# Object-Place Recognition Learning Triggers Rapid Induction of Plasticity-Related Immediate Early Genes and Synaptic Proteins in the Rat Dentate Gyrus

Authors: Jonathan Soulé; Zsuzsa Penke; Tambudzai Kanhema; Maria Nordheim Alme; Serge Laroche; Clive R. Bramham
Journal: Neural Plasticity
 (2008)

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## Abstract

Long-term recognition memory requires protein synthesis, but little is known about the coordinate regulation of specific genes. Here, we examined expression of the plasticity-associated immediate early genes (Arc, Zif268, and Narp) in the dentate gyrus following long-term object-place recognition learning in rats. RT-PCR analysis from dentate gyrus tissue collected shortly after training did not reveal learning-specific changes in Arc mRNA expression.  In situ hybridization and immunohistochemistry were therefore used to assess possible sparse effects on gene expression. Learning about objects increased the density of granule cells expressing Arc, and   to a lesser extent Narp, specifically in the dorsal blade of the dentate gyrus, while Zif268 expression was elevated across both blades. Thus, object-place recognition triggers rapid, blade-specific upregulation of plasticity-associated immediate early genes. Furthermore, Western blot analysis of dentate gyrus homogenates demonstrated concomitant upregulation of three postsynaptic density proteins (Arc, PSD-95, andα-CaMKII) with key roles in long-term synaptic plasticity and long-term memory.


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## Body

## 1. Introduction

Memory consolidation is thought
to rely on long-lasting, activity-dependent modifications of synaptic strength
and remodeling of neural network connectivity. For example, both
hippocampal-dependent learning and long-term potentiation (LTP) are associated
with cytoarchitectural reorganization of synapses, including thickening of the
postsynaptic density and expansion of the dendritic spine head. Such stable
structural alterations typically require new gene expression, protein
synthesis, as well as local actin polymerization [1–5].
Several lines of evidence implicate rapid, activity-dependent expression of immediate early genes (IEGs) in
consolidation of memory and long-term synaptic plasticity.IEGs encode a diverse
set of gene products that include secreted proteins, cytoplasmic enzymes, and
inducible transcription factors. Critical roles in consolidation of memory and
LTP have been identified for two IEGs, activity-regulated cytoskeleton
associated protein/activity-regulated gene 3.1 (Arc/Arg3.1), and Zif268 (also
known as Egr1, Krox24, and NGFI-A). Thus, gene
knockout or knockdown (antisense) of Arc [6, 7]
or Zif268 [8, 9] produces selective defects in
diverse types of long-term memory as well as in maintenance of late phase LTP
in the dentate gyrus (DG). Upon induction, Arc mRNA is rapidly
transported to dendrites where it undergoes local translation [10–12].
The Arc protein is implicated in control of actin polymerization at synapses
and regulation of AMPA-type glutamate receptor trafficking [13–16]. Zif268, a zinc-finger transcription factor of the Egr family, is implicated in
the control of gene networks [17, 18].
Arc and Zif268 are now widely used as markers of neuronal activation and
plasticity during memory formation [19–21].The neurotrophin, brain-derived neurotrophic factor (BDNF), is a major
regulator of protein synthesis-dependent consolidation of hippocampal memory [22–25]. For example, a BDNF-dependent de
novo protein-synthesis phase is necessary for memory formation,
consolidation, and persistence of hippocampus-dependent inhibitory avoidance
learning [26–29]. Recent work has revealed a
stringent requirement for Arc
synthesis in LTP elicited by either BDNF infusion or high-frequency stimulation
(HFS) in the dentate gyrus [13]. Another IEG induced by BDNF
infusion into the dentate gyrus is neuronal activity-regulated pentraxin (Narp)
[30].
Narp has been implicated in synapse formation and maturation during development
and induces clustering of AMPA receptors at excitatory synapses [31–33]. Interestingly, however, the immediate early
gene Zif268, which plays a critical role in HFS-induced LTP and long-term
memory, is not upregulated in response to in vivo infusion of BDNF [30, 34].Recognition memory can be assessed in rodents in various behavioral tasks
such as novel object recognition [35] or object-place recognition,
tasks based on rats’ innate propensity to explore novel rather than familiar
objects or to preferentially explore displaced objects. The involvement of the
hippocampal formation in the neural circuitry supporting recognition memory has
been shown by lesion studies [36–40]. In terms of molecular
mechanisms, certain molecules
such as the MAPK/ERK [41], the transcription factor CREB [42, 43] as well as the IEGs Arc [7], Zif268 [9, 44], and Egr3 [45] have been shown to be crucial for
the formation of long-term object recognition memory.Studies on the molecular
mechanisms of recognition memory have relied mainly on the behavioral analysis
of knockout mice. Thus, little is known about the coordinate regulation and
dynamics of gene expression and protein synthesis. Here, we studied the coordinate
expression of Arc, Narp, and Zif268 in the dentate gyrus after training rats in
an object-place recognition task. In this task leading to the formation of
long-term object-place recognition memory, rats explored three different
objects in a familiar environment. Animals remember the nature of the
encountered objects as well as their location in the environment, thus placing
a demand on spatial memory and hippocampal function [44, 46].A key feature of dendritic remodeling occurring during learning is likely
to be the coordinate synthesis and integration of protein constituents of the
postsynaptic density (PSD) complex of excitatory synapses. Arc localizes to the
PSD and is thought to play a key role in LTP by stabilizing nascent filamentous
actin [3, 13, 47–49]. In addition to Arc, the PSD proteins PSD-95 and α-CaMKII play major roles in
regulating the composition and function of the postsynaptic element during LTP
and memory formation [50–53].
All of these proteins can also be synthesized from local mRNAs in dendrites [11, 12, 54–56]. We therefore investigated coordinate regulation of key protein
constituents of the PSD, Arc, α-CaMKII, and PSD-95, in the dentate gyrus
following recognition learning.


## 2. Experimental Procedures

### 2.1. Animals

Adult male Sprague-Dawley rats (n=80; Iffa-Credo, France)
weighing 300–350 g at the
beginning of the experiment (mean age 8 weeks, range 7.5–9 weeks) were
used as subjects. After arrival in the laboratory, they were housed in pairs
under constant temperature and lighting conditions (22°C, light/dark cycle of 12:12 hours, lights on at 07:00). Rat chow and tap
water were provided ad libitum. All
efforts were made to minimize the number of animals and their suffering
throughout the experiments. Experiments were performed in accordance with the
European Communities Council Directive of November the 24th 1986 (86/609/EEC)
and the French National Committee (87/848). All experiments were conducted
during the light phase.


#### 2.1.1. Long-Term Memory for Spatial Configuration  of Objects

To test long-term object-place recognition
memory, we used a modified version of the standard object recognition task [35], based on the discrimination between a novel and a familiar spatial
location of an object [44]. Fifteen rats were handled twice daily for 4 days, followed by a 3-day
rest, before the beginning of the experiments. The experimental apparatus was a
cylindrical open field made of metal and painted black (diameter 90 cm, height
40 cm), with wood shavings on the floor, and located in a room with dim
lighting and constant background noise. A cue card was placed at a fixed location
on the top of the wall of the open field to facilitate spatial mapping of each
object. Rats were habituated to the open field in the absence of objects for
2×5-minute exploration a day for 3 days. The next day (acquisition session),
three objects were placed in the open field, and rats were allowed to explore
them for four 5-minute sessions with 5-minute intervals. The objects consisted
of assembled interlocking plastic block pieces (Lego-blocks) of different
shapes and colors. Retention testing, lasting 5 minutes, was conducted 2 or 3
days after the acquisition session in the same arena with the spatial position
of one object changed to a new position. Care was taken to displace objects in
a counterbalanced manner across animals, so that each of the objects was
displaced in a randomized manner in terms of nature of the object and position
to avoid any bias that could arise if some animals would have shown a
preference for an object or a place. To examine retention performance at the
two delays in the same animals, rats were tested twice in the
acquisition-retention sequence with different sets
of objects. There was a 2-day rest interval between the retention session and the next acquisition session of the first sequence and the acquisition
session of the second sequence. During the acquisition and retention phase, the
time spent exploring each object was recorded. The criteria for exploration
were based strictly on active exploration, where rats had both forelimbs within
a circle of 5 cm around an object, head oriented toward it or touching it with
their noses.Time spent exploring each object was expressed
in percent of total time spent exploring all the objects. Exploration
time for each object during the acquisition session was analyzed using ANOVA. For the retention session, exploration of the displaced
object was expressed as a percentage of the total time of object exploration
and compared with chance level (33.33%) using Student’s one-samplet-test.
The significance level was set at P<.05.


#### 2.1.2. Experimental Groups for In Situ Hybridization and Immunohistochemistry

Rats were submitted to one of five different
treatments. Cage control rats (CC,n=4) were handled daily as described in the
methods (on the same days as the other animals), and were taken directly from
their home cage and sacrificed on the same days as rats from the other groups.
Trained rats were submitted to habituation and the object recognition acquisition
session as described above, and sacrificed 10 minutes (L10, n=4) or 60 minutes
(L60, n=4) after the end of the acquisition session. Control rats matched to
the trained rats were handled and habituated as described above, and on the day
following the last habituation session, they were re-exposed to the same open
field without objects, which they explored according to the same time schedule
as L10 and L60 rats (four 5-minute sessions with 5-minute intervals) and were
killed 10 minutes (C10, n=4) or 60 minutes (C60, n=4) later.Rats were perfused transcardially under urethane anesthesia (1 mg/kg body weight) with
0.1 M phosphate buffer (PB; pH 7.4) containing 1 mM orthovanadate, then with
phosphate buffer containing 4% paraformaldehyde. Brains were postfixed in the
same fixative solution overnight at 4°C, transferred to a phosphate buffer
containing 0.1% sodium azide, and stored at 4°C. Brains were incubated
in PB containing 30% sucrose overnight at room temperature. On the following
day, coronal sections (30 μm-thick) were obtained on a Leica CM3050S cryostat
equipped with a Richard-Allan Sec35e blade. Chamber and object temperatures were
set to −20°C and −14°C, respectively. Sections were
immediately stored in PB containing 0.1% sodium azide at 4°C. For immunohistochemistry
and in situ hybridization,
sections corresponding to the dorsal hippocampus (between approximately −3.3 mm
and −4.5 mm from Bregma) were selected.


#### 2.1.3. Experimental Groups for RT-PCR and Western Blotting

Rats were submitted to the same behavioral protocols as for in situ hybridization and immunohistochemistry (n=9 for each group).
Animals were decapitated under urethane anesthesia; their brain was quickly
removed and rinsed with ice-cold,  sterile 0.9% saline. The hippocampus
was quickly removed and the dentate gyrus was dissected on ice, frozen in
liquid nitrogen and stored at −80°C.


#### 2.1.4. Poly(A) RNA and cDNA Preparation

Poly(A) RNA was isolated using the Dynabeads mRNA direct kit
(Dynal, Oslo, Norway) according to the
manufacturer’s protocol with minor modifications.
70μl magnetic beads
were used per sample and  the isolated poly(A) RNA fraction was eluted in 2×30μl of 10 mM Tris/HCl, pH 8.0. The yield and quality of the poly(A) RNA
were determined by measuring the absorbance at 260/280 nm. 60 ng poly(A) RNA
was reversed-transcribed using the Superscript First-Strand Synthesis
Kit (Invitrogen) and the resulting cDNA was diluted 20-fold.


#### 2.1.5. Semiquantitative Real-Time PCR and Normalization Strategies

Semiquantitative real-time PCR was performed on an iCycler
(Bio-Rad) using cDNA from individual animals and the iQ SYBR Green Supermix. 5μl cDNA were added to the PCR reaction mix to yield a total of 25 μl. PCR
quantification was performed in triplicate, and the fluorescence signal was
quantified by the second derivative maximum method using the iCycler iQ
Real-Time detection system software. Primers used are given in Table 1. Data
were normalized with the geometric mean of the three normalization genes polyubiquitin, Cyclophilin,
and HPRT. Primer sequences in 5′ to 3′ direction and annealing temperatures are
also given in Table 1.Table 1
Overview over primer sequences and accession numbers for the analyzed genes.
GenePrimer sequenceAnn temp. (C°)Acc. numberArcFw: CCCAGTCTGTGGCTTTTGTCA60NM019361Bw: GTGTCAGCCCCAGCTCAATCCyclophilinFw: AGCACTGGGGAGAAAGGATT60BC059141Bw: GATGCCAGGACCTGTATGCTPolyubiquitinFw: GGCAAGACCATCACCCTAGA60BC070919Bw: GCAGGGTTGACTCTTTCTGGHPRTFw: GCAGACTTTGCTTTCCTTGG60NM_012583Bw: TCCACTTTCGCTGATGACAC


### 2.2. Riboprobes

Arc riboprobes were prepared
from a cDNA insert matching the first 2975 nucleotides of the Arc mRNA (GenBank
accession number NM_019361) cloned into the pCRII-TOPO  vector (Invitrogen). Antisense and sense
probes were transcribed from linearized plasmids using T7 and SP6 polymerases
in the presence of DIG labelling mix according to the manufacturer’s
instructions (Roche).


### 2.3. In Situ Hybridization

Floating sections were placed in PBS for 5 minutes, treated with
proteinase K (10μg/mL) for 5 minutes at 37°C, and postfixed (5 minutes with 4%
PFA/PBS). After postfixation, sections
were treated with 0.25% acetic anhydride in 0.1 M TEA (pH = 8.0) for 10 minutes,
washed twice in 2xSSC, and placed for 10 minutes in prehybridization buffer.
Riboprobes were applied onto the sections and hybridization was performed in a
humidified chamber at 60°C for at least 16 hours. Sections were washed twice
with 2xSSC at RT for 30 minutes, once with 50% formamide in 2xSSC at 65°C,
rinsed in 2xSSC at 37°C, incubated with 20 μg/mL RNase A at 37°C for 30 minutes
and incubated in RNase A buffer for at 65°C for 30 minutes. After blocking in
2% blocking reagent for one hour at RT, AP-coupled anti-DIG antibody (1:2000,
Roche) was applied. Visualization was accomplished with the chromogenic
substrates NBT and BCIP (Roche). Control performed with the Arc sense riboprobes did not provide any staining. Arc-positive
cells exhibited characteristic staining in the soma, the perinuclear region
and/or in nuclear foci.


### 2.4. Antibodies

Primary antibodies used for immunoblotting were as follows: Arc H-300 (sc-15325, 1:200,
Santa Cruz),β-actin (clone AC-15, 1:5000, Sigma), PSD-95 (MA1-045, 1:500, Affinity BioReagents)
and α-CaMKII (mouse monoclonal, IgG1 MA1-048, 1:2000).
For immunochemistry, the antibodies were as follows: Zif268 (sc-110,
1:200, Santa Cruz) and Narp (polyclonal
antibody, 1:250, gift from Richard O’Brien, Johns Hopkins University).


### 2.5. Immunohistochemistry

Sections were first treated with PB containing 100 mM glycine (Sigma),
then washed in PBT (PB containing 0.1% Tween 20), incubated in 0,3% H2O2
diluted in PBT, permeabilized for 20 minutes with 0.5% Triton X-100 diluted in
PBT, rinsed and immersed for 30 minutes in blocking buffer (4% BSA and 4%
donkey serum in PBT). They were then incubated overnight at 4°C with
the primary antibody diluted in blocking buffer. After three washes in PBT,
biotinylated secondary antibody was applied for 1 hour at RT. Sections were
then washed in PBT, incubated for 1 hour in Streptavidin-HRP diluted in PBT,
washed in PBT and finally processed for DAB staining. Zif268-positive cells
were defined by their characteristic nuclear staining whereas Narp-positive
cells presented somatic staining.


#### 2.5.1. Image Acquisition and Analysis

Pictures were taken on a Nikon Eclipse 80i microscope coupled to a Nikon
DS-5M camera. Representative
pictures were acquired with a 4× objective whereas evaluation of the density of
granule cells positively marked by in
situ hybridization and immunohistochemistry was carried out using 10×
and 20× objectives. The NIS-elements Ar2.3 software (Nikon) was used for determination
of positively stained cells and the area covered by the granule cell layer.
Counting of stained cells was accomplished by systematic scanning of the entire
thickness of nonconsecutive sections to avoid under- and overestimation of the
cell densities. The density calculation was based on the number of positive
cell bodies or nuclei within the area bounded by the granule cell layer of the
upper or lower blade of the dentate gyrus.


#### 2.5.2. SDS-PAGE and Western Blotting

Protein levels in homogenate samples were determined using the BCA
Protein Assay kit (Pierce). Equal amounts of protein were loaded onto SDS-PAGE
gels (10%) and run overnight at constant 10 mA. Separated proteins were
transferred to a nitrocellulose membrane (Hybond-C, Amersham- GE Healthcare, Oslo, Norway)
at a constant voltage of 20 V overnight or 100 V for one hour. Membranes were
blocked on a gyro-rocker for 1 hour at room temperature (RT). Blocking buffer (BB) consisted of TBST
(Tris-buffered saline/0.1% Tween-20)
and 5% BSA or 5% nonfat dry milk. The primary antibodies were dissolved in BB
containing 5% BSA and the blots incubated for 2 hours at RT or 4°C overnight
with constant shaking. Following three washes with TBST, blots were incubated
for 1 hour in horseradish peroxidase-conjugated secondary antibody dissolved in
TBST. The blots were washed three times with TBST and proteins were visualized
using enhanced chemiluminescence (ECL Western Blotting Analysis System, Pierce
ECL Western Blotting Substrate). Blots were stripped with Restore Plus Western
Blot Stripping buffer (Pierce, Rockford,
USA) at room
temperature for 20 minutes and reprobed with another antibody detecting the
protein of interest. Optical density values for each protein were normalized
relative to values obtained with β-actin antibody.


### 2.6. Statistical Analysis

All data are presented as mean ± SEM. Statistical analysis was based on ANOVA
and Tukey’s post hoc test was used for further comparisons between the C10,
L10, C60, and L60 groups. The CC group used for normalization was independently
compared with the other groups using ANOVA. The significance level was set atP≤.05.


## 2.1. Animals

Adult male Sprague-Dawley rats (n=80; Iffa-Credo, France)
weighing 300–350 g at the
beginning of the experiment (mean age 8 weeks, range 7.5–9 weeks) were
used as subjects. After arrival in the laboratory, they were housed in pairs
under constant temperature and lighting conditions (22°C, light/dark cycle of 12:12 hours, lights on at 07:00). Rat chow and tap
water were provided ad libitum. All
efforts were made to minimize the number of animals and their suffering
throughout the experiments. Experiments were performed in accordance with the
European Communities Council Directive of November the 24th 1986 (86/609/EEC)
and the French National Committee (87/848). All experiments were conducted
during the light phase.


### 2.1.1. Long-Term Memory for Spatial Configuration  of Objects

To test long-term object-place recognition
memory, we used a modified version of the standard object recognition task [35], based on the discrimination between a novel and a familiar spatial
location of an object [44]. Fifteen rats were handled twice daily for 4 days, followed by a 3-day
rest, before the beginning of the experiments. The experimental apparatus was a
cylindrical open field made of metal and painted black (diameter 90 cm, height
40 cm), with wood shavings on the floor, and located in a room with dim
lighting and constant background noise. A cue card was placed at a fixed location
on the top of the wall of the open field to facilitate spatial mapping of each
object. Rats were habituated to the open field in the absence of objects for
2×5-minute exploration a day for 3 days. The next day (acquisition session),
three objects were placed in the open field, and rats were allowed to explore
them for four 5-minute sessions with 5-minute intervals. The objects consisted
of assembled interlocking plastic block pieces (Lego-blocks) of different
shapes and colors. Retention testing, lasting 5 minutes, was conducted 2 or 3
days after the acquisition session in the same arena with the spatial position
of one object changed to a new position. Care was taken to displace objects in
a counterbalanced manner across animals, so that each of the objects was
displaced in a randomized manner in terms of nature of the object and position
to avoid any bias that could arise if some animals would have shown a
preference for an object or a place. To examine retention performance at the
two delays in the same animals, rats were tested twice in the
acquisition-retention sequence with different sets
of objects. There was a 2-day rest interval between the retention session and the next acquisition session of the first sequence and the acquisition
session of the second sequence. During the acquisition and retention phase, the
time spent exploring each object was recorded. The criteria for exploration
were based strictly on active exploration, where rats had both forelimbs within
a circle of 5 cm around an object, head oriented toward it or touching it with
their noses.Time spent exploring each object was expressed
in percent of total time spent exploring all the objects. Exploration
time for each object during the acquisition session was analyzed using ANOVA. For the retention session, exploration of the displaced
object was expressed as a percentage of the total time of object exploration
and compared with chance level (33.33%) using Student’s one-samplet-test.
The significance level was set at P<.05.


### 2.1.2. Experimental Groups for In Situ Hybridization and Immunohistochemistry

Rats were submitted to one of five different
treatments. Cage control rats (CC,n=4) were handled daily as described in the
methods (on the same days as the other animals), and were taken directly from
their home cage and sacrificed on the same days as rats from the other groups.
Trained rats were submitted to habituation and the object recognition acquisition
session as described above, and sacrificed 10 minutes (L10, n=4) or 60 minutes
(L60, n=4) after the end of the acquisition session. Control rats matched to
the trained rats were handled and habituated as described above, and on the day
following the last habituation session, they were re-exposed to the same open
field without objects, which they explored according to the same time schedule
as L10 and L60 rats (four 5-minute sessions with 5-minute intervals) and were
killed 10 minutes (C10, n=4) or 60 minutes (C60, n=4) later.Rats were perfused transcardially under urethane anesthesia (1 mg/kg body weight) with
0.1 M phosphate buffer (PB; pH 7.4) containing 1 mM orthovanadate, then with
phosphate buffer containing 4% paraformaldehyde. Brains were postfixed in the
same fixative solution overnight at 4°C, transferred to a phosphate buffer
containing 0.1% sodium azide, and stored at 4°C. Brains were incubated
in PB containing 30% sucrose overnight at room temperature. On the following
day, coronal sections (30 μm-thick) were obtained on a Leica CM3050S cryostat
equipped with a Richard-Allan Sec35e blade. Chamber and object temperatures were
set to −20°C and −14°C, respectively. Sections were
immediately stored in PB containing 0.1% sodium azide at 4°C. For immunohistochemistry
and in situ hybridization,
sections corresponding to the dorsal hippocampus (between approximately −3.3 mm
and −4.5 mm from Bregma) were selected.


### 2.1.3. Experimental Groups for RT-PCR and Western Blotting

Rats were submitted to the same behavioral protocols as for in situ hybridization and immunohistochemistry (n=9 for each group).
Animals were decapitated under urethane anesthesia; their brain was quickly
removed and rinsed with ice-cold,  sterile 0.9% saline. The hippocampus
was quickly removed and the dentate gyrus was dissected on ice, frozen in
liquid nitrogen and stored at −80°C.


### 2.1.4. Poly(A) RNA and cDNA Preparation

Poly(A) RNA was isolated using the Dynabeads mRNA direct kit
(Dynal, Oslo, Norway) according to the
manufacturer’s protocol with minor modifications.
70μl magnetic beads
were used per sample and  the isolated poly(A) RNA fraction was eluted in 2×30μl of 10 mM Tris/HCl, pH 8.0. The yield and quality of the poly(A) RNA
were determined by measuring the absorbance at 260/280 nm. 60 ng poly(A) RNA
was reversed-transcribed using the Superscript First-Strand Synthesis
Kit (Invitrogen) and the resulting cDNA was diluted 20-fold.


### 2.1.5. Semiquantitative Real-Time PCR and Normalization Strategies

Semiquantitative real-time PCR was performed on an iCycler
(Bio-Rad) using cDNA from individual animals and the iQ SYBR Green Supermix. 5μl cDNA were added to the PCR reaction mix to yield a total of 25 μl. PCR
quantification was performed in triplicate, and the fluorescence signal was
quantified by the second derivative maximum method using the iCycler iQ
Real-Time detection system software. Primers used are given in Table 1. Data
were normalized with the geometric mean of the three normalization genes polyubiquitin, Cyclophilin,
and HPRT. Primer sequences in 5′ to 3′ direction and annealing temperatures are
also given in Table 1.Table 1
Overview over primer sequences and accession numbers for the analyzed genes.
GenePrimer sequenceAnn temp. (C°)Acc. numberArcFw: CCCAGTCTGTGGCTTTTGTCA60NM019361Bw: GTGTCAGCCCCAGCTCAATCCyclophilinFw: AGCACTGGGGAGAAAGGATT60BC059141Bw: GATGCCAGGACCTGTATGCTPolyubiquitinFw: GGCAAGACCATCACCCTAGA60BC070919Bw: GCAGGGTTGACTCTTTCTGGHPRTFw: GCAGACTTTGCTTTCCTTGG60NM_012583Bw: TCCACTTTCGCTGATGACAC


## 2.1.1. Long-Term Memory for Spatial Configuration  of Objects

To test long-term object-place recognition
memory, we used a modified version of the standard object recognition task [35], based on the discrimination between a novel and a familiar spatial
location of an object [44]. Fifteen rats were handled twice daily for 4 days, followed by a 3-day
rest, before the beginning of the experiments. The experimental apparatus was a
cylindrical open field made of metal and painted black (diameter 90 cm, height
40 cm), with wood shavings on the floor, and located in a room with dim
lighting and constant background noise. A cue card was placed at a fixed location
on the top of the wall of the open field to facilitate spatial mapping of each
object. Rats were habituated to the open field in the absence of objects for
2×5-minute exploration a day for 3 days. The next day (acquisition session),
three objects were placed in the open field, and rats were allowed to explore
them for four 5-minute sessions with 5-minute intervals. The objects consisted
of assembled interlocking plastic block pieces (Lego-blocks) of different
shapes and colors. Retention testing, lasting 5 minutes, was conducted 2 or 3
days after the acquisition session in the same arena with the spatial position
of one object changed to a new position. Care was taken to displace objects in
a counterbalanced manner across animals, so that each of the objects was
displaced in a randomized manner in terms of nature of the object and position
to avoid any bias that could arise if some animals would have shown a
preference for an object or a place. To examine retention performance at the
two delays in the same animals, rats were tested twice in the
acquisition-retention sequence with different sets
of objects. There was a 2-day rest interval between the retention session and the next acquisition session of the first sequence and the acquisition
session of the second sequence. During the acquisition and retention phase, the
time spent exploring each object was recorded. The criteria for exploration
were based strictly on active exploration, where rats had both forelimbs within
a circle of 5 cm around an object, head oriented toward it or touching it with
their noses.Time spent exploring each object was expressed
in percent of total time spent exploring all the objects. Exploration
time for each object during the acquisition session was analyzed using ANOVA. For the retention session, exploration of the displaced
object was expressed as a percentage of the total time of object exploration
and compared with chance level (33.33%) using Student’s one-samplet-test.
The significance level was set at P<.05.


## 2.1.2. Experimental Groups for In Situ Hybridization and Immunohistochemistry

Rats were submitted to one of five different
treatments. Cage control rats (CC,n=4) were handled daily as described in the
methods (on the same days as the other animals), and were taken directly from
their home cage and sacrificed on the same days as rats from the other groups.
Trained rats were submitted to habituation and the object recognition acquisition
session as described above, and sacrificed 10 minutes (L10, n=4) or 60 minutes
(L60, n=4) after the end of the acquisition session. Control rats matched to
the trained rats were handled and habituated as described above, and on the day
following the last habituation session, they were re-exposed to the same open
field without objects, which they explored according to the same time schedule
as L10 and L60 rats (four 5-minute sessions with 5-minute intervals) and were
killed 10 minutes (C10, n=4) or 60 minutes (C60, n=4) later.Rats were perfused transcardially under urethane anesthesia (1 mg/kg body weight) with
0.1 M phosphate buffer (PB; pH 7.4) containing 1 mM orthovanadate, then with
phosphate buffer containing 4% paraformaldehyde. Brains were postfixed in the
same fixative solution overnight at 4°C, transferred to a phosphate buffer
containing 0.1% sodium azide, and stored at 4°C. Brains were incubated
in PB containing 30% sucrose overnight at room temperature. On the following
day, coronal sections (30 μm-thick) were obtained on a Leica CM3050S cryostat
equipped with a Richard-Allan Sec35e blade. Chamber and object temperatures were
set to −20°C and −14°C, respectively. Sections were
immediately stored in PB containing 0.1% sodium azide at 4°C. For immunohistochemistry
and in situ hybridization,
sections corresponding to the dorsal hippocampus (between approximately −3.3 mm
and −4.5 mm from Bregma) were selected.


## 2.1.3. Experimental Groups for RT-PCR and Western Blotting

Rats were submitted to the same behavioral protocols as for in situ hybridization and immunohistochemistry (n=9 for each group).
Animals were decapitated under urethane anesthesia; their brain was quickly
removed and rinsed with ice-cold,  sterile 0.9% saline. The hippocampus
was quickly removed and the dentate gyrus was dissected on ice, frozen in
liquid nitrogen and stored at −80°C.


## 2.1.4. Poly(A) RNA and cDNA Preparation

Poly(A) RNA was isolated using the Dynabeads mRNA direct kit
(Dynal, Oslo, Norway) according to the
manufacturer’s protocol with minor modifications.
70μl magnetic beads
were used per sample and  the isolated poly(A) RNA fraction was eluted in 2×30μl of 10 mM Tris/HCl, pH 8.0. The yield and quality of the poly(A) RNA
were determined by measuring the absorbance at 260/280 nm. 60 ng poly(A) RNA
was reversed-transcribed using the Superscript First-Strand Synthesis
Kit (Invitrogen) and the resulting cDNA was diluted 20-fold.


## 2.1.5. Semiquantitative Real-Time PCR and Normalization Strategies

Semiquantitative real-time PCR was performed on an iCycler
(Bio-Rad) using cDNA from individual animals and the iQ SYBR Green Supermix. 5μl cDNA were added to the PCR reaction mix to yield a total of 25 μl. PCR
quantification was performed in triplicate, and the fluorescence signal was
quantified by the second derivative maximum method using the iCycler iQ
Real-Time detection system software. Primers used are given in Table 1. Data
were normalized with the geometric mean of the three normalization genes polyubiquitin, Cyclophilin,
and HPRT. Primer sequences in 5′ to 3′ direction and annealing temperatures are
also given in Table 1.Table 1
Overview over primer sequences and accession numbers for the analyzed genes.
GenePrimer sequenceAnn temp. (C°)Acc. numberArcFw: CCCAGTCTGTGGCTTTTGTCA60NM019361Bw: GTGTCAGCCCCAGCTCAATCCyclophilinFw: AGCACTGGGGAGAAAGGATT60BC059141Bw: GATGCCAGGACCTGTATGCTPolyubiquitinFw: GGCAAGACCATCACCCTAGA60BC070919Bw: GCAGGGTTGACTCTTTCTGGHPRTFw: GCAGACTTTGCTTTCCTTGG60NM_012583Bw: TCCACTTTCGCTGATGACAC


## 2.2. Riboprobes

Arc riboprobes were prepared
from a cDNA insert matching the first 2975 nucleotides of the Arc mRNA (GenBank
accession number NM_019361) cloned into the pCRII-TOPO  vector (Invitrogen). Antisense and sense
probes were transcribed from linearized plasmids using T7 and SP6 polymerases
in the presence of DIG labelling mix according to the manufacturer’s
instructions (Roche).


## 2.3. In Situ Hybridization

Floating sections were placed in PBS for 5 minutes, treated with
proteinase K (10μg/mL) for 5 minutes at 37°C, and postfixed (5 minutes with 4%
PFA/PBS). After postfixation, sections
were treated with 0.25% acetic anhydride in 0.1 M TEA (pH = 8.0) for 10 minutes,
washed twice in 2xSSC, and placed for 10 minutes in prehybridization buffer.
Riboprobes were applied onto the sections and hybridization was performed in a
humidified chamber at 60°C for at least 16 hours. Sections were washed twice
with 2xSSC at RT for 30 minutes, once with 50% formamide in 2xSSC at 65°C,
rinsed in 2xSSC at 37°C, incubated with 20 μg/mL RNase A at 37°C for 30 minutes
and incubated in RNase A buffer for at 65°C for 30 minutes. After blocking in
2% blocking reagent for one hour at RT, AP-coupled anti-DIG antibody (1:2000,
Roche) was applied. Visualization was accomplished with the chromogenic
substrates NBT and BCIP (Roche). Control performed with the Arc sense riboprobes did not provide any staining. Arc-positive
cells exhibited characteristic staining in the soma, the perinuclear region
and/or in nuclear foci.


## 2.4. Antibodies

Primary antibodies used for immunoblotting were as follows: Arc H-300 (sc-15325, 1:200,
Santa Cruz),β-actin (clone AC-15, 1:5000, Sigma), PSD-95 (MA1-045, 1:500, Affinity BioReagents)
and α-CaMKII (mouse monoclonal, IgG1 MA1-048, 1:2000).
For immunochemistry, the antibodies were as follows: Zif268 (sc-110,
1:200, Santa Cruz) and Narp (polyclonal
antibody, 1:250, gift from Richard O’Brien, Johns Hopkins University).


## 2.5. Immunohistochemistry

Sections were first treated with PB containing 100 mM glycine (Sigma),
then washed in PBT (PB containing 0.1% Tween 20), incubated in 0,3% H2O2
diluted in PBT, permeabilized for 20 minutes with 0.5% Triton X-100 diluted in
PBT, rinsed and immersed for 30 minutes in blocking buffer (4% BSA and 4%
donkey serum in PBT). They were then incubated overnight at 4°C with
the primary antibody diluted in blocking buffer. After three washes in PBT,
biotinylated secondary antibody was applied for 1 hour at RT. Sections were
then washed in PBT, incubated for 1 hour in Streptavidin-HRP diluted in PBT,
washed in PBT and finally processed for DAB staining. Zif268-positive cells
were defined by their characteristic nuclear staining whereas Narp-positive
cells presented somatic staining.


### 2.5.1. Image Acquisition and Analysis

Pictures were taken on a Nikon Eclipse 80i microscope coupled to a Nikon
DS-5M camera. Representative
pictures were acquired with a 4× objective whereas evaluation of the density of
granule cells positively marked by in
situ hybridization and immunohistochemistry was carried out using 10×
and 20× objectives. The NIS-elements Ar2.3 software (Nikon) was used for determination
of positively stained cells and the area covered by the granule cell layer.
Counting of stained cells was accomplished by systematic scanning of the entire
thickness of nonconsecutive sections to avoid under- and overestimation of the
cell densities. The density calculation was based on the number of positive
cell bodies or nuclei within the area bounded by the granule cell layer of the
upper or lower blade of the dentate gyrus.


### 2.5.2. SDS-PAGE and Western Blotting

Protein levels in homogenate samples were determined using the BCA
Protein Assay kit (Pierce). Equal amounts of protein were loaded onto SDS-PAGE
gels (10%) and run overnight at constant 10 mA. Separated proteins were
transferred to a nitrocellulose membrane (Hybond-C, Amersham- GE Healthcare, Oslo, Norway)
at a constant voltage of 20 V overnight or 100 V for one hour. Membranes were
blocked on a gyro-rocker for 1 hour at room temperature (RT). Blocking buffer (BB) consisted of TBST
(Tris-buffered saline/0.1% Tween-20)
and 5% BSA or 5% nonfat dry milk. The primary antibodies were dissolved in BB
containing 5% BSA and the blots incubated for 2 hours at RT or 4°C overnight
with constant shaking. Following three washes with TBST, blots were incubated
for 1 hour in horseradish peroxidase-conjugated secondary antibody dissolved in
TBST. The blots were washed three times with TBST and proteins were visualized
using enhanced chemiluminescence (ECL Western Blotting Analysis System, Pierce
ECL Western Blotting Substrate). Blots were stripped with Restore Plus Western
Blot Stripping buffer (Pierce, Rockford,
USA) at room
temperature for 20 minutes and reprobed with another antibody detecting the
protein of interest. Optical density values for each protein were normalized
relative to values obtained with β-actin antibody.


## 2.5.1. Image Acquisition and Analysis

Pictures were taken on a Nikon Eclipse 80i microscope coupled to a Nikon
DS-5M camera. Representative
pictures were acquired with a 4× objective whereas evaluation of the density of
granule cells positively marked by in
situ hybridization and immunohistochemistry was carried out using 10×
and 20× objectives. The NIS-elements Ar2.3 software (Nikon) was used for determination
of positively stained cells and the area covered by the granule cell layer.
Counting of stained cells was accomplished by systematic scanning of the entire
thickness of nonconsecutive sections to avoid under- and overestimation of the
cell densities. The density calculation was based on the number of positive
cell bodies or nuclei within the area bounded by the granule cell layer of the
upper or lower blade of the dentate gyrus.


## 2.5.2. SDS-PAGE and Western Blotting

Protein levels in homogenate samples were determined using the BCA
Protein Assay kit (Pierce). Equal amounts of protein were loaded onto SDS-PAGE
gels (10%) and run overnight at constant 10 mA. Separated proteins were
transferred to a nitrocellulose membrane (Hybond-C, Amersham- GE Healthcare, Oslo, Norway)
at a constant voltage of 20 V overnight or 100 V for one hour. Membranes were
blocked on a gyro-rocker for 1 hour at room temperature (RT). Blocking buffer (BB) consisted of TBST
(Tris-buffered saline/0.1% Tween-20)
and 5% BSA or 5% nonfat dry milk. The primary antibodies were dissolved in BB
containing 5% BSA and the blots incubated for 2 hours at RT or 4°C overnight
with constant shaking. Following three washes with TBST, blots were incubated
for 1 hour in horseradish peroxidase-conjugated secondary antibody dissolved in
TBST. The blots were washed three times with TBST and proteins were visualized
using enhanced chemiluminescence (ECL Western Blotting Analysis System, Pierce
ECL Western Blotting Substrate). Blots were stripped with Restore Plus Western
Blot Stripping buffer (Pierce, Rockford,
USA) at room
temperature for 20 minutes and reprobed with another antibody detecting the
protein of interest. Optical density values for each protein were normalized
relative to values obtained with β-actin antibody.


## 2.6. Statistical Analysis

All data are presented as mean ± SEM. Statistical analysis was based on ANOVA
and Tukey’s post hoc test was used for further comparisons between the C10,
L10, C60, and L60 groups. The CC group used for normalization was independently
compared with the other groups using ANOVA. The significance level was set atP≤.05.


## 3. Results

### 3.1. Long-Term Memory for the Spatial Configuration  of Objects

The training procedure involved habituation to the test arena, followed
by exposure to three objects at fixed locations on four consecutive 5-minute
sessions with 5-minute intervals (acquisition phase), and a retention test,
which was performed 2 or 3 days later. In the retention test, one of the
objects was displaced and the amount of time exploring the displaced object
relative to the total time of object exploration was determined. This paradigm
has been previously shown to induce long-term memory for  objects and
location of objects [44]. During the acquisition phase,
ANOVA did not show
significant differences between the time spent exploring the three objects
(time spent exploring the three objects for the 2-day delay: 33.0±2.0%,
38.9±2.2%, 28.1±1.3% of total time; F2,42=0.90, P=.41, for the 3-day
delay: 32.7±1.5%, 21.1±1.2%, 46.2±1.4% of total time; F2,42=2.68, P=.08). During
retention testing, rats spent significantly more time exploring
the displaced object than chance level (33.33%) at both the 2 and 3-day
retention intervals (time spent exploring the displaced object for the 2-day
delay: 45.9±1.3%
of total time; t14=3.27, P<.01; for the 3-day
delay: 39.3±2.3%
of total time; t14=2.54, P<.05), as shown in
Figures 1(a) and 1(b). This behavioral analysis shows that rats in our experimental
conditions were able to form a long-term object-place recognition memory.Performance at the 2-day and 3-day retention
intervals of the object-place recognition memory task. At both (a) 2-day
and (b) 3-day  delays after acquisition, rats (n=15 in each case) showed
preferential exploration of the displaced object. (c) Schematic representation
of the task. Asterisks indicate P≤.05 compared with chance level
(dashed line, 33.3%). Data are presented as mean ± SEM.
(a)(b)(c)


### 3.2. Object Recognition Training Induces Arc mRNA Expression in Granule Cells of the Dorsal Blade of the DG

We hypothesized that acquisition of different types of information about
the objects and their spatial location would be associated with rapid induction
of the immediate early gene Arc. This
issue was first addressed by semiquantitative RT-PCR analysis of Arc mRNA
levels in the microdissected DG. Surprisingly, no significant change in Arc mRNA levels could be observed
in C10, C60, L10, and L60 animals when compared with caged control (CC) animals
(Figure2, P>.05), indicating that Arc expression in the dentate
gyrus was not significantly affected by exploration of the arena with or without
the objects.Figure 2
Expression levels of Arc in the dentate gyrus
after object recognition. Fold change in mRNA levels (relative to the CC
group) is presented for Arc in the dentate gyrus of animals from all five
groups (n=8 for all groups, except L60, n=7). Data are presented as mean ± SEM.
Gene expression was normalized to control genes (see methods).Endogenous Arc-expressing granule cells represent a very low percentage (1–2%)
of the  total number  of granule cells in the DG. Following spatial behavioral
experience, the density of Arc-expressing cells increases specifically in the
dorsal (inner) blade, while the density in ventral (outer) blade remains nearly
unchanged [57]. We considered that such
sparse, blade-specific changes in gene expression may not be detected by PCR
analysis of whole DG homogenate samples. We therefore re-examined the effect of
learning about objects and their configuration on Arc mRNA expression using in situ hybridization (Figure 3). As
previously described, Arc-expressing cells were dispersed along both the dorsal
and the ventral blades of the DG of both the CC and trained groups (Figures 3(a)–3(d)). The granule
cell layer of CC animals presented an average density of 152.2±10.5
Arc-positive cells per mm2 in the dorsal blade whereas the ventral
blade presented an average density of 138.8±13.8  Arc-positive cells per
mm2. Figure 3(e) shows the normalized density of Arc mRNA-positive
granule cells in the dorsal and ventral blades of the DG following performance
of the recognition task. ANOVA revealed a blade effect (F(1,31)=64.310; P<.001), a time effect (F(1,31)=14.181; P<.001)
and a learning effect (F(1,31)=10.417; P=.004). In the
dorsal blade, a significant 2-fold increase in density was detected in L10
animals, relative to the CC group (P<.01), while the C10 group
exhibited a nonsignificant 1.4-fold increase relative to CC. The density of Arc
mRNA-positive cells in the dorsal blade remained elevated up to one hour after
training in the learning group. L60 animals displayed a significant 1.4-fold
increase when compared with CC levels (P<.01) and a 1.5-fold increase
in comparison to C60 levels (P=.02). In the ventral blade, a surprising
decrease in the density of Arc-positive cells was observed in the C10 and C60
group, relative to caged controls (P<.01), indicating that the
exploration of the environment induced a rapid and sustained decrease in Arc
expression that was specific to the ventral blade. Nonetheless, exposure to the
objects resulted in a 2-fold increase in Arc-expression at 10 minutes (P=.05)
relative to rats exposed only to the test arena. Interestingly, no effect of the presence of
the three objects was observed in the ventral blade at the 60-minute time
point. Thus, learning about objects in this recognition task resulted in rapid
and sparse increase in Arc mRNA expression in both blades of the DG. However, only the dorsal blade of the DG
exhibited a sustained increase in Arc mRNA expression up to one-hour
posttraining.Object recognition training increases Arc
mRNA expression in the dentate gyrus. Arc mRNA in situ hybridization reveals sparse expression of Arc in both
dorsal and ventral blades of the rat dentate gyrus of (a) CC, (b) C10,
and (c) L10   animals. (d) Higher magnification shows typical Arc mRNA localization in the cell body and
dendrites of granule cells. (e) Change in Arc-positive granule cell densities
(relative to the CC group) in the dorsal and the ventral blade of the dentate
gyrus across the C10, L10, C60, and L60 groups. Data are presented as mean
± SEM (n=4 for all groups). Asterisks indicate P≤.05 (if not indicated
otherwise, relative to CC group). Scale bars represent 200 μm in  (a)–(c) and 50 μm in
(d).
(a)(b)(c)(d)(e)


### 3.3. Object Recognition Training Increases Zif268 Protein Expression in Granule Cells of the Dorsal and Ventral Blades of the DG

Zif268 protein expression in the DG
was monitored by immunohistochemistry (Figure4, left panel). Zif268 protein
showed typical nuclear localization in the granule cells of both blades of the
DG (Figures 4(a)–4(d)). In caged
control animals, the dorsal blade presented an average density of 295.9 ± 42,8
positive cells per mm2 whereas the ventral blade presented an
average density of 336.3 ± 68 positive cells per mm2. Comparison of
C60 and L60 animals by ANOVA showed a learning effect (F(1,15)=24.183; P<.001) as well as a blade effect (F(1,15)=23,061; P<.001).
In the dorsal blade, a significant 1.8-fold increase in the density of
Zif268-positive granule cells was observed in the L60 group (P=.01) when
compared with the expression in the C60 group which was equivalent to that of
the caged controls (Figure 4(e)). In the ventral blade, a similar recognition
learning-specific increase was seen when L60 animals were compared with the C60
controls (P=.01). However, as also observed for Arc, the density of
Zif268-expressing cells in the ventral blade was significantly reduced in the
C60 group relative to caged controls (P=.02, Figure 4(e)). These results
show that object recognition induces Zif268 expression in both blades
of the DG.Figure 4
Object recognition training induces Zif268
and Narp protein expression in the dentate gyrus. Zif268 immunohistochemistry
reveals the presence of Zif268 protein in granule cells in both blades
of the dentate gyrus of (a) CC, (b) C60, and (c)  L60 animals. (d) Higher
magnification shows the presence of Zif268 in the nucleus of granule cells.
(e) Change in density of Zif268-positive nuclei (relative to the CC group) in the
dorsal and the ventral blade of the dentate gyrus across the C60 and L60 groups. Narp immunohistochemistry reveals the presence of Narp protein in granule
cells in both blades of the dentate gyrus of (f) CC, (g) C60, and (h) L60  animals. (i) Higher magnification shows the presence of Narp in the cell body of
granule cells.  (j) Change in density of Narp-positive cells (relative to the CC
group) in the dorsal and the ventral blade of the dentate gyrus across the C60
and L60 groups. Data are presented as mean ± SEM (n=4 for all groups).
Asterisks indicate P≤.05 (if not indicated otherwise, relative to CC
group). Scale bars represent 200 μm in  (a)–(c) and (f)-(h) and 50 μm in
(d) and (i).


### 3.4. Object Recognition Training Increases Narp  Protein Expression in Granule Cells of the  Dorsal Blade of DG

Narp staining was obvious in cells of both blades of the DG and was
restricted to the cell bodies (Figures4(f)–4(i)). Caged control
animals exhibited an average density of 582.5±52.9 Narp-positive cells per mm2 in the dorsal blade and 510.9±93.26 positive cells per mm2 in the
ventral blade. A modest increase of Narp-positive granule cells was detected in
the dorsal blade of C60 and L60 groups, relative to caged controls (Figure 4(j)).
The increase observed in the group of animals exposed to the objects (L60) was
significant (P=.03), whereas expression in the C60 group was not
significantly different from caged controls. In contrast to Arc and Zif268, no
changes in Narp expression were detected in the ventral blade in the C60 and
L60 groups. However, ANOVA did not detect any learning-specific change
indicating that the task had an effect on Narp protein expression specific to
the dorsal blade of the DG, which cannot be attributed solely to acquisition of
the object-place configuration.


### 3.5. Object Recognition Training Increases Levels of Arc,α-CaMKII, and PSD-95 Protein Expression in the DG

Western blot was used to assess the expression levels of Arc protein in
the DG of trained animals (Figure5(a)). Arc levels were elevated more than
2.5-fold in the L60 group compared with C60 (P=.05). This
learning-associated increase matches the changes in Arc mRNA as revealed by in situ hybridization (Figure 3(d)). We then asked whether this increase in
Arc expression is paralleled by altered expression of other proteins involved
in synaptic plasticity and memory consolidation. For this purpose we chose two
core constituents of the postsynaptic density complex, the scaffolding protein
PSD-95 and the enzyme α-CaMKII. Both
proteins undergo local dendritic synthesis, regulate the structure and receptor
composition of the PSD, and have important functions in synaptic plasticity and
memory [50–53, 58–61].
Like Arc, α-CaMKII (Figure 5(b)) and PSD-95 (Figure 5(c)) were both upregulated in
the L60 group relative to the C60 group, which was exposed to the arena without
objects (P=.03 and P=.02). Another intriguing aspect of the
protein response was the decrease in expression of Arc and α-CaMKII in the C60
group to as much as 50% of the caged controls, although this effect was not
significant.Object recognition training induces an increase in the expression of Arc,α-CaMKII, and PSD-95 proteins in the dentate gyrus. Representative blots and comparison
of normalized protein levels of  (a) Arc, (b) α-CaMKII, and (c) PSD-95
proteins are presented for the C60 and L60 groups (relative to CC). Data are
presented as mean ± SEM (n=7 for all groups). Protein levels were normalized to
β-actin. Asterisks indicate P≤.05.
(a)(b)(c)


## 3.1. Long-Term Memory for the Spatial Configuration  of Objects

The training procedure involved habituation to the test arena, followed
by exposure to three objects at fixed locations on four consecutive 5-minute
sessions with 5-minute intervals (acquisition phase), and a retention test,
which was performed 2 or 3 days later. In the retention test, one of the
objects was displaced and the amount of time exploring the displaced object
relative to the total time of object exploration was determined. This paradigm
has been previously shown to induce long-term memory for  objects and
location of objects [44]. During the acquisition phase,
ANOVA did not show
significant differences between the time spent exploring the three objects
(time spent exploring the three objects for the 2-day delay: 33.0±2.0%,
38.9±2.2%, 28.1±1.3% of total time; F2,42=0.90, P=.41, for the 3-day
delay: 32.7±1.5%, 21.1±1.2%, 46.2±1.4% of total time; F2,42=2.68, P=.08). During
retention testing, rats spent significantly more time exploring
the displaced object than chance level (33.33%) at both the 2 and 3-day
retention intervals (time spent exploring the displaced object for the 2-day
delay: 45.9±1.3%
of total time; t14=3.27, P<.01; for the 3-day
delay: 39.3±2.3%
of total time; t14=2.54, P<.05), as shown in
Figures 1(a) and 1(b). This behavioral analysis shows that rats in our experimental
conditions were able to form a long-term object-place recognition memory.Performance at the 2-day and 3-day retention
intervals of the object-place recognition memory task. At both (a) 2-day
and (b) 3-day  delays after acquisition, rats (n=15 in each case) showed
preferential exploration of the displaced object. (c) Schematic representation
of the task. Asterisks indicate P≤.05 compared with chance level
(dashed line, 33.3%). Data are presented as mean ± SEM.
(a)(b)(c)


## 3.2. Object Recognition Training Induces Arc mRNA Expression in Granule Cells of the Dorsal Blade of the DG

We hypothesized that acquisition of different types of information about
the objects and their spatial location would be associated with rapid induction
of the immediate early gene Arc. This
issue was first addressed by semiquantitative RT-PCR analysis of Arc mRNA
levels in the microdissected DG. Surprisingly, no significant change in Arc mRNA levels could be observed
in C10, C60, L10, and L60 animals when compared with caged control (CC) animals
(Figure2, P>.05), indicating that Arc expression in the dentate
gyrus was not significantly affected by exploration of the arena with or without
the objects.Figure 2
Expression levels of Arc in the dentate gyrus
after object recognition. Fold change in mRNA levels (relative to the CC
group) is presented for Arc in the dentate gyrus of animals from all five
groups (n=8 for all groups, except L60, n=7). Data are presented as mean ± SEM.
Gene expression was normalized to control genes (see methods).Endogenous Arc-expressing granule cells represent a very low percentage (1–2%)
of the  total number  of granule cells in the DG. Following spatial behavioral
experience, the density of Arc-expressing cells increases specifically in the
dorsal (inner) blade, while the density in ventral (outer) blade remains nearly
unchanged [57]. We considered that such
sparse, blade-specific changes in gene expression may not be detected by PCR
analysis of whole DG homogenate samples. We therefore re-examined the effect of
learning about objects and their configuration on Arc mRNA expression using in situ hybridization (Figure 3). As
previously described, Arc-expressing cells were dispersed along both the dorsal
and the ventral blades of the DG of both the CC and trained groups (Figures 3(a)–3(d)). The granule
cell layer of CC animals presented an average density of 152.2±10.5
Arc-positive cells per mm2 in the dorsal blade whereas the ventral
blade presented an average density of 138.8±13.8  Arc-positive cells per
mm2. Figure 3(e) shows the normalized density of Arc mRNA-positive
granule cells in the dorsal and ventral blades of the DG following performance
of the recognition task. ANOVA revealed a blade effect (F(1,31)=64.310; P<.001), a time effect (F(1,31)=14.181; P<.001)
and a learning effect (F(1,31)=10.417; P=.004). In the
dorsal blade, a significant 2-fold increase in density was detected in L10
animals, relative to the CC group (P<.01), while the C10 group
exhibited a nonsignificant 1.4-fold increase relative to CC. The density of Arc
mRNA-positive cells in the dorsal blade remained elevated up to one hour after
training in the learning group. L60 animals displayed a significant 1.4-fold
increase when compared with CC levels (P<.01) and a 1.5-fold increase
in comparison to C60 levels (P=.02). In the ventral blade, a surprising
decrease in the density of Arc-positive cells was observed in the C10 and C60
group, relative to caged controls (P<.01), indicating that the
exploration of the environment induced a rapid and sustained decrease in Arc
expression that was specific to the ventral blade. Nonetheless, exposure to the
objects resulted in a 2-fold increase in Arc-expression at 10 minutes (P=.05)
relative to rats exposed only to the test arena. Interestingly, no effect of the presence of
the three objects was observed in the ventral blade at the 60-minute time
point. Thus, learning about objects in this recognition task resulted in rapid
and sparse increase in Arc mRNA expression in both blades of the DG. However, only the dorsal blade of the DG
exhibited a sustained increase in Arc mRNA expression up to one-hour
posttraining.Object recognition training increases Arc
mRNA expression in the dentate gyrus. Arc mRNA in situ hybridization reveals sparse expression of Arc in both
dorsal and ventral blades of the rat dentate gyrus of (a) CC, (b) C10,
and (c) L10   animals. (d) Higher magnification shows typical Arc mRNA localization in the cell body and
dendrites of granule cells. (e) Change in Arc-positive granule cell densities
(relative to the CC group) in the dorsal and the ventral blade of the dentate
gyrus across the C10, L10, C60, and L60 groups. Data are presented as mean
± SEM (n=4 for all groups). Asterisks indicate P≤.05 (if not indicated
otherwise, relative to CC group). Scale bars represent 200 μm in  (a)–(c) and 50 μm in
(d).
(a)(b)(c)(d)(e)


## 3.3. Object Recognition Training Increases Zif268 Protein Expression in Granule Cells of the Dorsal and Ventral Blades of the DG

Zif268 protein expression in the DG
was monitored by immunohistochemistry (Figure4, left panel). Zif268 protein
showed typical nuclear localization in the granule cells of both blades of the
DG (Figures 4(a)–4(d)). In caged
control animals, the dorsal blade presented an average density of 295.9 ± 42,8
positive cells per mm2 whereas the ventral blade presented an
average density of 336.3 ± 68 positive cells per mm2. Comparison of
C60 and L60 animals by ANOVA showed a learning effect (F(1,15)=24.183; P<.001) as well as a blade effect (F(1,15)=23,061; P<.001).
In the dorsal blade, a significant 1.8-fold increase in the density of
Zif268-positive granule cells was observed in the L60 group (P=.01) when
compared with the expression in the C60 group which was equivalent to that of
the caged controls (Figure 4(e)). In the ventral blade, a similar recognition
learning-specific increase was seen when L60 animals were compared with the C60
controls (P=.01). However, as also observed for Arc, the density of
Zif268-expressing cells in the ventral blade was significantly reduced in the
C60 group relative to caged controls (P=.02, Figure 4(e)). These results
show that object recognition induces Zif268 expression in both blades
of the DG.Figure 4
Object recognition training induces Zif268
and Narp protein expression in the dentate gyrus. Zif268 immunohistochemistry
reveals the presence of Zif268 protein in granule cells in both blades
of the dentate gyrus of (a) CC, (b) C60, and (c)  L60 animals. (d) Higher
magnification shows the presence of Zif268 in the nucleus of granule cells.
(e) Change in density of Zif268-positive nuclei (relative to the CC group) in the
dorsal and the ventral blade of the dentate gyrus across the C60 and L60 groups. Narp immunohistochemistry reveals the presence of Narp protein in granule
cells in both blades of the dentate gyrus of (f) CC, (g) C60, and (h) L60  animals. (i) Higher magnification shows the presence of Narp in the cell body of
granule cells.  (j) Change in density of Narp-positive cells (relative to the CC
group) in the dorsal and the ventral blade of the dentate gyrus across the C60
and L60 groups. Data are presented as mean ± SEM (n=4 for all groups).
Asterisks indicate P≤.05 (if not indicated otherwise, relative to CC
group). Scale bars represent 200 μm in  (a)–(c) and (f)-(h) and 50 μm in
(d) and (i).


## 3.4. Object Recognition Training Increases Narp  Protein Expression in Granule Cells of the  Dorsal Blade of DG

Narp staining was obvious in cells of both blades of the DG and was
restricted to the cell bodies (Figures4(f)–4(i)). Caged control
animals exhibited an average density of 582.5±52.9 Narp-positive cells per mm2 in the dorsal blade and 510.9±93.26 positive cells per mm2 in the
ventral blade. A modest increase of Narp-positive granule cells was detected in
the dorsal blade of C60 and L60 groups, relative to caged controls (Figure 4(j)).
The increase observed in the group of animals exposed to the objects (L60) was
significant (P=.03), whereas expression in the C60 group was not
significantly different from caged controls. In contrast to Arc and Zif268, no
changes in Narp expression were detected in the ventral blade in the C60 and
L60 groups. However, ANOVA did not detect any learning-specific change
indicating that the task had an effect on Narp protein expression specific to
the dorsal blade of the DG, which cannot be attributed solely to acquisition of
the object-place configuration.


## 3.5. Object Recognition Training Increases Levels of Arc,α-CaMKII, and PSD-95 Protein Expression in the DG

Western blot was used to assess the expression levels of Arc protein in
the DG of trained animals (Figure5(a)). Arc levels were elevated more than
2.5-fold in the L60 group compared with C60 (P=.05). This
learning-associated increase matches the changes in Arc mRNA as revealed by in situ hybridization (Figure 3(d)). We then asked whether this increase in
Arc expression is paralleled by altered expression of other proteins involved
in synaptic plasticity and memory consolidation. For this purpose we chose two
core constituents of the postsynaptic density complex, the scaffolding protein
PSD-95 and the enzyme α-CaMKII. Both
proteins undergo local dendritic synthesis, regulate the structure and receptor
composition of the PSD, and have important functions in synaptic plasticity and
memory [50–53, 58–61].
Like Arc, α-CaMKII (Figure 5(b)) and PSD-95 (Figure 5(c)) were both upregulated in
the L60 group relative to the C60 group, which was exposed to the arena without
objects (P=.03 and P=.02). Another intriguing aspect of the
protein response was the decrease in expression of Arc and α-CaMKII in the C60
group to as much as 50% of the caged controls, although this effect was not
significant.Object recognition training induces an increase in the expression of Arc,α-CaMKII, and PSD-95 proteins in the dentate gyrus. Representative blots and comparison
of normalized protein levels of  (a) Arc, (b) α-CaMKII, and (c) PSD-95
proteins are presented for the C60 and L60 groups (relative to CC). Data are
presented as mean ± SEM (n=7 for all groups). Protein levels were normalized to
β-actin. Asterisks indicate P≤.05.
(a)(b)(c)


## 4. Discussion

The main findings of the present study are as follows. (1) Object recognition
training induces sparse IEG expression in the granule cell layer of the DG as
shown histochemically by the upregulation of Arc, Zif268, and to a lesser
extent, Narp. (2) Object exploration induces Zif268 expression across both
blades of the dentate gyrus, whereas Arc and Narp expression are selectively
induced in the dorsal blade. (3) The levels of Arc,α-CaMKII, and PSD-95, three
synaptically located proteins that are crucial for long-term memory are
concomitantly increased in DG homogenates one hour after object recognition
training.


### 4.1. Object Recognition Training Enhances Immediate Early Gene Expression in the DG

RT-PCR did not show significant up- or downregulation of Arc mRNA (Figure2). While this negative result suggested that granule cells are unresponsive,
RT-PCR may fail to detect changes that are restricted to subpopulations of
granule cells, or possible bidirectional changes within the population.
Our in situ
hybridization and immunohistochemistry approach revealed that Arc, Zif268, and
Narp are all upregulated in dentate granule cells shortly after completion of
the object recognition task (Figures 3 and 4). Furthermore, the distinct
spatial patterns of activation testify to a strong differential control of IEG
expression across the dorsal and ventral blade of the DG. Arc mRNA was only
transiently increased in the ventral blade, but showed sustained expression in
the dorsal blade. Narp protein showed the same dorsal blade-specific pattern,
whereas Zif268 was elevated equally in both DG blades.Chawla et al. [57] have previously demonstrated
sparse expression of Arc in the dorsal, but not ventral, blade of the DG
following a spatial behavioral experience in a novel environment. In that
study, rats exploring two different arenas exhibited environment-specific
increase of Arc expression in the dorsal blade. Thus, enhancement of Arc expression in the dorsal blade of the DG
is common to spatial exploration of a novel environment as well as object-place recognition
learning. Interestingly, no significant increase of Arc expression was observed
in our C10 and C60 group after exploration of a known environment, which
suggests that Arc induction in the DG is specific to novel spatial experience.
As discussed in the paper of Chawla et al., blade-specific alterations in gene
expression might be related to differences in the density of excitatory
synapses onto granule cells or differences in local circuitry between the
blades. Additionally, Fevurly and Spencer reported that stress also has an opposite
effect on Fos expression in the two blades of the dentate
gyrus [62].
Previous work has shown that Arc and Narp, but not Zif268, are strongly
upregulated during BDNF-LTP [30, 34, 63]. Further work is needed to determine if
effects of learning about new objects on Arc and Narp expression reflect
selective activation of endogenous BDNF signaling in the dorsal blade of the
dentate gyrus.Interestingly, Fos immunostaining in the DG is higher in rats presented
with familiar, but not novel arrangements of familiar items [64, 65].
This work involved the display of items on remote pictures whereas rats in our
study were free to explore the objects in an unchanged configuration.
Nevertheless, our results showing increases in Arc and Zif268 expression in the
DG after object-place recognition are in line with the proposal that the DG is
involved in the discrimination of the relative familiarity of spatial
arrangements [65]. By showing the regulated expression of several genes and proteins,
the present results confirm the responsiveness of the DG in the context of
object-place recognition memory.


### 4.2. Rapid Expression of Synaptic Proteins in the DG after Object Recognition Training

Dendritic spines are subject to activity-driven synaptic reorganization
and growth through mechanisms involving BDNF signaling, local protein
synthesis, and actin polymerization. We have observed parallel regulation of
Arc,α-CaMKII, and PSD-95 in the DG following recognition learning (Figure 5).
These proteins are all constituents of the PSD, they can be synthesized from
dendritic mRNA, and each of them has important functions in long-term
modification of synaptic structure and efficacy [47–49, 51, 53, 66].
Recent evidence suggests that conversion of short-term to long-term memory
requires a protein synthesis phase in a limited posttraining time window in the
hippocampus and that persistence of memory is BDNF-dependent [27, 28].
BDNF-induced LTP in the DG requires Arc synthesis, which serves to stabilize
the newly polymerized actin [13]. Arc and α-CaMKII are also
both locally translated in response to BDNF application to synaptoneurosomes [55, 67, 68].
Our data therefore support the model that recognition memory involves rapid and
coordinate regulation of plasticity-related PSD proteins.Besides the object learning-specific increases in protein expression,
there was a trend toward decreased gene and protein expression in animals
exposed to the empty arena. The mechanisms
underlying these decreases are unknown at present. There appears to be a
blade-specific component to this as the density of Arc- and Zif268-expressing
granule cells was significantly decreased only in the ventral blade. Arc andα-CaMKII protein expression were similarly reduced to below 50% in DG
homogenates obtained from rats repeatedly exposed to the empty arena. This is
interesting given recent evidence that memory formation and LTP maintenance
require proteasomal degradation of proteins [69–72],
especially in the context of memory reactivation, which presumably occurred in
our control rats that were repeatedly exposed to the arena [69–72]. The
current view of synaptic modification combines highly regulated protein
synthesis with specific proteasomal
degradation. It is therefore conceivable
that degradation of Arc and α-CaMKII following repeated exposure to the empty
arena plays some role in preparing synapses for subsequent protein
synthesis-dependent remodeling. Alternatively,
it has been previously demonstrated that prolonged exposure of animals to an
open-field results in decreased levels of phosphorylated CREB, which may act to
decrease CREB responsive genes [73]. There is  recent evidence for the presence of a CRE site in the Arc promoter.
Thus, downregulation of Arc expression could be the result of CREB hypophosphorylation in control animals
[74].In conclusion, we have provided evidence that the granule cells of the DG
are responsive to learning about, and forming a long-term memory of objects and
that the formation of this type of memory triggers upregulation of the
synaptic-plasticity related IEGs Arc and Zif268 along with enhanced expression
of the synaptic proteins PSD-95 andα-CaMKII. Interestingly, in some cases upregulation
associated with object-place learning appeared to be superimposed on
downregulation of expression induced by the known context. Further work is
needed to define the precise behavioral roles of gene and protein regulation in
the object-recognition paradigm. Pollak and colleagues [75]
recently reported coordinate expression of BDNF, Zif268, PSD-95, and pCaMKII in
the hippocampus after spatial training in the Morris water maze. The
similarities to the present study of object-place recognition memory give support
to the notion that similar molecular mechanisms underlie diverse forms of
hippocampus-dependent long-term memory.


## 4.1. Object Recognition Training Enhances Immediate Early Gene Expression in the DG

RT-PCR did not show significant up- or downregulation of Arc mRNA (Figure2). While this negative result suggested that granule cells are unresponsive,
RT-PCR may fail to detect changes that are restricted to subpopulations of
granule cells, or possible bidirectional changes within the population.
Our in situ
hybridization and immunohistochemistry approach revealed that Arc, Zif268, and
Narp are all upregulated in dentate granule cells shortly after completion of
the object recognition task (Figures 3 and 4). Furthermore, the distinct
spatial patterns of activation testify to a strong differential control of IEG
expression across the dorsal and ventral blade of the DG. Arc mRNA was only
transiently increased in the ventral blade, but showed sustained expression in
the dorsal blade. Narp protein showed the same dorsal blade-specific pattern,
whereas Zif268 was elevated equally in both DG blades.Chawla et al. [57] have previously demonstrated
sparse expression of Arc in the dorsal, but not ventral, blade of the DG
following a spatial behavioral experience in a novel environment. In that
study, rats exploring two different arenas exhibited environment-specific
increase of Arc expression in the dorsal blade. Thus, enhancement of Arc expression in the dorsal blade of the DG
is common to spatial exploration of a novel environment as well as object-place recognition
learning. Interestingly, no significant increase of Arc expression was observed
in our C10 and C60 group after exploration of a known environment, which
suggests that Arc induction in the DG is specific to novel spatial experience.
As discussed in the paper of Chawla et al., blade-specific alterations in gene
expression might be related to differences in the density of excitatory
synapses onto granule cells or differences in local circuitry between the
blades. Additionally, Fevurly and Spencer reported that stress also has an opposite
effect on Fos expression in the two blades of the dentate
gyrus [62].
Previous work has shown that Arc and Narp, but not Zif268, are strongly
upregulated during BDNF-LTP [30, 34, 63]. Further work is needed to determine if
effects of learning about new objects on Arc and Narp expression reflect
selective activation of endogenous BDNF signaling in the dorsal blade of the
dentate gyrus.Interestingly, Fos immunostaining in the DG is higher in rats presented
with familiar, but not novel arrangements of familiar items [64, 65].
This work involved the display of items on remote pictures whereas rats in our
study were free to explore the objects in an unchanged configuration.
Nevertheless, our results showing increases in Arc and Zif268 expression in the
DG after object-place recognition are in line with the proposal that the DG is
involved in the discrimination of the relative familiarity of spatial
arrangements [65]. By showing the regulated expression of several genes and proteins,
the present results confirm the responsiveness of the DG in the context of
object-place recognition memory.


## 4.2. Rapid Expression of Synaptic Proteins in the DG after Object Recognition Training

Dendritic spines are subject to activity-driven synaptic reorganization
and growth through mechanisms involving BDNF signaling, local protein
synthesis, and actin polymerization. We have observed parallel regulation of
Arc,α-CaMKII, and PSD-95 in the DG following recognition learning (Figure 5).
These proteins are all constituents of the PSD, they can be synthesized from
dendritic mRNA, and each of them has important functions in long-term
modification of synaptic structure and efficacy [47–49, 51, 53, 66].
Recent evidence suggests that conversion of short-term to long-term memory
requires a protein synthesis phase in a limited posttraining time window in the
hippocampus and that persistence of memory is BDNF-dependent [27, 28].
BDNF-induced LTP in the DG requires Arc synthesis, which serves to stabilize
the newly polymerized actin [13]. Arc and α-CaMKII are also
both locally translated in response to BDNF application to synaptoneurosomes [55, 67, 68].
Our data therefore support the model that recognition memory involves rapid and
coordinate regulation of plasticity-related PSD proteins.Besides the object learning-specific increases in protein expression,
there was a trend toward decreased gene and protein expression in animals
exposed to the empty arena. The mechanisms
underlying these decreases are unknown at present. There appears to be a
blade-specific component to this as the density of Arc- and Zif268-expressing
granule cells was significantly decreased only in the ventral blade. Arc andα-CaMKII protein expression were similarly reduced to below 50% in DG
homogenates obtained from rats repeatedly exposed to the empty arena. This is
interesting given recent evidence that memory formation and LTP maintenance
require proteasomal degradation of proteins [69–72],
especially in the context of memory reactivation, which presumably occurred in
our control rats that were repeatedly exposed to the arena [69–72]. The
current view of synaptic modification combines highly regulated protein
synthesis with specific proteasomal
degradation. It is therefore conceivable
that degradation of Arc and α-CaMKII following repeated exposure to the empty
arena plays some role in preparing synapses for subsequent protein
synthesis-dependent remodeling. Alternatively,
it has been previously demonstrated that prolonged exposure of animals to an
open-field results in decreased levels of phosphorylated CREB, which may act to
decrease CREB responsive genes [73]. There is  recent evidence for the presence of a CRE site in the Arc promoter.
Thus, downregulation of Arc expression could be the result of CREB hypophosphorylation in control animals
[74].In conclusion, we have provided evidence that the granule cells of the DG
are responsive to learning about, and forming a long-term memory of objects and
that the formation of this type of memory triggers upregulation of the
synaptic-plasticity related IEGs Arc and Zif268 along with enhanced expression
of the synaptic proteins PSD-95 andα-CaMKII. Interestingly, in some cases upregulation
associated with object-place learning appeared to be superimposed on
downregulation of expression induced by the known context. Further work is
needed to define the precise behavioral roles of gene and protein regulation in
the object-recognition paradigm. Pollak and colleagues [75]
recently reported coordinate expression of BDNF, Zif268, PSD-95, and pCaMKII in
the hippocampus after spatial training in the Morris water maze. The
similarities to the present study of object-place recognition memory give support
to the notion that similar molecular mechanisms underlie diverse forms of
hippocampus-dependent long-term memory.



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*Source: 269097-2009-01-26.xml*