In vitro studies
Primary cultures of cortical neurons were infected with A-GFP-NR1(+) and A-GFP-NR1(–) vectors at high multiplicity of infection (MOI = 5–7). About 50% of the cells expressed GFP, showing that both vectors transduced the cells (Figs 1b and d). By immunocytochemistry (Figs 1a and c), a high expression of NR1 was observed in cells infected with A-GFP-NR1(+), whereas there was an evident decrease in the immunostaining when the culture had been infected with A-GFP-NR1(–), compared to non-transduced cells. Accordingly, the density of bands in the immunoblot (Fig. 1e) of cells infected with A-GFP-NR1(+) showed a significant increase in NR1 (433.9% ± 17.7, p < 0.001, n = 4), whereas those infected with A-GFP-NR1(–) showed a significant decrease (26.1% ± 1.5, p < 0.01, n = 4) compared to non-infected cells (100% ± 8.5) (one-way anova and Tukey's Multiple Comparison Test).
Figure 1. Immunocytochemistry of primary cultures of cortical neurons infected with either A-GFP-NR1(+) or A-GFP-NR1(−) vectors at high multiplicity of infection (MOI) (5 particles/cell). The transgene expression was revealed by the corresponding antibodies. There are two cells in the same picture (a), one infected and expressing GFP (b), with background levels of red fluorescence (a), and the other, presumably not infected, without green fluorescence, expressing endogenous levels of NR1 (a). There was a very high expression of both GFP (d) and NR1 (c) in cells infected with A-GFP-NR1(+). (400 ×). (e) Immunoblot: bands correspond to NR1 and to actin (as revealed by the corresponding antibodies) from primary cultures infected either with A-GFP-NR1(+) or A-GFP-NR1(−) or not infected. Cells infected with the vector only expressing GFP (A-GFP) were also included as control. A hippocampal homogenate was run in parallel to allow comparison with the endogenous subunit and to determine the specificity of the band. GFP, green fluorescent protein.
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We had previously shown that cells tranfected with A-GFP-NR1(–) from the same stock produced the NR1 antisense RNA (Adrover et al. 2003); here we show the decrease of NR1 in neurons infected with A-GFP-NR1(–). NR1 was overexpressed in neurons infected with A-GFP-NR1(+), whereas GFP was conspicuously expressed from both vectors. Therefore, the constructed amplicon vectors, practically devoid of helper virus, were efficient at expressing the transgenes and modifying endogenous levels of NR1 in neurons; hence, they were appropriate to be used for our in vivo assays.
In vivo studies
Rats injected with the vectors were tested in an open field 5 days later. The naive animals showed no differences compared to sham-operated rats in every assay for all the recorded parameters (not shown). The number of crossings and rearings showed a normal distribution (Wilk-Shapiro modified test, Mahibbur and Govindarajulu 1997). Media ± standard error of mean (SEM) of number of crossings and rearings were, for sham operated, 122.2 ± 5.4 and 22.6 ± 1.4, for A-GFP-NR1(+) injected, 113.3 ± 8.9 and 19.3 ± 1.8, and for A-GFP-NR1(–), 106.9 ± 6.4 and 19.3 ± 0.8 (n = 12 for each group). There were no significant differences between groups (one-way anovaF = 0.91, p > 0.05 for crossings, F = 2.67, p > 0.05 for rearings). These results strongly indicated that there were no alterations in locomotor activity or in exploratory behaviour of injected rats.
Then, the animals were trained in the inhibitory avoidance task. As test latencies were limited to 120 s and they were not normally distributed (Wilk–Shapiro modified test), the data were analysed by non-parametric statistics (Conover 1999). There were no differences between groups for training latencies (Kruskal–Wallis); median with interquartile ranges [25; 75] were, for sham-operated rats, 3 s [2.00; 3.49], for A-GFP-NR1(+), 3.32 s [2.90; 6.27], and for A-GFP-NR1(–), 2.72 s [1.61; 4.59] (n = 12 for each group). In the test session 24 h later, A-GFP-NR1(+) injected rats showed no differences compared to sham-operated animals; test latencies were significantly higher than training latencies (p < 0.05, Wilcoxon test). Median of test latencies for sham-operated animals was 16.57 s [10.54; 65.73] and for A-GFP-NR1(+) injected rats, 15.22 s [9.93; 57.27]. In other words, they learned and remembered to spend longer on the platform. Although there was an overexpression of NR1 in primary culture of neurons transduced with the A-GFP-NR1(+) vector, in our in vivo experiments neither significant improvements in performance nor overexpression of the subunit were detected. Genetic enhancement of NMDAR function in transgenic mice resulted in an enhancement of both synaptic coincidence detection and learning/memory (Tang et al. 1999; Wong et al. 2002). Barria and Malinow (2002) reported that exogenously expressed NR1 in culture neurons transfected with the corresponding plasmid was not assembled as NMDAR unless coexpressed with NR2. If NR1 protein expressed from the vectors was not properly assembled in vivo, an improvement in learning and memory would not be expected.
On the other hand, rats injected with A-GFP-NR1(–) did not show a significant increase in test latencies. Median for training latencies was 2.72 s [1.61; 4.59] and for test latencies it was 5.46 s [4.25; 9.43]. These results corroborated previous findings and strongly supported the involvement of hippocampal NMDAR in recording the trace for this task (Jerusalinsky et al. 1992; Adrover et al. 2003). Furthermore, we have demonstrated previously that rats injected with A-NR1(–) vectors did not habituate to the open field, and there was a slight decrease in the NR1 subunit in the hippocampus (Cheli et al. 2002). Those behavioural tests have been reproduced in the present work with similar results (not shown).
We used confocal microscopy to determine and quantify the cells transduced by the vectors in vivo. GFP expression was restricted to a small region in the dorsal hippocampus (Fig. 2a); most of the ‘green cells’ were pyramidal neurons. The volume for quantification (1000 µm thick × 400 µm wide × 70 µm high) was calculated from serial brain slices. Total cell number (Hoechst), neurons (NeuN) and transduced cells (GFP) were quantified (Mouton 2002) (Figs 2b and c). About 6–7% of pyramidal neurons in CA1 and only about 1–1.5% of granule cells just below those pyramidal neurons were transduced, as judged by GFP expression. This implies that reduction of NMDAR in only a small proportion of CA1 pyramidal neurons affects ability to learn and/or remember the task. When larger hippocampal regions were affected, i.e. with NMDAR antagonists (Jerusalinsky et al. 1992), the performance in similar tasks did not appear to be different from the results in this study.
Figure 2. (a) Immunohistochemistry with GFP and GFAP antibodies in brain slices from rats injected with A-GFP-NR1(-) vectors into CA1. Green cells are CA1 pyramidal neurons and red cells correspond to actived microglia (GSA-B4). (b) Immunohistochemistry with anti-NeuN, and anti-GFP in hippocampal slices, stained with Hoechst 33342. Images were obtained every 3 µm (Leica confocal microscope). Total cells (blue), neurons (red) and infected cells (green) were counted. (c) Table: proportion of green cells (anti-GFP) respect to total cells (Hoechst 33342) and neurons in CA1 and dentate gyrus (anti-NeuN).
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Cain (1998) reported that rats receiving NMDAR antagonists displayed sensorimotor disturbances. Inada et al. (2003), delivering NR1-antisense to the rat hippocampus, caused a mild knockdown that did not affect spatial memory but it appeared to produced a deficit in sensorimotor gating. From the behavioural point of view, neither the conditional CA1 NR1-KO mice (Tsien et al. 1996), nor our infected rats appeared to have sensorimotor alterations or disturbances in exploratory behaviour. The learning deficits for CA1 NR1-KO mouse have been extended to non-spatial tasks (Rampon et al. 2000; Shimizu et al. 2000). Since most of CA1 pyramidal neurons in that mouse suffered the deletion, it was not clear which cells and how many were needed to support the trace.
The injection of a neurotropic amplicon vector expressing NR1 antisense RNA seems a rapid and efficient procedure to modify the receptor, with temporal and regional restrictions. Here we showed that the knockdown of the NR1 subunit in as few as 6–7% of CA1 pyramidal cells was sufficient to impair learning of an inhibitory avoidance task. Interestingly, Rumpel et al. (2005) have reported that memory of fear conditioning was reduced if AMPA receptor synaptic incorporation was blocked in about 10% of lateral amygdala neurons.
A-GFP-NR1(+) vector caused the same average rate of infection as A-GFP-NR1(–); this strongly suggests that expression of NR1 antisense RNA, rather than any other related variable, was responsible for learning impairment.
Although the hippocampus appears crucial for memory acquisition in rodents, taking into account that severe neuropathology or large surgical lesions do not seem to easily perturb memory formation, it is at least surprising that dysfunction of just a few neurons in CA1 could cause significant impairments in recognizing a new environment (Cheli et al. 2002) and in learning an inhibitory avoidance task. Assuming that our amplicon vectors, which mainly transduced pyramidal neurons, did not preferentially infect certain of them (they were administered 5 days before the behavioural assays, reaching a ‘plateau’ in expression on the third day), one must conclude that affecting a small fraction of pyramidal neurons in CA1 is sufficient to disturb memory formation. It might be speculated that neurons and their synapses underlying learning-related synaptic plasticity were more susceptible to the expression level of the transgene and, therefore, NMDAR are particularly required in those neurons undergoing synaptic plasticity during learning. But assuming that CA1 neurons were infected at random, the results suggest that there would not be a high level of redundancy in the circuits involved, leading to the idea that a certain level of NR1 subunit expression/availability is required during memory formation in most of CA1 pyramidal neurons bearing NMDAR.