished
both PTP (411.8 ± 38.8%) and LTP (115.1 ± 9.6%) of the
recorded fEPSP (Fig. 7c). We conclude that NMDA
receptor-independent, PKA-mediated LTP in mossy fiber-CA3 synapses is
normal in TN-C-deficient mutants.
Normal HFS-induced LTP in the dentate gyrus
To complete the analysis of the trisynaptic circuit in the
hippocampus, we recorded NMDA receptor-dependent LTP in the synapses formed by the medial and lateral perforant pathways in the dentate gyrus. The fEPSPs evoked by paired-pulse stimulation of the medial perforant pathway (with a 50 msec interval) exhibited paired-pulse depression independently of the genotype (79.9 ± 5.9%,
n = 7 in TN-C-deficient mice and 86.6 ± 4.4%,
n = 8 in wild-type littermates). Paired-pulse
stimulation of the lateral perforant pathway elicited facilitation of
similar magnitude in both TN-C-deficient (124.4 ± 4.8%;
n = 8) and wild-type (122.5 ± 5.9%;
n = 6) mice. SHFS applied in the presence of the
GABAA receptor antagonist picrotoxin induced
similar levels of LTP in the slope of fEPSPs measured 50-60 min after
stimulation. SHFS of the medial perforant pathway potentiated the slope
of EPSPs to 135.1 ± 5.1% in wild-type mice and 134.0 ± 4.0% in TN-C-deficient mutants (Fig.
8a,c). SHFS of the
lateral perforant pathway potentiated the slope of EPSPs to 135.4 ± 9.8% in TN-C-deficient mice and 137.1 ± 7.9% in wild-type littermates (Fig. 8b,d). Thus, there was no
difference between genotypes in synaptic plasticity in the lateral and
medial perforant path connections to the dentate
gyrus.
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Figure 8.
Normal LTP in the dentate gyrus of TN-C-deficient
mice. a, b, Short high-frequency
stimulation (SHFS) of the medial
(a) or lateral (b)
perforant pathway (applied at time point 0) evoked a similar
potentiation in slices from wild-type (tn-C +/+) and
TN-C-deficient mice (tn-C /) in the presence of 100 µM picrotoxin. Mean slope of fEPSPs recorded 0-10 min
before TBS was taken as 100%. Four points recorded during the period
marked by SHFS represent potentiation recorded between five trains (10 sec after a train). Data represent mean + SEM; n
indicates the number of tested slices; N indicates the
number of tested mice. Right panels show fEPSPs recorded
before and 60 min after TBS. Calibration: 10 msec, 250 µV.
c, d, Cumulative plots representing
levels of LTP measured 50-60 min after beginning of high-frequency
stimulation of medial (a) or lateral
(b) perforant pathway. Each symbol
represents a single experiment. Cumulative probability at any given
value X is the probability to observe a potentiation
less than or equal to X. No significant difference
between genotypes was found.
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Reduced LTP in the CA1 region of TN-C-deficient mice is not caused
by increased levels of inhibition or impaired NMDA receptor-mediated
transmission
Because LTP recorded in the dentate gyrus of TN-C mutants in
the presence of picrotoxin was normal and abnormal levels of perisomatic inhibition had been revealed in mice deficient for the closely related tenascin family member TN-R (Saghatelyan et al.,
2001), we hypothesized that TN-C mutants could have elevated levels of
GABAA receptor-mediated inhibition impeding
induction of LTP in CA1 in the absence of picrotoxin. We therefore
recorded CA1 LTP in the presence of picrotoxin with the hope of
rescuing LTP in TN-C-deficient mice. Indeed, the average level of LTP
in mutants was increased in the presence of picrotoxin to 145.1 ± 4.1%, remaining significantly lower than LTP seen in wild-type littermates after picrotoxin application (167.5 ± 6.2%) (Fig. 9a,c). These
findings indicate that mechanisms distinct from GABAergic inhibition
mediate most, if not all, reduction of LTP in TN-C-deficient mutants.
STP values were slightly but not significantly higher in wild-type mice
than in mutants (193.0 ± 24.1 vs 172.3 ± 11.3%, respectively) (Fig. 9a,b).
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Figure 9.
Blockade of GABAA receptor-mediated
inhibition does not fully rescue LTP in the CA1 region of
TN-C-deficient mice. a, TBS of Schaffer collaterals in
the presence of picrotoxin evoked an increase in the slopes of fEPSPs
recorded in the CA1 region of slices from wild-type mice
(tn-C +/+). In slices from TN-C-deficient littermates
(tn-C /), potentiation appeared significantly lower
than in wild-type mice. Mean slope of fEPSPs recorded 0-10 min before
TBS was taken as 100%. Data represent mean + SEM; n
indicates the number of tested slices; N indicates the
number of tested mice. Right panels show fEPSPs
recorded before and 60 min after TBS. Calibration: 20 msec, 500&n |
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TOP
ABSTRACT
INTRODUCTION
