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| massed training |
www.pnas.org/cgi/doi/10.1073/pnas.1413335111
Significance
There are no treatments for congenital intellectual disabilities.
Here we show that newly discovered timing rules for maximizing hippocampal long-term potentiation predict training
regimens that offset defects in synaptic chemistry and memory
in the fragile X mental retardation 1 (Fmr1) KO model of fragile
X syndrome. Wild-type mice required far less training to form
stable memories when given three training trials separated by
1 hour as opposed to one extended session; shorter or longer
intervals were ineffective. The same spaced training protocol
rescued memory in Fmr1 KO mice and restored activation of
synaptic ERK1/2, a kinase critical for both LTP and learning.
These results suggest a readily implementable, neurobiologically
based therapeutic strategy for a prevalent form of intellectual
disability.
Author contributions: R.R.S., G.L., and C.M.G. designed research; R.R.S., K.W., and Y.Q.Y.
performed research; R.R.S., G.L., and C.M.G. contributed new reagents/analytic tools; R.R.S.,
K.W., G.L., and C.M.G. analyzed data; and R.R.S., G.L., and C.M.G. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
NEUROSCIENCE
Recent studies have shown that short, spaced trains of afferent stimulation produce much greater long-term potentiation (LTP) than that
obtained with a single, prolonged stimulation episode. The present
studies demonstrate that spaced training regimens, based on these
LTP timing rules, facilitate learning in wild-type (WT) mice and can
offset learning and synaptic signaling impairments in the fragile X
mental retardation 1 (Fmr1) knockout (KO) model of fragile X syndrome. We determined that 5 min of continuous training supports
object location memory (OLM) in WT but not Fmr1 KO mice. However, the same amount of training distributed across three short
trials, spaced by one hour, produced robust long-term memory in
the KOs. At least three training trials were needed to realize the
benefit of spacing, and intertrial intervals shorter or longer than 60
min were ineffective. Multiple short training trials also rescued novel
object recognition in Fmr1 KOs. The spacing effect was surprisingly
potent: just 1 min of OLM training, distributed across three trials,
supported robust memory in both genotypes. Spacing also rescued
training-induced activation of synaptic ERK1/2 in dorsal hippocampus of Fmr1 KO mice. These results show that a spaced training
regimen designed to maximize synaptic potentiation facilitates recognition memory in WT mice and can offset synaptic signaling and
memory impairments in a model of congenital intellectual disability.
Fig. 1. Long-term object location memory (OLM) and novel object recognition (NOR) are impaired in Fmr1 KOs. (A) For OLM, mice were given 5 min of
continuous (massed) training with two identical objects (A1 and A2). For retention testing 24 h later, one object was in the familiar location (A3) and one was
placed in a novel location (A4); for control (con) mice, objects were in the familiar location. (B) With 5-min training, OLM was robust in WT mice but absent
in KOs (***P < 0.001; n 8 per group). (C) Short-term memory (STM) was comparable between genotypes (P = 0.57; n 10 per group). (D) KOs and
WTs expressed long-term OLM when trained for 10 min (P = 0.88; n 8 per group). (E) Fmr1 KOs on the C57BL/6 background had deficient long-term OLM
(*P < 0.05; n 7 per group) but control level short-term OLM (P = 0.68; n 7 per group) with 5-min massed training. (F) KOs traveled greater distance (meters)
than WTs on habituation days 25 (P < 0.01 for each day), but not during training or retention trials (P > 0.50 both; n 8 per group). (G) KOs trained and
tested in their dark cycle did not express OLM (***P < 0.001; n 10 per group). (H) KO mice given 5-min massed training on two or six successive days failed to
express long-term OLM although memory was robust in WTs (***P < 0.001 vs. WTs; n 8 per group). (I) With 5-min training, WTs showed robust long-term
NOR whereas KOs did not (***P < 0.001; n 12 per group).
interaction times during training and retention trials were comparable in WTs and KOs (Fig. S1). Fmr1 KOs are also hyperactive in open-field tests (16). In accord with this, distance
traveled for KOs decayed more slowly than for WTs across habituation sessions, resulting in greater locomotion on pretraining
days 25. However, with objects present, the genotypes traveled
the same distance (Fig. 1F).
To assess the robustness of the OLM impairment, we tested
mice during the dark phase of their day/night cycle. Long-term
OLM was still absent in KOs (Fig. 1G) although total object
exploration times were not different between genotypes during
training or retention testing.
Next we tested whether 5 min of continuous (i.e., massed)
training produces a memory trace that is too weak to elicit
quantifiable retention. If so, then additional training should increase trace strength, producing measurable discrimination indices. Mice were trained for 5 min and 24 h later for 5 min more.
Tested 1 d later, WT mice showed strong OLM but KOs did not
(Fig. 1H). We gave additional mice 5-min training daily for 6 d:
OLM was still absent in the KOs indicating that they do not form
a partial memory trace after 5 min of training.
Fmr1 KOs Have Impaired, Long-Term Novel Object Recognition (NOR).
We focused on OLM because its retrieval depends on hippocampal field CA1 (11, 14), which exhibits an elevated LTP
threshold in Fmr1 KOs (7, 8) and enhanced potentiation with
spaced stimulation in WTs (10, 17). However, Fmr1 KOs have
more robust LTP impairments in other regions including cortex
(6, 1821). Therefore, we tested if the KOs have deficits in enduring NOR memory which depends, at least in part, on perirhinal
and insular cortices for retrieval (14, 22, 23). Short-term NOR
impairments have been described for Fmr1 KOs (6, 24). Mice
were trained as for OLM for 5 min. For retention testing 24 h
later, one of the familiar objects was replaced with a novel object
but there was no change in arena-context or object location. WT
16908 | www.pnas.org/cgi/doi/10.1073/pnas.1413335111
mice spent more time sampling the novel object but KOs did not
(Fig. 1I). The total time spent exploring objects was comparable
for WTs and KOs during training but somewhat longer for KOs
during retention testing (Fig. S2). NOR deficits in the KOs persisted after 10 min of training (Fig. S3). We conclude that in Fmr1
KO mice impairments in OLM and NOR reflect defects in longterm object recognition memory and, as with LTP impairments,
the effects of genotype are more robust for measures with greater
dependence upon cortical function.
Spaced Training Facilitates WT Learning and Rescues Memory in Fmr1
KOs. As WT mice do not form OLM with 3 min of continuous
NEUROSCIENCE
Fig. 3. Increases in synaptic ERK1/2 activation are associated with object location memory (OLM) in WT mice and absent in KOs given massed training. (AC)
Deconvolved images of immunolabeling for PSD95 (green) and p-ERK1/2 (red) in CA1; overlap appears yellow (arrow). (Scale bars: 5 m, 1.25 m, and 0.5 m in
A, B, and C, respectively.) (D) In CA1 stratum radiatum (SR), Total numbers of densely p-ERK1/2+ elements did not differ between genotypes (P = 0.73;
normalized to WT means) but those colocalized with PSD95 (PSD95+) were more numerous in KOs than WTs (*P < 0.05; N 10 per group). (E) Mice were
handled and habituated and then given 5-min massed training with (train) and without (con) objects present; p-ERK1/2+ PSDs was quantified for CA1 SR
sample fields (Fig. S6). (F) Numbers of densely p-ERK1/2+ PSDs were greater in section 7, 2.16 mm posterior to Bregma (2 way ANOVA P = 0.002, interaction
between section and group; P < 0.001, section 7 vs. others) of WT mice given spaced training. The dashed box around images shows the approximate region
of further analysis (images from Allen Institute for Brain Science). (G) After 5-min massed training, numbers of densely p-ERK1/2+ PSDs were increased in WTs
(**P < 0.01) but decreased in Fmr1 KOs (*P < 0.05, **P < 0.01; n 10 per group; normalized to WT control mean). Note: KOs had constitutively greater
numbers of p-ERK1/2 enriched contacts. (H) Ten minutes of massed training increased numbers of densely p-ERK1/2+ PSDs in KOs (*P < 0.05; n 10 per group:
normalized to KO controls).
Fig. 4. Synaptic ERK1/2 is activated with spaced training, and required for OLM in Fmr1 KOs. (A) Experimental Fmr1 KOs were given three 100 s OLM training
trials separated by 1 h; control (con) mice were placed in the apparatus without objects present. Brains were harvested after the third arena session. (B)
When quantified for the three planes (massed region) evaluated in WTs, numbers of p-ERK1/2+ PSDs in KO spaced training mice were not different from
those in KO control mice (P = 0.22). However, numbers of p-ERK1/2+ PSDs were elevated in the caudal two-thirds of this span (*P < 0.05; n 9 per group;
normalized to KO control mean). (C) Intensity frequency distribution for p-ERK1/2+ PSDs in CA1 planes activated by spaced training shows a rightward-shift in
immunolabeling intensities of p-ERK1/2+ contacts in trained KOs. (D) Paradigm for testing effects of ERK1/2 activation inhibitor SL327 (50 mg/kg) on OLM
with spaced training in KOs: injections were given 30 min before arena sessions on days 510 and 30 min before the last trial on the training day. (E) In KOs,
OLM was blocked by SL327 but was robust with vehicle-treatment (*P < 0.05; n = 10 per group). (F) Exploration times (seconds) were comparable between
vehicle- and SL327-treated KOs during spaced training and retention testing (P > 0.05 all vehicle vs. SL327 comparisons; n = 10 per group).
Seese et al.
NEUROSCIENCE
16912 | www.pnas.org/cgi/doi/10.1073/pnas.1413335111
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Seese et al.
Supporting Information
Seese et al. 10.1073/pnas.1413335111
Materials and Methods
Animals. Adult male Fmr1 KO mice in the FVB129 and C57BL/6
backgrounds, and sex, age, and background matched wild-type
(WT) controls were used. Mice were standard group housed with
litter mates (two to five per cage) in rooms at 68 F and 55% humidity; 12 h on/12 h off light cycle; and food and water ad libitum.
Behavioral Analyses. For object location memory (OLM) and novel
object recognition (NOR) studies, mice were handled for 2 min
daily for 5 d and then habituated to a white chamber (24 30 30
cm) containing sawdust bedding for 5 min daily for 5 d (1, 2). On
the following day, mice were subject to OLM or NOR training.
For both paradigms, mice were placed in the chamber containing
identical 100 mL glass beakers in two corners of the apparatus,
1 inch from the perimeter, for periods described in Results.
OLM retention testing of experimental mice involved returning
the mouse to the chamber with one beaker in its original (familiar) location and the second in closer proximity to the center
of the apparatus (novel) with the novel location alternated
across each experimental group. For OLM retention testing of
control mice involved placing the mouse in the arena in which
the two objects were in their original/training locations. For
NOR retention testing, experimental mice returned to the arena
containing two objects in the original training locations: one of
these was identical to the training-object whereas the second was
replaced with a novel object.
Experiments were performed in an otherwise dark room with
direct overhead lighting (235 lx) during the animals light cycle
(unless otherwise specified). Some experiments were conducted
on the animals dark cycle: In these instances, all procedures
(handling, training, testing) were conducted under red light and
12 h later than the light-cycle testing time. In between arena
sessions, animal feces were removed from the apparatus, sawdust
was thoroughly mixed, and beakers were cleaned with 70%
ethanol and dried. All training and testing videos are scored
offline by raters, blind to genotype and experimental group.
For both tasks, mice were scored as interacting with (i.e., exploring) an object when sniffing with their nose touching or within
0.5 cm from the object. Interaction was not scored when the
animal (i) was within this radius and was grooming or digging or
(ii) touched the object or was within this radius but did not show
intent to interact (e.g., they fell within this zone when turning).
Total exploration time was quantified as the time interacting
with both objects. To assess preferential attention to an object,
a discrimination index was calculated as 100 (tnovel tfamiliar)/
(tnovel + tfamiliar). As plotted, a positive discrimination index
represents a preference for the novel object or novel object location for NOR and OLM, respectively. Total distance traveled
was calculated via ANY-Maze.
ERK1/2 Inhibition. In some studies, starting on the final day of
handling, mice were primed with i.p. injections of saline (2 d) and
then DMSO vehicle (4 d). On the day of training, activation of
ERK1/2 was blocked using SL327 [50 mg/kg at 2.5 mg/m (3);
Tocris Bioscience], an inhibitor of the ERK1/2 activator MEK;
control mice received vehicle (DMSO, Fisher Scientific). On
pretreatment days, saline/vehicle injections were given 30 min
before handling or habituation. On the day of training, animals
underwent OLM training with three 100-s trials, each separated
by 1 h. The injection of SL327 or vehicle was administered 30
min before the third training trial. Mice were then tested for
retention 24 h posttraining. As in other behavioral experiments,
total exploration times and total distances traveled were calculated for all training and retention trials. For immunolabeling
analyses, SL327 (50 mg/kg) was administered to a separate cohort of KOs and brains were harvested 30 min later (e.g., the
time at which the third spaced trial would occur in the separate
behavioral experiments) for tissue processing as described below.
Tissue Collection and Immunofluorescence. To assess effects of behavior on synaptic ERK1/2 activation, tissue sections through
hippocampus were processed for dual immunofluorescence and
then FDT to quantify labeling within the postsynaptic compartment. Immediately at the end of the behavioral session, mice were
euthanized with isoflurane 2 min following training. As described
in detail elsewhere (4, 5), brains were rapidly harvested, fast
frozen in 2-methylbutane (50 C), and cryostat sectioned (20
m, coronal) through the rostrocaudal extent of hippocampus;
the sections were thaw mounted onto microscope slides and
fixed in methanol (20 C, 15 min). Series containing every 10th
tissue section were washed through several changes of 0.1 M
phosphate buffer and incubated (48 h, room temperature) in
a mixture of mouse anti-PSD95 (1:1,000; Thermo Scientific
MA1-045) and rabbit antisera to either phosphorylated (p-)
ERK1/2 Thr202/Tyr204 (1:500; Cell Signaling 4370) or total
ERK1/2 (1:500; Cell Signaling 9102). The tissue was again
washed in phosphate buffer and then incubated with donkey
anti-mouse AlexaFluor 488 and donkey anti-rabbit AlexaFluor
594 (1:1,000; Invitrogen). After final washes slides were coverslipped with VectaShield with DAPI (Vector Labs).
Fluorescence Deconvolution Tomography (FDT). Using a Leica
DM6000 epifluorescence microscope, a 63 oil-immersion objective (NA 1.4), and an ORCA-ER (Hamamatsu) camera,
digital image z-stacks were collected through a depth of 3 m (at
0.2-m steps which is below the Nyquist rate for the imaging
conditions used) for 136 105 m sample fields that were centered on hippocampal field CA1 stratum radiatum (SR; offset
from the pyramidal cell layer by about 100 m). For each section,
two or three z-stacks were collected from fields within the middle
depths of SR as shown in Fig. S6; these z-stacks were individually
processed for restorative deconvolution (Volocity 5.0, PerkinElmer) and through in-house software to normalize background
fluorescence intensity and construct 3D montages of each 42,840-m3
sample field (i.e., each z-stack generated one 3D sample field).
Automated systems then counted and measured in 3D the size
and fluorescence intensities of single- and double-labeled elements within size constraints of synapses. Approximately 40,000
PSDs were quantified for each sample field. Objects were counted as double-labeled if there was any overlap in the boundaries
of immunolabeling with the two fluorophores as evaluated in 3D.
Quantification was limited to objects that satisfied size and eccentricity constraints of synapses, were detected across multiple
intensity thresholds, and were fully contained within the image
z-stack (4, 5). Objects detected by only one threshold, and thus
likely being fluorescent artifacts, were excluded from quantification. Counts of double-labeled PSDs were normalized to the
total number of PSD95-immunopositive elements within a given
sample field; normalized values from each z-stack were averaged
to obtain a mean value for each brain.
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Fig. S1. Total times (in seconds) exploring objects during 5-min massed object location memory training. Retention trials were not different between WT and
Fmr1 KO mice (P > 0.40; n 8 per group).
Fig. S2. Plot shows that total times (in seconds) exploring objects during 5-min continuous novel object recognition (NOR) training was comparable between
genotypes whereas, during NOR retention testing, KOs spent more time than WTs in object exploration (*P = 0.03). Thus, in comparing training to retention
sessions, the WTs show a greater reduction in object exploration time relative to the KOs. n 12 per group.
Fig. S3. After 10 min of massed training, wild-type (WT) mice exhibited novel object recognition (NOR, i.e., had a positive discrimination index) whereas Fmr1
KOs did not. Mice were tested 24 h after training. Performance of the KOs was significantly different from that of WTs (*P < 0.05; n 9 per group).
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Fig. S4. Fmr1 KO mice were injected with the ERK1/2 activation inhibitor SL327 (50 mg/kg) or DMSO/vehicle and then euthanized 30 min later to assess effects
of treatment on numbers of p-ERK1/2+ synapses (double labeled with anti-PSD95) using fluorescence deconvolution tomography (as applied in Fig. 3).
Quantification of densely p-ERK1/2+ PSDs showed that SL327 significantly reduced synaptic p-ERK1/2 levels (**P < 0.01 vs. KO vehicle; n = 10 per group).
Fig. S5. Bar graph shows total distance traveled by Fmr1 KO mice that were treated with vehicle (DMSO) or SL327 (50 mg/kg) in DMSO (see Fig. 4D for
treatment paradigm). SL327 was administered in between the second and third training session (i.e., 30 min before the final training trial; see arrow). For all
trials as well as for retention testing, total distance traveled for WT and Fmr1 KO animals were comparable (P > 0.05; n = 10 per group).
Fig. S6. Schematic showing the placement of image z-stacks (boxes with asterisks) in CA1 str. radiatum (SR). SG, str. granulosum; SLM, str. lacunosummoleculare; SP, str. pyramidale.
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