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 environment (Vazdarjanova and Guzowski, 2004
; Guzowski et al
). 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 (C). 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 (Arc
Nuclei with Arc-foci (yellow) can be observed in A and A, and reflect those cells active within ~5 min of tissue collection. Nuclei containing Arc-cyto (green) can be observed in the same figures, which indicate neurons active within ~30 min 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; C).
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 (more ...)
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 (more ...)
Lipopolysaccharide affects information processing in CA3 networks: restoration by memantine treatment
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% (B), which was significantly higher than the 2.5%–5% observed for caged control animals (P < 0.001; 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 (B). In lipopolysaccharide-infused animals there was a significant increase in the percentage of Arc positive neurons in epochs 1 and 2 and under both exploration conditions compared to artificial cerebrospinal fluid animals [ANOVA: F(7,38) = 7.5, P < 0.001, for epoch 1; F(7,38) = 7.1, P < 0.001 for epoch 2; all post hoc Bonferroni comparisons P < 0.05]. 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 (B).
The percentages of neurons active during both epochs 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, P < 0.001] (C). This suggests that in CA3, the same neurons were activated 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 environments (AB). A similar relationship was observed in animals infused with aCSF + memantine (C). In contrast, after lipopolysaccharide infusion, the fraction of activated neurons during the AA and AB paradigms was similar (C), 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, P < 0.001; C].
Information processing in the CA1 area is unaltered by lipopolysaccharide or memantine infusion
For all experimental conditions used here, the fractions of CA1 neurons showing behaviourally induced Arc averaged 35%–40%, which was significantly higher than for the caged control animals in which only 5%–6% showed Arc expression (P < 0.001; 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 conditions [ANOVA: F(7,38) = 3.8, P < 0.02 for epoch 1; F(7,38) = 1.5, P < 0.24 for epoch 2, B]. 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 the AB paradigm in all treatment conditions [ANOVA: F(7,38) = 35.8, P < 0.001; C], suggesting normal information processing in the CA1 region even during neuroinflammation.
The per cent of double-labelled neurons activated during epochs 1 and 2 (C and C) can be expressed in summary form using a similarity score analysis that normalizes activity values to range from 0 to 1 (A; see Methods section). Similarity scores 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 A). In contrast, artificial cerebrospinal fluid rats allowed to explore two different environments (AB), showed two statistically independent 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 (A). 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, P
< 0.05; for CA1: F
(7,38) = 32.5, P
< 0.05; all appropriate post hoc
comparisons had P
< 0.05]. 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 (A). The observed similarities and differences among the groups are best understood via graphical display of the relationship between similarity scores between CA1 and CA3 (B). 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 (B). 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 B 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
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 (more ...)
Memantine does not directly affect microglia
Chronic infusion of lipopolysaccharide in vivo
resulted in the selective activation of microglia within CA3 area and this was significantly reduced with memantine treatment (A 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
), 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 cultured N9 microglial cells in the presence of memantine and/or lipopolysaccharide. Treatment with memantine did not result in any changes from control levels (). Treatment with lipopolysaccharide did stimulate these microglial cells to significantly increase transcription of the pro inflammatory mediators Tumor Necrosis Factor α (TNFα) and inducible nitric oxide synthase (iNOS), as measured by qPCR (). When cells were treated with memantine and lipopolysaccharide at the same time, microglial cells increased the transcription of TNFα and iNOS similarly to the lipopolysaccharide treatment alone. Taken together, the data suggest that memantine treatment alone did not directly affect microglial cells.
Figure 5 Representative flat images of reconstructed CA3 area from the dorsal hippocampus (~−3.6 mm from bregma) from animals chronically infused with LPS (A) and animals chronically infused with LPS and treated with memantine (B). Activated microglia (more ...)
Figure 6 Quantitative real-time PCR (qPCR) analysis of TNFα (A) and iNOS mRNA (B) levels in cultured N9 microglial cells. Cells treated with LPS for 24 h showed significantly increased expression of both TNFα and iNOS mRNA compared to vehicle (macrophage (more ...)