A JOURNAL OF NEUROLOGY
Accuracy of hippocampal network activity is
disrupted by neuroinflammation: rescue by
S. Rosi,1,2,3V. Ramirez-Amaya,4A. Vazdarjanova,5E. E. Esparza,4P. B. Larkin,6,7J. R. Fike,1,3
G. L. Wenk8and C. A. Barnes9,10
1 Brain and Spinal Injury Center, University of California, San Francisco, CA, USA
2 Department of Physical Therapy and Rehabilitation Science, University of California, San Francisco, CA, USA
3 Department of Neurological Surgery, University of California, San Francisco, CA, USA
4 Instituto de Neurobiologia, Universidad Nacional Autonoma de Mexico, Juriquilla, Queretaro, Mexico
5 Medical College of Georgia, Augusta, GA, USA
6 Neuroscience Graduate Program, University of California, San Francisco, CA, USA
7 Gladstone Institute of Neurological Disease, University of California, San Francisco, CA, USA
8 Department of Psychology, Ohio State University, Columbus, OH, USA
9 Arizona Research Laboratories, Neural Systems Memory and Aging, University of Arizona, Tucson, AZ, USA
10 Evelyn F. McKnight Brain Institute, University of Arizona, Tucson, AZ, USA
Correspondence to: S. Rosi, Ph.D.,
Brain and Spinal Injury Center,
San Francisco General Hospital,
Building 1, Room 101,
1001 Potrero Avenue,
San Francisco, CA 94110,
Understanding how the hippocampus processes episodic memory information during neuropathological conditions is important
for treatment and prevention applications. Previous data have shown that during chronic neuroinflammation the expression of
the plasticity related behaviourally-induced immediate early gene Arc is altered within the CA3 and the dentate gyrus; both of
these hippocampal regions show a pronounced increase in activated microglia. Low doses of memantine, a low to moderate
affinity open channel uncompetitive N-Methyl-D-aspartate receptor antagonist, reduce neuroinflammation, return Arc expression
to control levels and attenuate cognitive deficits induced by lipopolysaccharide. Here we investigate whether neuroinflammation
affects the accuracy of information processing in the CA3 and CA1 hippocampal regions and if this is modified by memantine
treatment. Using the immediate early gene-based brain-imaging method called cellular analysis of temporal activity by fluo-
rescence in situ hybridization, it is possible to detect primary transcripts at the genomic alleles; this provides exceptional
temporal and cellular resolution and facilitates the mapping of neuronal activity. Here, we use this method to compare the
neuronal populations activated by two separate experiences in CA1 and CA3 and evaluate the accuracy of information proces-
sing during chronic neuroinflammation. Our results show that the CA3 pyramidal neuron activity is not stable between two
exposures to the same environment context or two different contexts. CA1 networks, however, do not differ from control
conditions. These data suggest that during chronic neuroinflammation, the CA3 networks show a disrupted ability to encode
spatial information, and that CA1 neurons can work independently of CA3. Importantly, memantine treatment is able to partially
normalize information processing in the hippocampus, suggesting that when given early during the development of the
pathology memantine confers neuronal and cognitive protection while indirectly prevents pathological microglial activation.
doi:10.1093/brain/awp148 Brain 2009: 132; 2464–2477 |
Received November 6, 2008. Revised April 23, 2009. Accepted May 2, 2009. Advance Access publication June 16, 2009
? The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
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Keywords: hippocampal networks; immediate early gene; neuroinflammation; NMDARs modulation
Abbreviations: aCSF=artificial cerebrospinal fluid; Arc=activity-regulated cytoskeletal associated protein; catfish=cellular
analysis of temporal activity by fluorescence in situ hybridization; Cy3=cyanine 3; LPS=lipopolysaccharide; NMDARs=N-Methyl-
The chronic neuroinflammation present in the early stages of
many neurodegenerative conditions, such as Alzheimer’s disease,
traumatic brain injury and HIV dementia, may contribute to the
severe cognitive decline associated with these disorders (McGeer
and McGeer, 1998; Akiyama et al., 2000; Morganti-Kossmann
et al., 2001; Fischer-Smith et al., 2004). Activated microglia and
their products are key mediators of the neuroinflammatory process
that contributes to neuronal damage (Barger and Basile, 2001).
There is evidence that during the early stages of Alzheimer’s
disease, before neuronal degeneration occurs, activated microglia
are found in the hippocampal formation, a region that plays a
critical role in associative learning and memory (Cagnin et al.,
2001). Further support for the role of microglia in altered
hippocampal function comes from laboratory studies that show
that chronic neuroinflammation increases activated microglia in
the dentate gyrus and CA3 region and alters the expression
activity-regulated cytoskeleton-associated protein (Arc), in these
same regions (Rosi et al., 2005, 2006). This may result in altered
coupling of neuronal activity with macromolecular synthesis
(transcription and translation) implicated in learning and memory
(Rosi et al., 2005).
Arc is expressed in cortical and hippocampal glutamatergic
neurons, and is required for engaging durable plasticity processes
that may underlie memory consolidation (Lyford et al., 1995;
Guzowski et al., 2000). For example, reduction in Arc through
genetic manipulation or antisense oligonucleotides leads to
(Guzowski et al., 2000; Plath et al., 2006). Following behavioural
foraging experiences, Arc is activated in hippocampal neurons in
proportions similar to those recorded electrophysiologically under
similar conditions (Lee et al., 2004; Leutgeb et al., 2004). The
correspondence in circuit dynamics between electrophysiology
and measurements of Arc has led to the suggestion that expres-
sion of this gene can serve as a reliable monitor of cellular activity
reflecting spatial and contextual information processing (Guzowski
et al., 1999).
Hippocampal network function is essential for discrimination and
retrieval of spatial information and enables effective navigational
behaviour. Understanding how the disruption of this function is
affected by neuroinflammation is critical for the development
treatments/approaches to prevent or attenuate cognitive dysfunc-
tions. It has been previously shown that the low-to-moderate
affinity open channelnon-competitive
receptor (NMDAR) antagonist memantine can rescue navigational
behaviour during chronic neuroinflammation while preventing
activation of microglia (Rosi et al., 2006). Given its unique
1999), memantine at low doses can selectively block pathological
influx of calcium ions without affecting the voltage-dependence of
NMDAR transmission that is critical for learning and memory. By
stochastically blocking open neuronal NMDA channels, memantine
may indirectly affect microglial activation. It is not known,
however, if the cognitive improvement conferred by memantine
is due to restoring proper hippocampal network function, and if
memantine affects microglial activation directly or indirectly
through neuronal NMDARs.
To study the hippocampal network function during chronic
neuroinflammation, we used a novel IEG-based brain-imaging
method called cellular analysis of temporal activity by fluorescence
in situ hybridization (catFISH), which monitors the activity history
of neurons after two given learning experiences separated by
compartmentalization for localization of mRNA with temporal
specificity, and because the dynamics of Arc expression are
different in the dentate gyrus (Ramirez-Amaya et al., 2005), our
study only focused on the CA areas. In addition, to determine if
memantine acts directly on microglia, we studied its affect on
microglial cells in culture.
Subjects and surgical procedures
Indianapolis, IN, USA) rats were used in this study. All rats were
individually caged with food and water freely available. As previously
described, artificial cerebrospinal fluid (aCSF, n=36) or lipopolysac-
charide (LPS, n=29) (Sigma, St Louis, MO, USA E. coli, serotype
055:B5, TCA extraction, 1.0mg/ml dissolved in aCSF) were loaded
into an osmotic minipump (Alzet model #2004, to deliver 0.25ml/h;
Durect Corp., Cupertino, CA, USA) and chronically infused for 28 days
through a cannula implanted into the fourth ventricle of the brain
(Rosi et al., 2005). On the same day of the ventricular implant, a
second osmotic minipump was inserted subcutaneously in the back
of 14 rats from the lipopolysaccharide infused groups and 16 rats
from the aCSF-infused group (Alzet model #2ML4, to deliver
2.5ml/h) to release subcutaneous memantine (Tocris Bioscience,
Ellisville, MO, USA) at a dose of 10mg/kg/day (Rosi et al., 2006).
Body weights were determined daily and general behaviour was
To habituate rats, they were handled daily for 10 days before the
novel environment exploration. Twenty-nine days after the initiation
of the artificial cerebrospinal fluid, lipopolysaccharide or memantine
infusions, the rats were separated in two cohorts: exploration (LPS,
n=10; LPS+M, n=10; aCSF, n=11 and aCSF+M, n=10) and
caged control (LPS, n=5; LPS+M, n=4; aCSF, n=9; aCSF+M,
n=6). Each rat from the exploration cohort was exposed to novel
Accuracy of hippocampal networks Brain 2009: 132; 2464–2477 |
environment A for 5min (epoch 1), returned to its home cage in
the colony room for 25min, and then exposed for 5 more minutes
(epoch 2) either to the same environment (A) or different environment
(B; Fig. 1A and B). Environment A was an open box (61?61cm) with
24-cm high walls divided into nine grids, and environment B was a
cylinder (75cm diameter, 20cm high, nine grids) that was located in a
different room from environment A but equidistant from the colony
room. Between subjects, the environments were cleaned thoroughly
with 20% ethyl alcohol (environment A) or 5% bleach (environment
B). All behavioural testing was conducted during the dark half of the
light/dark daily cycle, which is the active period for rodents. Each rat
was manually moved into the centre of a different grid every 15s in a
pseudorandom schedule during the 5-min exploration session. Under
this paradigm the proportions of Arc expressing neurons was compa-
rable to those obtained with free moving rats (Vazdarjanova and
Guzowski, 2004). By manually moving the animals, we reduced the
variability and insured that all animals fully explored the environments.
Light intensity and distal spatial cues were maintained throughout
all behavioural exploration sessions. Immediately after the second
exploration session, rats were deeply anaesthetized with isoflurane
gas and killed by decapitation. The brain was quickly removed
(5120s) and frozen in ?70?C isopentane (2-methyl butane, Sigma).
Caged control animals that did not explore the novel environments
were killed directly from their home cages interspersed with the rats
that were given exploration treatment.
The brains were divided at the midline and blocked together such that
each slide contained sections from one rat from each of the experi-
mental groups (behaviour and non-behaviour, LPS, aCSF and LPS+M,
aCSF+M) (Rosi et al., 2006). All slides were cryosectioned and stored
at ?70?C until processed for immunocytochemistry or fluorescence in
situ hybridization (FISH).
Three slides (containing all the conditions described above) were
selected fromthe medialportion
(anterior–posterior, ??3.6mm from Bregma) and were stained for
activated microglia OX-6 positive, as previously described in detail
(Rosi et al., 2005, 2006).
Figure 1 Schematic representation of the experimental procedure (A) and types of novel environments (B) used in this study. Animals
were allowed to explore the novel environment A for 5min (epoch 1). After 25-min rest period in the home cage, the rats were
exposed to either environment A again or environment B (epoch 2). Environment A was an open box (61?61cm) with 24-cm high
walls. Environment B was a cylinder (75-cm diameter), and was located in a different room than environment A. (C) Schematic
representation of the neuronal populations that express Arc (cytoplasmic, intranuclear foci or both) following exploration of identical
(AA, top illustration) or different (AB, bottom illustration) environments. Nuclei are indicated by light blue circles, Arc transcription foci
(Arc-foci) are indicated by two reds spots in the nuclei, cytoplasmic Arc mRNA (Arc-cyto) is indicated by green coloured nuclei and
green shading. The green open circle contains the neuronal population activated during epoch 1 and the red open circle contains the
neuronal population activated during epoch 2. The overlap between these two circles represents the neuronal population activated
during both epoch 1 and epoch 2. In rats exposed to the same environment twice (AA, top illustration) the majority of Arc positive cells
contain Arc-cyto and Arc-foci; indicating that Arc activation occurs in the same cell population of neurons during each exposure.
Rats exposed to two different environments (AB, bottom illustration) show an equal proportion of stained for Arc-cyto or Arc-foci
and smaller proportion containing both; suggesting that different environments activate independent populations of neurons.
Brain 2009: 132; 2464–2477S. Rosi et al.
Fluorescence in situ hybridization
Sections processed for FISH were prepared and handled as described
previously (Vazdarjanova and Guzowski, 2004; Rosi et al., 2005,
2006). To unambiguously identify Arc transcription induced by the
first exploration (epoch 1) and to distinguish it from the recent
transcription induced by the second exploration (epoch 2), we used
the detection of Arc intron riboprobe digoxigenin-labelled and Arc full-
length riboprobe labelled with fluorescein (for detail see: Guzowski
et al., 2006). This allowed visualization of the initial transcription
(cytoplasmic mRNA form the first exploration) with one fluorophor
(FITC, PerkinElmer, Life sciences, Emeryville, CA, USA) and the later
transcription (intranuclear foci mRNA from the second exploration)
with another fluorophor (cyanine-3, Cy3, PerkinElmer). This approach
improves reliability and efficiency and is the basis of the catFISH
technique. Digoxigenin- or fluorescein-labelled riboprobes (for Arc
generated using commercial transcription kits (MaxiScript, Ambion,
Austin,TX, USA) and RNAlabelling
Hertforshire, UK). Briefly, digoxigenin-labelled Arc intranuclear foci
(Arc-foci) was detected with anti-digoxigenin-HRP (Roche products)
and was revealed with Cy3. After the detection of Arc-foci, the
slides were quenched with 2% H2O2 to quench the residual HRP
activity. Fluorescein-labelled Arc full-length probe was then detected
with antifluorescein HRP (Roche Products) and revealed with FITC.
Nuclei were counterstained with TOPRO (Molecular Probes, Eugene,
OR, USA). The control conditions, which were run with no RNA
probe, showed no staining.
Image acquisition and analysis
CA3 and CA1 area images were taken using a Zeiss 510 Metaseries
laser scanning confocal microscope with a 400X water immersion lens
(Thornwood, NY, USA). Three Z-stacks from each brain region (1mm
optical thickness/plane) were imaged and three slides per rat were
imaged. Thus, comparable numbers of cells from each rat were
analysed. Offline analyses were performed using MetaMorph imaging
software (Universal Image Corporation, West Chester, PA, USA) as
described previously (Guzowski et al., 1999; Vazdarjanova and
Guzowski, 2004; Rosi et al., 2005, 2006). Neuronal nuclei from the
pyramidal cell layers of CA1 and CA3 were classified as negative
(containing no transcription foci or cytoplasmic Arc), Arc-foci posi-
tive (containing only Arc intranuclear foci staining), Arc-cyto positive
(containing only cytoplasmic staining) or double-labelled Arc-foci/
Arc-cyto positive (containing both intranuclear foci transcript and
cytoplasmic). For analysis, the investigator was blind to the experimen-
Similarity score analysis
When analysing the Arc catFISH images it is helpful to reduce the
complex cell staining data to a value that can be used to compare
cell activity across multiple brain regions. The similarity score takes
the four cell staining values defined above (Arc-foci, Arc-cyto,
double-labelled Arc-foci/Arc-cyto and negative) and reduces them
to a single value (Vazdarjanova and Guzowski, 2004). A value of
1 represents a single neuronal population activated in both exploration
sessions (epoch 1 and 2). A value of 0 indicates that two statistically
independent cell populations were activated during the two explora-
tion sessions (epochs 1 and 2). The similarity score was derived
as follows: (i) Epoch 1 active cells=fraction of total Arc-positive
[(Arc-foci/Arc-cyto + Arc-cyto only)/total cells]; (ii) Epoch 2 active
cells=fraction of total fraction of total Arc- positive [(Arc-foci/
Arc-cyto +Arc-foci only)/total cells]; (iii) p(E1E2)=epoch 1 active
cell fraction?epoch 2 active cell fraction. This represents the
probability of cellsbeingactivated
Arc-cyto), assuming that the two epochs activated statistical indepen-
dent neuronal population; (iv) diff(E1E2)=(Arc-foci/Arc-cyto)-p(E1E2).
This represents the measure of the deviation from the independent
hypothesis; (v) Least epoch=the smaller of the ensembles activated
by epoch 1 or epoch 2; and (vi) Similarity score=diff(E1E2)/
(least epoch-p(E1E2)). This normalizes the diff(E1E2) fraction to a
perfect condition where A/A is 1 and A/B is 0 (Vazdarjanova and
Microglia cell culture
N9 microglial cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 5% fetal bovine serum and
2% L-glutamine. Cultures were maintained in an incubator at 37?C,
5% CO2, 95% humidity. Medium was changed every 3–4 days and
cells were passaged weekly using 0.05% trypsin. Cells used in all
experiments had been passaged fewer than 17 times.
For experiments with LPS and memantine treatment, N9 cells were
plated into 24 well plates at 40 000 cells/well in DMEM. Two hours
after plating, DMEM was removed and replaced with macrophage
serum free medium (MSFM). Twenty-four hours after plating,
MSFM was removed and replaced with fresh MSFM containing LPS
and/or memantine (LPS 100ng/mL, memantine 10 or 50mM).
Twenty-four hours after treatment with LPS and/or memantine, cells
were lysed and RNA was harvested using a Qiagen RNeasy Mini kit
according to the manufacturer’s instructions. RNA was immediately
reverse transcribed to cDNA using Multiscribe? reverse transcriptase
performed using SYBR Green Mastermix (Applied Biosystems) as
provided by the manufacturer. qPCR was performed using an ABI
Prism 7700 Sequence Detector with the following program: UNG
activation (50?C for 2min), initial denaturation (95?C for 10min),
and then 40 cycles of denaturation (95?C for 15s) then annealing
and extension (60?C for 1min). Primer specificity was confirmed
using melting curve analysis. Amplification efficiency was calculated
for each primer pair using serially diluted standards. This efficiency
was used to convert cycle threshold (number of PCR cycles required
to reach an arbitrary threshold fluorescence value) into relative gene
expression. For each sample, expression of the gene of interest was
normalized to beta actin expression. Samples run on separate qPCR
plates were calibrated to each other using standards that were
common to all plates.
StatView Softaware was used to perform all the statistical analysis
related to catFISH, and Graphpad Prism statistical analysis software
was used to analyse gene expression data from four independent
treatment effect as well as similarity scores, were analysed with
ANOVA. When the overall ANOVA was significant (P50.001) further
comparison between groups was done using Bonferroni post hoc tests.
Only three comparisons, a priori, were planned (aCSF control versus
LPS, aCSF+M or LPS+M). For all tests, the null hypothesis was
rejected at the 0.05 level of significance.
Accuracy of hippocampal networksBrain 2009: 132; 2464–2477 |
The immediate early gene-based single cell brain imaging method
(Arc catFISH) was used to quantitatively assess the reliability of
information processing in hippocampal neuronal networks during
neuroinflammation and following therapeutic treatment with
memantine. The chronic infusion of lipopolysaccharide with or
without memantine was well-tolerated by all rats, although there
was a transient weight loss over the first few days of treatment.
No adverse neurological signs were observed, and counts of CA1
and CA3 neurons revealed no significant changes in the total
number of neurons per region in any of the experimental groups
when compared to artificial cerebrospinal fluid controls (data not
shown). The mean number of neurons analysed for each rat,
using the catFISH methods, was 365?18.8 for CA1 and
287?10.3 for CA3.
Operationally, neuronal activation was defined as the fraction of
neurons that expressed Arc mRNA, either in the nucleus (i.e. foci)
or cytoplasm after rats were allowed to explore a given novel
et al., 2006). The neuronal populations active during two distinct
exploration periods (epochs 1 and 2) of either the same (AA) or
different (AB) environments were detected using Arc catFISH,
which allowed the determination of which distinct epoch a given
cell was active (Fig. 1C). If the cell was active in the first
epoch only, Arc expression would be found exclusively only in
the cytoplasm (Arc-cyto). If the cell was active in the second
behavioural epoch only Arc expression would be found in the
nucleus (Arc-foci). If the cell was active in both epochs then
Arc would be located in both the nucleus and the cytoplasm
Nuclei with Arc-foci (yellow) can be observed in Figs 2A
and 3A, and reflect those cells active within ?5min of tissue
collection. Nuclei containing Arc-cyto (green) can be observed in
the same figures, which indicate neurons active within ?30min of
tissue collection. Neurons double labelled with both intranuclear
and cytoplasmic mRNA (Arc-foci/Arc-cyto) were active in both
exploration sessions (epoch 1 and 2). Therefore, the active cells
in epoch 1 are reflected by all the Arc-cyto positive neurons
(Arc-foci/Arc-cyto and Arc-cyto only) and the active cells in
epoch 2 are represented by all the Arc-foci positive neurons
(Arc-foci/Arc-cyto and Arc-foci only; Fig. 1C).
information processing in CA3
networks: restoration by memantine
To establish how lipopolysaccharide affected network processing,
and the subsequent effects of memantine treatment, it was
necessary to first determine the numbers of neurons expressing
behaviourally induced Arc. Under all the experimental conditions
used here, the fractions of CA3 neurons expressing Arc averaged
between 17% and 30% (Fig. 2B), which was significantly higher
than the 2.5%–5% observed for caged control animals (P50.001;
data not shown). In animals infused with artificial cerebrospinal
fluid, exploration in either the AA or AB environment conditions
resulted in similar fractions of activated neurons in both epoch 1
and epoch 2 (Fig. 2B). In lipopolysaccharide-infused animals there
was a significant increase in the percentage of Arc positive neu-
rons in epochs 1 and 2 and under both exploration conditions
F(7,38)=7.5, P50.001, for epoch 1; F(7,38)=7.1, P50.001 for
epoch 2; all post hoc Bonferroni comparisons P50.05]. Notably,
the proportions of neurons active during AA and AB in epochs
1 and 2 in animals infused with artificial cerebrospinal fluid were
similar to those seen in animals infused with aCSF+M and
LPS+M (Fig. 2B).
The percentagesof neurons
were identified by the double-labelled cells (Arc-foci/Arc-cyto).
In rats infused with artificial cerebrospinal fluid, the fraction of
double-labelled neurons (active during both epochs 1 and 2)
was significantly higher after the AA paradigm compared with
the AB exploration group [ANOVA: F(7,38)=17, P50.001]
(Fig. 2C). This suggests that in CA3, the same neurons were acti-
vated when the animals explored the same environment twice
(AA), but that a statistically independent and smaller group of
neurons was activated after exploration of two different environ-
ments (AB). A similar relationship was observed in animals infused
with aCSF + memantine (Fig. 2C). In contrast, after lipopolysac-
charide infusion, the fraction of activated neurons during the AA
and AB paradigms was similar (Fig. 2C), while, in LPS-infused rats
treated with memantine, the overall response was qualitatively
similar to that seen in the two artificial cerebrospinal fluid
groups [ANOVA: F(7,38)=17, P50.001; Fig. 2C].
Information processing in the CA1
area is unaltered by lipopolysaccharide
or memantine infusion
For all experimental conditions used here, the fractions of CA1
35%–40%, which was significantly higher than for the caged
control animals in which only 5%–6% showed Arc expression
(P50.001; data not shown). Similar to what was observed in
CA3, exploration (AA or AB) resulted in the activation of a similar
percentage of neurons during epochs 1 and 2 for all treatment
F(7,38)=1.5, P50.24 for epoch 2, Fig. 3B]. When considering
only double-labelled cells activated by the two exploration sessions
(Arc-foci/Arc-cyto) the fraction of activated neurons after the AA
paradigm was significantly higher than that seen in animals from
F(7,38)=35.8, P50.001; Fig. 3C], suggesting normal information
processing in the CA1 region even during neuroinflammation.
The per cent of double-labelled neurons activated during epochs
1 and 2 (Figs 2C and 3C) can be expressed in summary form using
a similarity score analysis that normalizes activity values to range
from 0 to 1 (Fig. 4A; see Methods section). Similarity scores
Brain 2009: 132; 2464–2477S. Rosi et al.
Figure 2 Summary of the Arc catFISH data in area CA3. (A) Confocal projection images showing Arc expression following exploration
of either identical (AA, top row) or different (AB, bottom row) environments in three of the treatment groups. Treatment groups
included animals chronically infused with artificial cerebrospinal fluid (aCSF); lipopolysaccharide (LPS); LPS and treatment with
memantine (LPS+M), and aCSF plus memantine (aCSF+M). Digoxigenin-labelled Arc-intron antisense probe was detected with
Cy3 (red, see Methods section). Fluorescein-labelled Arc antisense full probe was detected with FITC (green, see Methods section),
and cell nuclei were counterstained with in blue. The Arc-foci appear yellow as a result of the overlap of red (dogoxigenin) and the
green FITC-labelled Arc. Animals from the aCSF treatment group exposed to the AA exploration condition primarily exhibited neurons
stained for both Arc-foci and Arc-cyto with only a small number of neurons containing Arc-cyto or Arc-foci only. As expected,
Accuracy of hippocampal networks Brain 2009: 132; 2464–2477 |
approaching 1 suggest a high degree of overlap in activation
patterns, and scores closer to 0 suggest independent populations.
For animals infused with artificial cerebrospinal fluid that explored
the same environment twice (AA), both CA3 and CA1 cells
showed high similarity scores (?0.8), as predicted (Fig. 4A).
In contrast, artificial cerebrospinal fluid rats allowed to explore
two different environments (AB), showed two statistically indepen-
dent populations of neurons activated in CA3 and CA1, as
evidenced by a low similarity score (?0.2). Similar results were
observed for animals infused with artificial cerebrospinal fluid
and treated with memantine (Fig. 4A). In lipopolysaccharide-
treated animals, however, the CA3 similarity scores for the AA
group were significantly lower than for the artificial cerebrospinal
fluid-treated animals; the similarity scores for the AB group were
higher [ANOVA for CA3: F(7,38)=16.5, P50.05; for CA1:
F(7,38)=32.5, P50.05; all appropriate post hoc comparisons
had P50.05]. Interestingly, in lipopolysaccharide-treated rats,
treatment with memantine was able to largely restore the high
overlap in the AA condition and lower overlap in the AB treatment
group (Fig. 4A). The observed similarities and differences among
the groups are best understood via graphical display of the
relationship between similarity scores between CA1 and CA3
(Fig. 4B). The distribution of the two populations of animals AA
and AB is very well separated with AB close to 0 and AA close to 1
for artificial cerebrospinal fluid and aCSF+M animals (Fig. 4B).
After lipopolysaccharide treatment, the distribution of the two
populations of animals AA and AB is not well separated in either
of the two behavioural conditions. Following the LPS-memantine
treatment the distribution of the animals appear more separated.
As is shown in Fig. 4B there does not appear to be a strong
correlation between CA1 and CA3 similarity scores within animal
groups as might be expected from the literature (Vazdarjanova
and Guzowski, 2004). The calculated correlation figures are
aCSF AA, R2=0.164; aCSF AB, R2=0.347; LPS AA R2=0.032;
LPS AB, R2=0.027; aCSF+M AA, R2=0.16; aCSF+M AB,
R2=0.46; LPS+M AA, R2=0.647; LPS+M AB, R2=0.363.
Memantine does not directly affect
Chronic infusion of lipopolysaccharide in vivo resulted in the
selective activation of microglia within CA3 area and this was
significantly reduced with memantine treatment (Fig. 5A and B).
To determine if memantine affected microglial activation indirectly
through its action on neuronal NMDARs, we employed an in vitro
system and used quantitative Real Time PCR (qPCR) to examine
expression of NMDARs mRNA. Using primers specific for the NR1
subunit of the NMDA receptor, a subunit that is required for
formation of a functional channel (Monyer et al., 1992), no
NMDAR expression was detected in either untreated or LPS-
stimulated N9 microglial cells (not shown). Despite the lack of
NMDARs, the possibility remained that memantine acted directly
on microglial cells to alter their activation state by a mechanism
that did not depend on NMDARs. To test this possibility, we cul-
tured N9 microglial cells in the presence of memantine and/or
lipopolysaccharide. Treatment with memantine did not result in
any changes from control levels (Fig. 6). Treatment with lipopoly-
saccharide did stimulate these microglial cells to significantly
increase transcription of the pro inflammatory mediators Tumor
Necrosis Factor a (TNFa) and inducible nitric oxide synthase
(iNOS), as measured by qPCR (Fig. 6). When cells were treated
with memantine and lipopolysaccharide at the same time, micro-
glial cells increased the transcription of TNFa and iNOS similarly to
the lipopolysaccharide treatment alone. Taken together, the data
suggest that memantine treatment alone did not directly affect
The results of the present experiments provide novel insights into
hippocampal network impairment that may underlie the cognitive
deficits observed during chronic neuroinflammation. This work
relied upon the measurement of the IEG Arc, which has been
shown to be a reliable monitor of cellular activity reflecting spatial
and contextual information processing (Guzowski et al., 1999).
Two principal findings emerge from these studies. First, following
treatment with a proinflammatory agent (LPS), behaviourally
driven hippocampal network activity is preserved in CA1 pyramidal
cells, but the circuit activation patterns are profoundly disrupted in
CA3 cells. Second, memantine, a moderate-affinity uncompetitive
open NMDAR channel antagonist, administered concurrently with
LPS, is able to preserve normal activity patterns in the CA3 region.
A final important observation from these experiments is that the
anti-inflammatory effect of memantine appears to be due to its
effects on neurons rather than glia. The two observations that
support this conclusion are that NMDARs are not observed on
microglia and that memantine did not impact the expression of
pro-inflammatory genes in cultured microglia (Fig. 6).
when exposed to the AB exploration condition, the aCSF animals showed similar numbers of neurons stained for Arc-cyto only, Arc-foci
only or both. In contrast, animals from the LPS treatment group showed similar population of neurons stained with Arc-foci, Arc-cyto
or both in the AA and the AB exploration conditions. The staining profile observed during treatment with memantine with and without
LPS was similar to the aCSF group alone. Scale bar 50mm. (B) In the LPS treatment group, the percentage of neurons activated during
epoch 1 (double labelled for Arc-cyto/Arc-foci plus Arc/cyto only) and epoch 2 (double labelled for Arc-cyto/Arc-foci plus Arc/foci
only), was significantly higher compared to the aCSF treatment group, (*P50.05 aCSF AA versus LPS AA, aCSF AB versus LPS AB).
(C) The percentage of double-labelled neurons (Arc-cyto/Arc-foci) activated during both epochs 1 and 2. The highest percentage
of double-labelled neurons was observed during the AA exploration condition and the lowest during the AB exploration condition in
the aCSF, aCSF+M and LPS+M treatment groups. No difference was observed between the exploration condition AA and AB in the
LPS treatment group. (#P50.0001 AA versus AB for aCSF, LPS and LPS+M group).
Brain 2009: 132; 2464–2477S. Rosi et al.
Spatial information processing
accuracy is impaired in the CA3
network during neuroinflammation
Both electrophysiological (Lee et al., 2004; Leutgeb et al., 2004)
and Arc catFISH studies (Guzowski et al., 2004; Vazdarjanova and
Guzowski, 2004) indicate that overlapping CA3 and CA1 neuronal
ensembles are activated following two epochs of exploration of
thesame environment; this
Furthermore, those studies also indicate that after exploration of
two distinct environments, the ensembles engaged in CA3 and
CA1 have a low degree of overlap, and thus can be considered
to be statistically independent; this represents pattern separation.
represents pattern completion.
Figure 3 Summary of the Arc catFISH data in CA1 area. (A) Confocal projection images showing CA1 cell staining profiles detected
using Arc catFISH following exploration of either identical (AA, top row) or different (AB, bottom row) environments in three of the
treatment groups. Treatment groups included animals chronically infused with artificial cerebrospinal fluid (aCSF); aCSF and treatment
with memantine (aCSF+M); lipopolysaccharide (LPS); LPS and treatment with memantine (LPS+M). Following the AA exploration
condition the majority of the neurons showed double labelling (Arc-foci/Arc-cyto) and only a small number of neurons contained
only Arc-cyto or only Arc-foci. Conversely, animals exposed to the AB exploration condition showed similar numbers of neurons
with Arc-cyto only, Arc-foci only or both. (B) The percentages of Arc-positive neurons activated during epochs 1 and 2 were similar in
all the treatment conditions. (C) The AA exploration condition resulted in the highest percentage of double-labelled neurons (Arc-foci/
Arc-cyto) for all treatments (LPS, LPS+M and aCSF), indicating overlap of neurons activated during epochs 1 and 2. As predicted, the
AB exploration condition had lower overlap. (#P50.05 AB versus AA for aCSF, LPS and LPS+M group) in all treatment conditions.
Accuracy of hippocampal networks Brain 2009: 132; 2464–2477 |
Figure 4 Analysis of CA1 and CA3 neurons activated by epochs 1 and 2 using the similarity score analysis. A similarity score of 1
indicates that the neurons activated during epoch 1 are the same as those activated during epoch 2 (all the neurons are double labelled
for Arc-foci/Arc-cyto). A similarity score of 0 indicates that all the neurons activated during epoch 2 are different from epoch 1,
suggesting complete statistical independence of the two populations (see Discussion section). Treatment groups included animals
chronically infused with artificial cerebrospinal fluid (aCSF); lipopolysaccharide (LPS); aCSF and treatment with memantine (aCSF+M);
LPS treatment with memantine (LPS+M). (A) The similarity score for the aCSF treatment group showed high overlap of neurons
activated during epochs 1 and 2 in the AA exploration condition, and low overlap of neurons activated in the AB exploration condition
in both CA3 and CA1 area. In contrast, LPS treatment resulted in a similar overlap of neurons activated during the two behavioural
epochs in both AA and AB conditions only in CA3. This did not occur in the CA1 area. Interestingly, treatment with memantine, was
able to largely restore the high overlap in the AA condition and low overlap in AB condition (*P50.05, AA versus AB) in CA3. There
was a striking difference in overlap scores between the LPS treatment group compared to aCSF animals (#P50.05, aCSF AA versus LPS
AA, aCSF AB versus LPS AB). (B) Correlation analysis between CA3 and CA1 similarity scores for each animal within each treatment
and behavioural condition (aCSF AA, R2=0.164; aCSF AB, R2=0.027; LPS AA, R2=0.032; LPS AB, R2=0.027; aCSF+M AA,
R2=0.016; aCSF+M AB, R2=0.006; LPS+M AA, R2=0.647; LPS+M AB, R2=0.363). Within each group of animals there was no
significant correlation between CA1 and CA3 similarity scores; however, note the large separation between AA and AB in the aCSF and
aCSF+M group, and the mixed distribution in the LPS group; the LPS+M group shows a less sparse and more separate distribution
of CA1 and CA3 similarity scores.
Brain 2009: 132; 2464–2477S. Rosi et al.
The implication of these data is that the CA3 hippocampal system
forms stable, independent neural representations or spatial maps,
after a given behavioural experience (Leutgeb et al., 2004). In the
animals with chronic LPS-induced neuroinflammation, the size
of neuronal populations activated in CA3 after exploration was
significantly higher (?30%) than that activated in the aCSF-
infused control animals (?20%). The reduced sparsity of the
CA3 network, which refers to the abnormal number of neurons
that are inappropriately activated, may in part explain the
cognitive impairment thatresults
(Rosi et al., 2005). This is supported by modelling studies indicat-
ing that associative networks, such as those investigated here, are
worse at separating representations that are less sparse, and their
mnemonic storage capacity is diminished (McNaughton, 1987;
Treves and Rolls, 1992). Here we found direct evidence for this
effect, in that neuroinflammation led not only to impaired sparsity
Figure 5 Representative flat images of reconstructed CA3 area from the dorsal hippocampus (??3.6mm from bregma) from animals
chronically infused with LPS (A) and animals chronically infused with LPS and treated with memantine (B). Activated microglia are
showed in red and nuclei are counterstained in green. Scale bar=100mm. As previously reported (Rosi et al., 2006) animal infused with
LPS that received memantine treatment showed a significant reduction in activated microglia cells/mm2in the CA3 area as compared to
LPS alone (C).
Accuracy of hippocampal networksBrain 2009: 132; 2464–2477 |
of CA3 activation (Fig. 2B), but also to the inability of neurons to
discriminate different environments which is evident by the lack of
pattern separation (Fig. 2C). Moreover, the reduced sparsity
most likely reflects representations that are noisier (i.e. that are
reactivated with limited consistency across sessions). This noise
may be expressed by weaker pattern separation (additional
neurons that do not distinguish two distinct environments), and
by weaker pattern completion (a different set of additional
neurons may be activated when returning to the same environ-
ment). Consistent with this interpretation, in LPS-treated rats,
the degree of overlap of the CA3 neuronal ensembles during
exploration of the same environment (AA) is significantly lower,
and during exploration of distinct environments (AB) significantly
higher, than that observed in aCSF-AA control animals (Fig. 2C).
Dysfunction of pattern separation and completion processes in
CA3 may thus impair the normal ability of the system to discrim-
inate two different environments as distinct, and also to ignore
small differences in an environment, enabling the recollection
that the environment is familiar. This dysfunction can be
interpreted as a reduced reliability of information processing in
hippocampal networks. Thus, these data are congruent with
previously reported studies which showed that during chronic
neuroinflammation animals were unable to solve the Morris
water maze task (Rosi et al., 2006, Fig. 1A and B). In order to
properly solve the Morris water maze task, animals require proper
Redish and Touretzky, 1998; Nakazawa et al., 2002). The
catFISH methodology provides a unique opportunity to dissect
such pattern separation at the cellular level in LPS and LPS+M
These results are similar, in some respects, to the changes
observed in memory-impaired old animals (Barnes et al., 1997).
While neuronal ensemble recordings obtained from freely behav-
ing young rats suggest accurate retrieval of activity patterns in
neurons between the first and second of two identical experiences,
old rats do not always exhibit such retrieval (Barnes et al., 1997).
That is, spatial memory-impaired old rats probabilistically show in
CA1 either accurate retrieval (Leutgeb et al., 2004) or global
remapping, that is, an activity pattern typical of a different
environment condition (AB) even though animals are exposed to
the same environment twice (AA) (Barnes et al., 1997). On the
other hand, in the CA3 region of old rats, the activity patterns
observed after exploration of two different environments is the
same as if they were exposed to the same environment twice
(AA) (Tanila et al., 1997a,b; Redish et al., 1998). This dysfunction
of hippocampal system computations in ageing has been inter-
preted as a failure to accomplish pattern completion and pattern
separation operations that normally function in this system
(Burke and Barnes, 2006). While the changes in normal ageing
and under neuroinflammation
how changes in network function can contribute to changes in
information processing that can result in cognitive deficits.
are distinct,they illustrate
CA3 versus CA1 network accuracy
As discussed above, studies in normal animals using both
electrophysiological and Arc catFISH methods support the idea
that CA3 and CA1 perform distinct but complementary functions
in the processing of contextual and spatial information (Guzowski
et al., 2004; Lee et al., 2004; Leutgeb et al., 2004; Vazdarjanova
and Guzowski, 2004). The CA3 network is particularly efficient in
performing pattern completion when subtle environmental differ-
ences exist, and can perform unambiguous pattern separation
when the environmental features are notably distinct (Lee et al.,
2004; Vazdarjanova and Guzowski, 2004). The CA1 network, on
the other hand, performs pattern separation more gradually,
showing partial remapping when subtle changes in environmental
features occur (Leutgeb et al., 2004; Vazdarjanova and Guzowski,
The selective effects on network stability observed in CA3 could
result from anatomical and physiological differences between
the CA3 and CA1 hippocampal subregions. The present results
suggest that CA1 function is intact, in spite of the fact that
CA3 operations are disrupted. This indicates that accuracy of
Figure 6 Quantitative real-time PCR (qPCR) analysis of TNFa
(A) and iNOS mRNA (B) levels in cultured N9 microglial cells.
Cells treated with LPS for 24h showed significantly increased
expression of both TNFa and iNOS mRNA compared to vehicle
(macrophage serum free medium) treated cells. Twenty-four
hour of memantine treatment, alone or in combination with
LPS treatment, had no effect on gene transcription (*P50.05);
n=4 for separate experiments, error bars represent SEM.
Brain 2009: 132; 2464–2477 S. Rosi et al.
CA1 network function can be independent of the computations
performed in CA3. This finding is consistent with the observation
that CA1 firing characteristics can be preserved in the face of
disruptedCA3 activity following
et al., 1989). Additionally, recent results demonstrate that the
direct input to the CA1 received from entorhinal cortical layer III
neurons (Brun et al., 2008) contributes significantly to information
processing in CA1.
Memantine treatment partially restores
the accuracy of information processing
in the hippocampus
Treatment with memantine, a low-to-moderate affinity open-
channel non-competitive NMDARs antagonist (Parsons et al.,
1999; Danysz and Parsons, 2003), was able to counteract the
effect of chronic lipopolysaccharide treatment at least in terms
of the accuracy of spatial information processing in CA3. While
treatment with memantine alone did not affect activity in CA1
or CA3, animals with chronic neuroinflammation treated with
memantine did activate highly overlapping neuronal ensembles
during exploration of two identical environments, and largely
independent populations of neurons following exploration of two
distinct environments. Thus, pattern completion and separation
operations were normalized by the memantine treatment in ani-
mals subjected to lipopolysaccharide infusion. The mechanism
underling memantine’s effect on information processing may
involve postsynaptic calcium entry in the presence of elevated
extracellular glutamate. This could develop in response to the
pro-inflammatory environment produced by lipopolysaccharide.
Consistent with this hypothesis is the fact that Arc expression is
NMDAR-dependent and memantine is known to act only on
open NMDAR channels inhibiting inward depolarizing currents
(Danysz and Parsons, 2003).
There were differences between CA3 and CA1 information
processing that may be important in explaining the effect of
memantine on the behavioural performance of LPS-treated
animals. The similarity score analyses confirm that lipopolysacchar-
ide animals showed a pronounced decrease in the accuracy of the
hippocampal CA3 network as compared to the other experimental
groups. Interestingly, the similarity scores between CA3 and CA1
in the AA and AB groups are distinctly different (Fig. 4) in
the aCSF and aCSF+M groups, indicating that under normal
conditions the computations performed by the CA3 and the
CA1 networks are congruent with each other. In contrast,
the similarity score between these hippocampal regions in the
LPS-treated animals are not
computations performed in these two hippocampal regions in
animals with neuroinflammation. It is noteworthy that for lipopo-
lysaccharide animals treated with memantine, similarity scores for
CA3 and CA1 showed greater separation between the AA and
AB condition, compared to lipopolysaccharide alone, but were
intermediate relative to that observed in artificial cerebrospinal
fluid controls ? memantine. This suggests that, although meman-
tine treatment tends to restore network activity patterns within
CA3, it does not completely restore the congruence of information
distinct, revealing incongruent
processing between CA3 and CA1 regions in lipopolysaccharide
infused animals. Although the observed defect is subtle, this
may explain why, even when the number of behaviourally acti-
vated cells is restored, the performance of lipopolysaccharide
memantine-treated animals in the Morris water maze task is
only partially improved (Rosi et al., 2006, Fig. 1A and B). This
suggests that even if CA1 and CA3 can perform their computa-
tions independently, the integration of information between the
two hippocampal subregions is important for optimal spatial
Activated microglia and information
processing in the hippocampus
Previously it was shown that Arc expression in CA3 and CA1 was
related to the presence (CA3) or absence (CA1) of activated
microglia (Rosi et al., 2005). In the present study, we have
extended this observation by showing that information processing
in CA3 was disrupted, while it remained accurate in CA1.
Memantine decreased the number of activated microglia in CA3
(Fig. 5) and also largely restored the accuracy of information
processing in that region, but there were no discernible effects
in CA1. This suggests that at least for CA3, the presence of
How memantine restores accuracy of information processing
known. One possibility is that it acts on NMDARs expressed by
microglia, and another is that it acts directly on neuronal
NMDAR. Support for the first possibility is that the presence of
activated microglia correlates both with impaired memory and
accuracy of information processing (Rosi et al., 2005, 2006).
Additionally, it has been recently suggested that memantine may
act via non-neuronal NMDARs in the developing brain (Manning
et al., 2008). However, there are currently no data reporting
NMDARs expression on adult microglia. Here we provide support
for the second possibility, by demonstrating that even when
stimulated with lipopolysaccharide, microglia do not express
NMDARs. Furthermore, memantine does not influence the
activation of untreated or LPS-treated microglial cells in culture.
These findings support the idea that memantine modulates
NMDARs on neurons. This mechanism may underlie its anti-
inflammatory action and may be responsible for its ability to
restore hippocampal function and spatial cognitive performance.
The implication of these data is that activated microglia indirectly
disrupt neuronal information processing. The possible role of acti-
vated microglia on neuronal activity and vice versa has been, and
is, the focus of several recent studies (de Jong et al., 2005; Biber
et al., 2007, 2008; de Haas et al., 2007).
cytokines and nitric oxide, which in turn leads to a cascade of
(Robinson et al., 1993), and increased synthesis of glutamate by
astrocytes (Bezzi et al., 1998). The excess extracellular glutamate
induced by neuroinflammation (Fine et al., 1996) may result in an
excess of calcium entry in the postsynaptic neurons leading to
Accuracy of hippocampal networksBrain 2009: 132; 2464–2477 |
a positive feedback toxic cycle in which activated microglia release
inflammatory mediators such as cytokines (Bezzi et al., 2001).
Furthermore, elevated levels of glutamate may act on non-
NMDARs and cause chronic membrane depolarization that
would partially relieve the voltage-dependent Mg2+block of the
NMDARs. It is possible that subsequent activation of NMDARs
by ordinary glutamatergic synaptic activity may thus permit a
continuous influx of Ca2+ions into the neurons; at this time this
is only speculative. However, under this scenario, CA3 pyramidal
neurons would be unable to discriminate signals coming from the
mossy fibres and perforant path. The inflammation-induced
threshold change for neuronal activation (i.e. Arc expression),
may therefore reflect the observed altered pattern completion
and separation processes, and may be influenced by elevated
levels of cytokines and/or nitric oxide. Furthermore, elevated
levels of inflammatory cytokines enhance the NMDA-dependent
intracellular calcium levels (Viviani et al., 2003). Given that
memantine promotes the normal entry of calcium through
NMDARs, this could help explain how this agent stabilizes
Therefore, during the early stages of neuroinflammation,
dysfunctionat the NMDA-glutamate
accuracy of information processing, while in the later stages this
condition may contribute to neuronal degeneration. Overall, given
the role of activated microglia on neuronal activity and vice versa
(de Jong et al., 2005; Biber et al., 2007, 2008; de Haas et al.,
2007), it is possible that the specificity of the deficit is due to this
microglial activation and its effect on CA3 pyramidal cell function.
The direct input from the entorhinal cortex to CA1, on the other
hand, may allow relatively normal activity to occur in the CA1
pyramidal cells, in spite of the altered input from CA3.
While it is not possible to definitively answer the question of
whether memantine acts via
NMDARs (or both), recent data concerning this topic suggest
that it is less likely to work through alterations of interneuron
function. Computational models of hippocampal function (Treves
and Rolls, 1992) have shown that efficient pattern separation
requires parallel activation of two excitatory inputs to CA3, the
perforant path from layer two of the entorhinal cortex and the
granule cell axons (mossy fibres, MF). When multiple cortical input
patterns to CA3 arrive simultaneously, feed forward inhibition
driven by mossy fibres limit the number of pyramidal cells acti-
vated by the same input. This further increases the sparsity of the
network and the storage capacity in the CA3 (Treves and Rolls,
1992). Thus, the CA3 feed forward inhibitory interneurons
may play a role in regulating decorrelation of compressed
memory representations in CA3 area. Furthermore, it has been
demonstrated that theinhibitory
that contributes to hippocampal network plasticity. This plasticity,
however, is independent of NMDAR, and dependent on metabo-
tropic glutamate receptors (Perez et al., 2001; Galvan et al.,
2008). Given these data it is possible that the changes observed
during neuroinflammation and treatment with memantine at
the dose used here, are mostly mediated via principal cell
NMDARs. Future work will elucidate the precise mechanisms
guiding these processes.
We have demonstrated that LPS-induced neuroinflammation
disrupts accuracy of information processing in the hippocampus
and that this disruption is partially normalized by co-administration
of memantine. Memantine appears to exert its effect by acting
directly on neuronal NMDARs, and not on microglia themselves.
Thus, memantine treatment given early in the progression of
neurodegenerative diseases may confer neuronal and cognitive
protection by conserving accurate information processing and
indirectly preventing pathological microglia activation.
We would like to thank Dr Alessandro Treves for his critical
feedback on the article and Dr Kathleen Lamborn for her help
with the data analysis.
National Institutes of Health (AG009219 to C.A.B.; AG10546
to G.L.W.; AG030331 to G.L.W.); McKnight Brain Research
Foundation state of Arizona and Arizona Department of Health
Services (to C.A.B.); Consejo Nacional de Ciencia y Tecnologia
(51028 to V.R.A); Direccion General de Asuntos del Personal
Academico UNAM (IN213907 to V.R.A.).
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