Hippocampal function is not required for the precision of remote place memory
© Kitamura et al; licensee BioMed Central Ltd. 2012
Received: 11 January 2012
Accepted: 2 February 2012
Published: 2 February 2012
During permanent memory formation, recall of acquired place memories initially depends on the hippocampus and eventually become hippocampus-independent with time. It has been suggested that the quality of original place memories also transforms from a precise form to a less precise form with similar time course. The question arises of whether the quality of original place memories is determined by brain regions on which the memory depends.
To directly test this idea, we introduced a new procedure: a non-associative place recognition memory test in mice. Combined with genetic and pharmacological approaches, our analyses revealed that place memory is precisely maintained for 28 days, although the recall of place memory shifts from hippocampus-dependent to hippocampus-independent with time. Moreover, the inactivation of the hippocampal function does not inhibit the precision of remote place memory.
These results indicate that the quality of place memories is not determined by brain regions on which the memory depends.
The hippocampus is a key brain structure for learning and memory [1–3]. Recall of some associative and spatial memories initially depends on the hippocampus, but that hippocampal dependency progressively decays over time, a process that is associated with a gradual increase of neocortex-dependency [4–9]. It has been suggested that the quality of original memories also transforms from a precise (i.e., detailed) form to a less precise (i.e., more schematic or generic) form with similar time course [10–12]. The question arises of whether changes in the quality of the original memories depend on the shift in brain regions on which the recall of these memories relies, i.e., whether the hippocampus is always required for the precision of memories. This is an important question for understanding physiological significances of the hippocampal-cortical complementary memory systems.
There are several studies that address this issue in rodents. Using a contextual fear conditioning paradigm, which is an associative learning between a place and aversive experience, some studies demonstrated that the hippocampus is always necessary for the precision of place memories [13, 14], supporting the memory transformation view  in which the quality of place memory correlates with the brain region on which that memory depends. By contrast, another study demonstrated that the hippocampus is not required for memory precision after the passage of time , supporting the memory reorganization view  in which the quality of place memory does not correlate with the brain region. Importantly, this discrepancy can be attributed to differences in experimental protocols used for association with fear . Because association with fear modifies (i.e., strengthen or generalize) the precision [16, 17], we cannot rule out the possibility that fear association may mask the actual precision of place memory. Moreover, contextual fear conditioning indirectly evaluates the place memory by measuring fear responses. Analyses based on association procedures may not be suitable for evaluating the precision. A direct way to address this issue is to measure the place memory per se without association protocols. In this study, we introduced a new procedure: a one-trial and non-associative place recognition test in mice. The clear advantage is the absence of any explicit reinforcement. In this procedure, we simply placed mice in a previously experienced vs. novel place. In the previously experienced place, mice exhibited habituation (reduced motility). In the novel context, mice did not show this reduction in motility. Thus, we evaluated the adaptation level as an index of the successful retrieval of place memory. Mice quickly acquired place memory during a free exploration and clearly discriminate experienced place from novel place. Especially mice maintained these memories for one month, which is different from the case in rats . This procedure allows us to directly evaluate the precision of remote place memory. Using this procedure combined with genetic and pharmacological approaches, we examined the relationship between the precision of place memory and the brain region on which that memory depends.
Materials & methods
All animal procedures were conducted in compliance with the guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee of the University of Toyama and the Mitsubishi Kagaku Institute of Life Sciences. For WT experiments, male C57BL/6JSLC mice at 8 weeks of age were purchased from Sankyo Laboratory (Japan). For experiments using inducible cAMP response element binding protein (CREB) repressor transgenic mice [19, 20] and α-Calcium-calmodulin kinase II heterozygous null mutant mice [21–23], the progeny for each line was produced using in vitro fertilization and embryo transfer techniques to produce a number of animals sufficient for behavioral testing. Age- and gender-matched littermates were used for the tests. All behavioral experiments were conducted and analyzed by scientists blind to the genotypes of the animals. Food and water were provided ad libitum.
Cannulation and drug infusion
We used a surgical procedure described previously [24, 25]. Briefly, mice were implanted bilaterally with stainless steel guide cannulae (Eicom) using the following stereotactic coordinates: AP = -2.0 mm, ML = ± 1.5 mm, V = -2.1 mm from the bregma. Mice were allowed to recover for at least 14 days in individual home cages. To transiently inactivate hippocampus, a fluorescently-labeled γ-aminobutyric acid subtype A receptor agonist (FCM, fluorophore-conjugated muscimol; Molecular Probe) was used. Mice were briefly anesthetized with isoflurane to facilitate the insertion of the injection cannula. FCM (0.8 mM, 0.5 ul) or PBS alone was infused into the dorsal hippocampus at a rate of 0.20 μl/min 60 min before the retrieval test. The fluorescent signals of FCM were distributed in the dorsal hippocampus bilaterally. To inhibit protein synthesis in the hippocampus, anisomycin (ANI, 100 μg/μl, 0.75 μl; Sigma) was infused into the dorsal hippocampus bilaterally at a rate of 0.25 μl/min 10 minutes after the learning. For the i.p. injection of ANI, ANI (150 mg/kg, i.p.) was dissolved in PBS (pH adjusted to 7.0-7.4) and administered to mice 10 minutes after the learning. At this dose, ANI inhibits 90% of protein synthesis in the brain during the first 2 hours . For the i.p. injection of (R)-CPP (10 mg/kg, i.p.; Tocris), an antagonist of N-methyl-D-aspartate receptors, (R)-CPP was dissolved in PBS and administered to mice 4.5 hours before the learning. To examine the effects of disrupting CREB function on place memory formation, we used the transgenic mice that express an inducible CREB repressor in the forebrain, where a dominant-negative CREB protein is fused with the ligand binding domain of a mutant estrogen receptor . Tamoxifen (4-hydroxytamoxifen; 16 mg/kg, i.p. TAM; Sigma), which was dissolved in 10 ml of peanut oil (Sigma), or peanut oil only (OIL) was administered to WT or transgenic mice 6 hours before the learning session.
Place memory test
Contextual fear conditioning
Contextual fear conditioning was carried out as described previously [24, 27, 28]. Briefly, during the training session, mice were placed in the conditioning chamber (S) in Room A. After 3 minutes, animals were subjected to three unsignaled footshocks (2 sec duration, 0.5 mA, a min apart). After the last shock, mice remained in the chamber for 1 minute and were then returned to their home cages. During the testing session, mice were placed back into the conditioning chamber (S) or the novel chamber (C) for 3 minutes in Room A at 1 day or 28 days after training. At the end of each session, mice were returned to their home cages and the chambers were cleaned with water and 80% ethanol. All experiments were conducted using a video tracking system (Muromachi Kikai; Japan) to measure the freezing behavior of the animals. Freezing was defined as a complete absence of movement, except for respiration. Scoring of the duration of the freezing responses was started after 1 sec of sustained freezing behavior.
All data are presented as mean ± SEM. The number of animals used is indicated by "n". Comparisons between two-group data were analyzed by unpaired Student's t-tests or paired t-tests. Multiple group comparisons were assessed using a one-way, two-way, or repeated measures analysis of variance (ANOVA), followed by the post-hoc Scheffe's test when significant main effects or interactions were detected. The null hypothesis was rejected at the P < 0.05 level.
Mice were exposed to a novel square-type (S) chamber to learn this place (Figure 1A, B). One day later, mice were again exposed to the same chamber or a novel circle-type (C) chamber to test memory retrieval (Figure 1A, C). We monitored the motility of mice in the chambers and evaluated the adaptation level for the experienced or novel chamber as an index of the successful retrieval of place memory (Figure 1C, D). Mice showed a reduction in their motility when exposed to the same S chamber (paired t-test; t6 = 9.96, P < 0.001) (Figure 1D). By contrast, a six-minute exposure to the S chamber did not induce adaptation in the novel C chamber (paired t-test; t6 = -0.07, P > 0.9) (Figure 1C, D). We obtained a similar result when we changed the sequence (C-C paired t-test: t5 = 8.39, P < 0.001; C-S paired t-test: t6 = 1.16, P > 0.2) (Figure 1D). These results indicate that mice quickly learn the place and completely discriminate the experienced place from the novel place one day after learning. Thus, the adaptation can be used to index place memory.
Finally, we examined whether hippocampal function is required for the precision of remote place memory retrieval. In the retrieval test for 28-day memory, FCM-infused mice were placed in the experienced S chamber or novel C chamber (Figure 4E). FCM-infused mice showed adaptation for the experienced S chamber (paired t-test: t9 = 12.06, P < 0.001), whereas FCM-infused mice did not show adaptation for the novel C chamber (paired t-test: t9 = -0.68, P > 0.5) (Figure 4E). We further examined the variable-rooms/constant-chamber condition 28 days after learning (Figure 4E). Similar to naive mice (Figure 3A), FCM-infused mice did not show adaptation for the experienced S chamber in the novel room B (paired t-test: t11 = -0.84, P > 0.4) (Figure 4E). These results indicate that the pharmacological inactivation of hippocampal function does not inhibit the precision of remote place memory.
In this study, we carried out the contextual fear conditioning test and the non-associative place recognition test under the same experimental condition (same chambers and same exposure time to chamber) (Figure 3). These results clearly indicate that the association with fear masks the actual precision of place memory. Moreover, in contextual fear conditioning mice showed the freezing responses even for unconditioned place in recent memory test (Figure 3B) [13, 15, 24, 32–34], whereas in our non-associative place recognition test mice did not show any adaptation behavior even for similar place in remote memory test (Figure 3A). Therefore, the conclusions of previous studies using contextual fear conditioning [13–15] need to be validated by non-associative protocol. Thus, we examined the contribution of hippocampal function on the precision of remote place memory by non-associative place recognition test.
Using this procedure, we found that the place memory is precisely maintained for 28 days, which may require α-CaMKII-dependent plasticity in the cortex, and that the retrieval of remote place memory (not recent memory) does not require hippocampal function. These results indicate that the quality of a place memory does not correlate with the brain region on which that memory depends. Moreover, we found that the inactivation of hippocampal function does not inhibit the precision of remote place memory. These results indicate that the hippocampal function is not required for the precision of remote place memory. This is consistent with a human case study in which a patient with bilateral extensive hippocampal damage showed intact memories for places learned long ago, but not intact recent place memory . Eight patients with bilateral hippocampal damage were able to recall their remote autobiographical memories . Thus, the quality of original place memories is not determined by brain regions on which the memory depends.
We thank S. Kamijo, M. Matsuo and H. Hidaka for the care of the animals. We also thank B. Wiltgen and N. Ohkawa for reading the manuscript and for discussions, and all members of the Inokuchi laboratory for daily discussions. This work was supported by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Agency (JST) to K.I., a Grant-in-Aid for Scientific Research (S) to K.I., the Mitsubishi Foundation to K.I., the Uehara Memorial Foundation to K.I., a Grant-in-Aid for Young Scientists (B) to T.K., and the Sasagawa Scientific Research Grant to T.K.
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