Author's Accepted Manuscript

Hippocampal place cell responses to distal and proximal cue manipulations in dopamine D2 receptor-knockout mice Chien Le Nguyen, Anh Hai Tran, Jumpei Matsumoto, Etsuro Hori, Teruko Uwano, Taketoshi Ono, Hisao Nishijo

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S0006-8993(14)00521-6 http://dx.doi.org/10.1016/j.brainres.2014.04.023 BRES43524

To appear in: Brain Research

Accepted date: 13 April 2014 Cite this article as: Chien Le Nguyen, Anh Hai Tran, Jumpei Matsumoto, Etsuro Hori, Teruko Uwano, Taketoshi Ono, Hisao Nishijo, Hippocampal place cell responses to distal and proximal cue manipulations in dopamine D2 receptor-knockout mice, Brain Research, http://dx.doi.org/ 10.1016/j.brainres.2014.04.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hippocampal place cell responses to distal and proximal cue manipulations in dopamine D2 receptor-knockout mice

Chien Le Nguyen1, Anh Hai Tran1, Jumpei Matsumoto1, Etsuro Hori1, Teruko Uwano2, Taketoshi Ono2, Hisao Nishijo1

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System Emotional Science, and 2Integrative Neuroscience, Graduate School of Medicine and

Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan

Short title: Hippocampal place cells in D2R-KO mice

Correspondence to: Dr. H. Nishijo. System Emotional Science Graduate School of Medicine and Pharmaceutical Sciences University of Toyama Sugitani 2630, Toyama 930-0194, Japan Tel.: +81-76-434-7215; Fax: +81-76-434-5012 E-mail: [email protected]

 

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Abstract

The human hippocampus is critical for learning and memory. In rodents, hippocampal pyramidal neurons fire in a location-specific manner and form relational representations of environmental cues. The important roles of dopaminergic D1 receptors in learning and in hippocampal neural synaptic plasticity in novel environments have been previously shown. However, the roles of D2 receptors in hippocampal neural plasticity in response to novel and familiar spatial stimuli remain unclear. In order to clarify this issue, we recorded from hippocampal neurons in dopamine D2 receptor-knockout (D2RKO) mice and their wild-type (WT) littermates during manipulations of distinct spatial cues in familiar and novel environments. Here, we report that D2R-KO mice showed substantial deficits in place-cell properties (number of place cells, intra-field firing rates, spatial tuning, and spatial coherence). Furthermore, although place cells in D2R-KO mice responded to manipulations of distal and proximal cues in both familiar and novel environments in a manner that was similar to place cells in WT mice, place fields were less stable in the D2R-KO mice in the familiar environment, but not in the novel environment. The present results suggested that D2 receptors in the hippocampus are important for place response stability. The place-cell properties of D2R-KO mice were similar to aged animals, suggesting that the alterations of place-cell properties in aged animals might be ascribed partly to alterations in the D2R in the HF of aged animals.

Key words: mice; hippocampus; place-cell; dopamine; D2 receptor-knockout; environmental manipulation

 

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1. Introduction

The hippocampal formation (HF) is critical for episodic memory in humans and other primates (Eichenbaum et al., 1999; Ekstrom et al., 2000; Maguire et al., 1998; Matsumura et al., 1999; Roll and Xiang, 2005). Lesions or manipulations of the HF in rodents cause deficits in spatial functions (Gasbarri et al., 1996b; Whishaw et al., 1997; Wilkerson and Levin, 1999), and hippocampal neurons in rodents fire in a location-specific manner ( O’Keefe and Burgess, 1996; O’Keefe and Dostrovky, 1971; Wilson and McNaughton, 1993) in association with external and internal cues (Gothard et al., 1996; Hetherington and Shapiro, 1997; Knierim et al., 1998; Leutgeb et al., 2005a, 2005b; Lever et al., 2002; Muller and Kubie, 1987; Shapiro et al., 1997; Wiener et al., 1989; Zinyuk et al., 2000) or contextual information (Fanselow 2000; Gill and Mizumori, 2006; Zelikowski et al., 2012), indicating a role in spatial memory (Bannerman et al. 2012; Leutgeb et al. 2005a, 2005b; Wilson and McNaughton, 1993; Vann and Albasser, 2011). Furthermore, the HF appears to provide a neural representation of physical space, although broader functions have also been suggested (Eichenbaum et al., 1999; Maguire et al., 1998). The place-cell representation of place is thought to underlie certain forms of spatial learning (Cho et al., 1998; Dragoi et al., 2003; Eichenbaum et al., 1999; Kentros et al., 2004, 1998; McHugh et al., 2007, 1996; Rotenberg et al., 2000). The HF, which receives dopaminergic fibers that originate from the substantia nigra and ventral tegmental area (Gasbarri et al., 1996a, 1994a, 1994b), express dopamine (DA) D1 and D2 receptors (Goldsmith and Joyce 1994; Mansour et al., 1992, 1990; Meador-Woodruff et al., 1992, 1989; Missale et al., 1998). DA controls synaptic transmission in the HF (Hsu, 1996; Noriyama et al., 2006; Otmakhova and Lisman, 1998) and contributes to long-term potentiation (Frey et al., 1991; Otmakhova and Lisman,

 

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1996). Consistent with these findings, place cells in D1 receptor (D1R)-knockout (KO) mice were found to not respond to the manipulations of distal and proximal cues in a novel environment, suggesting that the D1R is crucially implicated in encoding spatial information in a novel environment and that it influences the plasticity of HF representations (Tran et al., 2008). There has been some evidence that D2 receptors (D2R) play a role in memory and synaptic plasticity in the HF; a positive correlation was found between the performances of tasks that were related to memory, verbal fluency, and executive cognitive function and D2R binding in the HF (Takahashi et al., 2008, 2007). HF D2R densities correlate with memory functions in Alzheimer’s disease (Kemppainen et al., 2003). However, the role of D2Rs in place-cell activities remains unknown. In the present study, we investigated place-cell activities in D2R-KO mice and their wild-type (WT) littermates in response to spatial cue manipulations in familiar and novel environments.

2. Results

2.1 Behavioral performance of the D2R-KO mice The existence of DA receptors in the retina has previously been reported (Courtière et al., 2003; Djamgoz et al., 1997; Nguyen-Legros et al., 1999). Their presence raised a concern regarding the visual acuity of D2R-KO mice. We therefore conducted a visual acuity test in the mice (Crawley, 2000; Fox, 1965). No differences were found in either the number of positive responses or in the latencies in response between the 2 types of mice (Table 1) (positive response: WT, 78/100 vs. D2R-KO, 71/90, P = 0.88; latency: WT, 63.00 ± 51.01 vs. D2R-KO, 52.00 ± 54.26, P = 0.14), showing that the visual perceptual skills in the D2R-KO mice was not markedly deficient compared to those in the WT mice.

 

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We have previously reported that D2R-KO mice display reduced locomotor activity, slower acquisition of a place-learning task, and an inefficient pattern of movement in the task (Tran et al., 2002). We reproduced these previous findings in the mice used in the present study, with the same behavioral experiments as those in the previous study (Supplementary Fig. 1).

2.2 Histology The positions of the recording electrodes were verified microscopically and mapped onto the appropriate tissue sections, and the sections were compared with the mouse brain atlas (Hof et al., 2000). All of the recording sites were located in the CA1 region in both types of mice (Fig. 1).

2.3 Place cells in D2R-KO and WT mice in the familiar environment We recorded neuronal activity from the CA1 region of the HF of the WT and D2R-KO mice performing random reward place search tasks (RRPST) in open fields (Fig. 2A and B). A total of 144 cells were recorded from the WT mice, and 117 cells were recorded from the D2R-KO mice (Table 2). Of these cells, 79 from the WT mice displayed place-related activity, and 39 from the D2R-KO mice displayed place-related activity. There was a significant difference in the number of recorded cells that displayed place-related activity between the 2 groups of mice (WT, 79/144, 54.9% vs. D2R-KO, 39/117, 33.3%, P = 0.01, χ2 test). Thus, the number of place cells was significantly decreased in the D2R-KO mice. For the place cells, we characterized the basic firing properties in the standard session in the familiar environment (Table 2). There were significant differences in the mean intra-field firing rates, spatial tuning, and spatial coherence between the 2 groups.

 

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2.4 D2R-KO in the familiar environment We consequently examined the neural plasticity of HF place cells during rotation manipulations of the distal and proximal cues in the familiar circular recording chamber (Fig. 2C). Only place cells that had at least 1 place field in each of the 5 sessions in each environment were analyzed. The responses of the place cells to manipulations of the environmental cues were categorized as being controlled by distal cues, proximal cues, both cue types, or neither cue type (Table 3). In the WT mice, the effects of the proximal cues predominated over the distal cues (Table 3), in that about half of the place cells (30/66, 45.5%) followed the rotation of the proximal cues (Fig. 3; B4), and fewer place cells (10/66, 15.2%) followed the rotation of the distal cues (Fig. 3; A2). There was a certain number of place cells (26/66, 39.4%) that responded to neither the distal nor proximal cues (Table 3), and none of the place cells was controlled by both cue types (0/66, 0%) (Table 3). It was noted that there were some units that were affected by either distal or proximal cues but that did not met the criterion (place field shift of more than 50% of the cues’ angle rotated -180°) for at least 1 type of cue rotation. In the D2R-KO mice, the effects of the proximal cues predominated, as was observed in the WT mice (Table 3). About half of the place cells (17/33, 51.5%) responded to the proximal cues (Table 3), and only 6% (2/33, 6.1%) followed the distal cues (Table 3). Figure 4 shows 3 examples of place cells. The place cell shown in Fig. 4B followed the proximal cue rotation (B4), while the place cell in Fig. 4A followed the distal cues in the familiar environment (A2). Consistent with the WT group, about 40% of the place cells (13/33, 39.4%) did not respond to the distal or proximal cues (Fig. 4C; 1–5), and only one place cell showed responses to either of the 2 cue types (1/33, 3.0%) in the D2R-KO mice. These results suggested that place-cell responses to the proximal and distal cues in the familiar environment were similar in the WT and D2R-KO mice.

 

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2.5 D2R-KO in the novel environment We performed further experiments in order to elucidate the flexibility of the HF place cells in processing environmental stimuli in the novel environment and to determine whether the D2R system is involved in this process (Fig 2D). When initially exposed to the novel square chamber in baseline session 1, 24 of the 66 WT place cells that were tested displayed firing changes compared with the familiar circular chamber (i.e., remapping). Of these place cells, 10 cells turned off their firing, 10 cells changed their firing field(s), and 4 cells showed increases in their firing rates (at least 2 times more than those in the familiar environment). Of the 33 place cells in the D2R-KO mice, 19 cells were remapped (5 cells turned off their firing, 13 cells changed their firing field locations, and 1 cell showed firing increments in the intra-field firing rates). There was no significant difference in the number of cells with these changes in the novel environment between the 2 groups of mice (WT, 24/66, 36.4% vs. D2R-KO, 19/33, 57.6%, P = 0.056, χ2 test). These results suggested that place cell responses to the novel environment were similar between the WT and D2R-KO mice. Next, we analyzed place cell responses to manipulations of the distal and proximal cues in the novel environment. In the novel environment, interestingly, place cells in both the WT and D2R-KO mice shifted their preference cues from the proximal to distal ones (Distal: WT, 24/46, 52.2%; D2R-KO, 10/23, 43.5%; Table 3) (P > 0.05, χ2 test). The 3 place cells in the WT mice that are shown in Fig. 3 followed the distal cues in the novel environment (A6–10, B6–10, C6–10), while the place cell in the D2R-KO mice that is shown in Fig. 4C followed the distal cues in the novel environment (C6–10). The percentages of place cells that did not respond to either of the 2 cue types were similar to those in the familiar environment and did not differ between the 2 groups of mice (WT, 19/46, 41.3%; D2R-KO,

 

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11/23, 47.8%) (P > 0.05, χ2 test). In addition, the total number of place cells that responded to environmental manipulations did not differ between the 2 groups of mice in either the familiar or the novel environments (P > 0.05, χ2 test).

2.6 Comparison of the stability of the place fields between the WT and D2R-KO mice In order to assess the stability of the place fields, the firing rate maps were compared between the baseline sessions (Table 4). The percentages of place cells with intra-field firing changes were similar between the 2 groups of mice in both the familiar environment (WT, 6/66, 9.1% vs. D2R-KO, 5/33, 15.2%, P > 0.05, χ2 test) and the novel environment (WT, 7/50, 14.0% vs. D2R-KO, 2/24, 8.3%, P > 0.05, χ2 test). However, the percentages of place cells with firing rate map changes between baseline sessions 1 and 2 were greater in the D2R-KO mice than in the WT mice in the familiar environment (WT, 7/66, 10.6% vs. D2R-KO, 15/33, 45.5%, P < 0.05, χ2 test). In addition, the percentages of place cells with firing rate map changes between baseline sessions 1 and 3 were greater in the D2R-KO mice than in the WT mice in the familiar environment (WT, 15/66, 22.7% vs. D2R-KO, 21/33, 63.6%, P < 0.05, χ2 test). However, there was no difference between the WT and D2R-KO mice in the novel environment (Table 4). Furthermore, some of the place cells with firing rate map changes displayed place field changes between the baseline sessions (Table 5). The percentages of place cells that displayed new place field(s) in baseline session 2 and/or 3 were greater in the D2R-KO mice than in the WT mice in the familiar environment (WT, 4/66, 6.1% vs. D2R-KO, 7/33, 21.2%, P < 0.05, χ2 test). In addition, the percentages of place cells that lost place field(s) in baseline session 2 and/or 3 were also greater in the D2R-KO mice than in the WT mice in the familiar environment (WT, 4/66, 6.1% vs. D2RKO, 9/33, 27.3%, P < 0.05, χ2 test). These results indicated that the place fields were less stable in the  

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familiar environment in the D2R-KO mice. 3. Discussion

3.1 Role of D2R-KO in spatial learning In the present study, we found that D2R-KO induced a reduction in the number of place cells in the familiar environment and affected the intra-field firing rate, spatial tuning, and spatial coherence in the HF in the familiar environment. The results of the comparable positive responses and latencies in the WT and D2R-KO mice in the visual acuity test suggest that visual perceptual skills in the D2R-KO mice were not markedly different from those in the WT mice. This further suggests that the alterations in spatial representation in the D2R-KO mice are ascribed to alterations in cognitive and/or motor functions in the D2R-KO mice. Our previous study reported impaired performance in a place-learning task in D2R-KO mice (Tran et al., 2002). In this task, a mouse could receive rewards by shuttling two specific reward places. Previous behavioral and neurophysiological studies that used the same place-learning task have reported that the HF is critical for formation of efficient approach responses (Hori et al., 2002; Kimura et al., 2009), and changes in HF place-cell activity are highly correlated with formation of efficient shuttling trajectories between the reward sites (Kobayashi et al., 2003). A pharmacological study has demonstrated that the infusion of a D2R antagonist into the HF disturbed spatial memory in a radial arm maze (Wilkerson and Levin, 1999). Furthermore, a D2R antagonist has been shown to induce disturbances in long-term potentiation, which is a key phenomenon that is involved in memory formation, in the rat HF (Frey et al., 1990; Yanagihashi et al., 1991). These findings suggested that the learning deficits in the place-learning task in the D2R-KO mice might be ascribed to functional deficits

 

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of HF place cells due to D2R-KO, found in the present study. However, a previous study that used the same place-learning task reported that D1R-KO mice also displayed similar, but more profound, deficits in learning this task (Tran et al., 2005). DA D1 and D2 receptors are generally thought to exert opposite effects on intracellular signals, but recent studies have reported a cooperative effect of D1 and D2 receptors (Pollack, 2004). The coactivation of D1 and D2 receptors has been shown to enhance glutamate-mediated cellular excitation (Hopf et al., 2003). These findings suggest that D1 and D2 receptors work cooperatively in the HF during spatial learning.

3.2 Role of D2R in remapping The crucial role of HF neurons in encoding spatial novelty has long been pronounced by many authors, and the term remapping has been used to describe these kinds of neuronal pattern adjustments that occur in response to changes in sensory or cognitive inputs (Hollup et al., 2001; Kobayashi et al., 1997; Leutgeb et al., 2005a, 2005b). In the present experiment, the depletion of the D2R did not result in significant changes in the remapping phenomenon in the novel environment. Furthermore, there was no significant difference in the remapping phenomenon in D1R-KO mice (Tran et al., 2008). These results were consistent with a previous study that suggested that updated place information is conveyed to the HF essentially through glutamate and that DA modulates this process and stabilizes place fields (Gill and Mizumori, 2008).

3.3 Role of D2R in the HF in environmental manipulation In order to test the hypothesis that the D2R contributes to the modulation of spatial representations in the HF in response to environmental changes, we recorded from HF place cells in

 

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D2R-KO and WT mice during the manipulation of environmental cues. Place-cell activities in both the D2R-KO and WT mice showed similar responses to cue manipulations in the familiar and novel environments. First, place-cell responses were dependent on proximal cues in the familiar environment. These results were consistent with recent studies, which have found that the exact location of placespecific activity may be determined relatively to more proximal cues, while distal cues may determine the orientation of spatial representations (Knierim and Rao, 2003; Lever et al., 2002; O’Keefe and Burgess, 1996; O’Keefe and Nadel, 1978; Save and Poucet, 2000; Siegel et al., 2008; Yoganarasimha and Knierim, 2005). Second, place-cell activities in both the D2R-KO and WT mice were dependent on distal cues in the novel environment. Another study also reported a similar phenomenon, in that place cells that did not remap in a novel environment were mostly controlled by distal cues (Shapiro et al., 1997). This predominant control by distal cues suggests that in this experiment, the animals tended to use information that was more stable and more reliable among the many kinds of cues in order to deal with the constantly changing environment. This difference in the predominant cues between the familiar and novel environments suggested that place cells flexibly process cue information depending on the context (Renaudineau et al., 2007). Furthermore, few place cells responded to manipulations of both the distal and proximal cues in both the D2R-KO and WT mice. This suggested the presence of 2 independent reference frames (distal cue-based and open field-based reference frames), which was consistent with previous studies that reported the existence of multiple independent spatial reference frames (Knierim, 2002; Tanila et al., 1997; Shapiro et al., 1997; Zinyuk et al., 2000). However, it is noted that the circular and square chambers were always used as the familiar and novel environments, respectively. Therefore, the distal cues could be encoded with respect to the corners of the square arena, and consequently have a more stronger polarizing effect in the novel environment. That is, the effects of

 

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distal cues might be ascribed to the shape of the chambers, but not to novelty of the environment. However, it might be unlikely, since a previous study reported that distal cues exerted less than the usual amount of influence over place fields in the presence of salient local cues (Shapiro et al., 1997). Further studies, in which the circular field is used as the novel environment, are required to prove or disprove this idea. Taken together, these findings suggest that place cells can respond to spatial information from the familiar and novel environments in D2R-KO mice. In contrast to the D2R-KO mice, our previous study showed that D1R-KO decreased the number of place cells that respond to changes in spatial cues in the novel environment (Tran et al., 2008). These results suggest that the D1R plays a key role in spatial learning in novel environments.

3.4 Comparison of place-cell properties between the WT and D2R-KO mice The fundamental role of hippocampal formation in the internal representation of space has been confirmed (Leutgeb et al., 2005b; Wilson and McNaughton, 1993), and our previous study suggested that dopamine D1 receptors are crucially implicated in spatial learning and memory (Tran et al., 2008). Moreover, HF expresses both D1 and D2 receptors (Goldsmith et al., 1994; Mansour et al., 1992, 1990; Meador-Woodruff et al., 1992, 1989; Missale et al., 1998) and they involve in memory and synaptic plasticity in the HF (Frey et al. 1991; Hsu, 1996; Kemppainen et al., 2003; Noriyama et al., 2006; Otmakhova and Lisman 1998, 1996; Takahashi et al., 2008, 2007; Tran et al., 2008). Therefore, deficits in place-cell properties found (number of place cells, intra-field firing rates, spatial tuning, and spatial coherence) in the present study could be attributed to D2R knock-out. Furthermore, place fields were less stable in the D2R-KO mice in the familiar environment but not in the novel environment.

 

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Interestingly, these characteristics of the place-cell properties in the D2R-KO mice were similar to those that have been observed in aged animals. The place fields of CA1 place cells in aged rats are less stable between repeated trials in a familiar environment (Barnes et al., 1997), and the spatial tuning and stability of CA1 place cells are decreased in aged mice (Yan et al., 2003). Furthermore, aged animals are slower at learning a spatial working memory task on a radial maze in a spatial environment that is familiar to the animals but not in a novel environment (Mizumori et al., 1996). It has been reported that D2R expression or densities in the HF are altered in aged animals and humans as well as in patients with Alzheimer’s disease (Amenta et al., 2001; Kaasinen et al., 2000; Kemppainen et al., 2003; Ryoo and Joyce, 1994), and that the infusion of a D2R antagonist into the HF impairs spatial working memory, while the infusion of a D2R agonist into the HF improves spatial working memory (Wilkerson and Levin, 1999). These findings suggest that the alterations of place-cell properties that have been observed in aged animals might be ascribed partly to alterations in the D2R in the HF of aged animals.

4. Materials and Methods 4.1 Animals Seventeen male WT mice (26–33 g) and 13 male D2R-KO mice (24–29 g) were used in this experiment. Animals were obtained from the Experimental animal division, RIKEN BioResource center, Ibaraki, Japan. D2R-KO mice were generated with the gene targeting method and confirmed with polymerase chain reaction genotyping and Western blot tests, as fully described in Yamaguchi et al. (1996). All experimental protocols were performed in accordance with the guidelines for the care and use of laboratory animals that were approved by the University of Toyama and the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Committee for Animal

 

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Experiments at the University of Toyama (License number: S-2009 MED-6). 

4.2 Electrode implantation Mice were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and monopolar stimulating electrodes (100 µm, stainless steel, Inter Medical Co., Ltd., Aichi, Japan) were implanted in the medial forebrain bundle on both sides at the level of the posterior lateral hypothalamic area for intracranial selfstimulation (ICSS) (anteroposterior, -2.1 to -2.4 mm; mediolateral, ± 0.7 mm; and dorsoventral, -4.6 to 5.2 mm) (Hof et al., 2000). The recording electrode assembly consisted of 2 tetrodes of twisted 20-µm Nichrome wires or a bundle of 8 Nichrome wires (Formvar insulated, California Fine Wire Company, Grover Beach, CA, USA) that were encased in a stainless-steel cannula (28 gauge), and they were mounted on a moveable microdrive, which enabled later adjustments in the position of the electrodes. The recording electrode was implanted into the dorsal part of the hippocampal CA1 region (2.1 mm posterior to Bregma, 1.8 mm lateral to Bregma, and 1.4 mm below the skull surface) (Hof et al., 2000) during the same surgery. A jeweler’s screw that was fixed to the skull in all mice served as a ground electrode for the ICSS. The microdrive was secured onto the skull with jeweler’s screws and dental cement. The electrode tips were gold-plated before the surgery in order to reduce the impedances to 100 −300 kΩ at 1 kHz.

2.3 Experimental apparatus for spatial tasks The apparatus for the spatial task training was a circular open field (80-cm diameter, 25-cm high wall). It was painted black on the inside and was enclosed by a black curtain (180-cm diameter and 200cm height). The apparatus was elevated 80 cm above the floor on a cart with casters that allowed the

 

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manual rotation and movement of the open field (Fig. 2A). The ceiling of the enclosure contained 4 small speakers, which were spaced 90° apart, near the circumference, 4 incandescent light bulbs that were individually mounted near the inner edge of each speaker, and a video camera at the center. Usually, a light bulb was lit at the 3 o'clock position, and a speaker continuously emitted white noise at the 9 o'clock position. The lit light bulb and emitting speaker served as distal cues. A small lightemitting diode (LED) was mounted on the head of the mouse during the experimental procedures. The video camera (CinePlex, Plexon Inc., Dallas, TX, USA) converted the real video image signal to a binary signal and tracked the horizontal motion of the LED. The X and Y coordinates of the position of the mouse head were recorded at 33 frames/sec in a laboratory computer (Precision T3400, Dell, Round Rock, TX, USA).

4.4 Behavioral procedure 4.4.1 Visual acuity testing A modified visual cliff test (Crawley, 2000; Fox, 1965) was used to test the visual acuity of our mice. A wooden box (46 cm × 46 cm) with a horizontal plane was connected to a vertical drop (48 cm), which in turn was connected to a lower horizontal plane at the nadir of the vertical drop. Paper with a black and white checkerboard pattern covered the surface of the horizontal planes and the vertical drop. A sheet of transparent Plexiglas covered the cliff. A ridge of aluminum (2.54 cm wide and 3.8 cm thick) was placed at the edge of the cliff. Both sides of the apparatus were highly illuminated. The whiskers of the mice were removed before the visual cliff test in order to eliminate tactile information. The mouse was placed on the center ridge at the start of each of 10 consecutive trials (after 5 trials, the apparatus was turned 180°, and 5 more trials were conducted). When the mouse chose to step down onto the

 

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horizontal checkered surface, it was regarded as a positive response, while the mouse stepping down onto the cliff-drop side was regarded as a negative response. The time taken for the mouse to step down from the center ridge was recorded as the latency of response.

4.4.2 Locomotor activity and performance of spatial tasks The locomotor activity and performance of spatial tasks were assessed with the same protocols as Tran et al. (2002). Briefly, spontaneous locomotion in the open field and the number of rewards acquired, distance traveled and duration of each trial in three spatial tasks [distance movement task where a mouse was rewarded when it moved a certain distance, random reward place search task (see below), and place-learning task where a mouse was rewarded when it shuttled two specific reward places] were measured and compared between WT and D2R-KO mice.

4.4.3 Random reward place search task (RRPST) In this task, a computer program delineated a circular zone with its center randomly chosen within a square that was circumscribed around the open field. The mouse was rewarded with ICSS when it entered the reward zone, which was then made inactive (Fig. 2B). After a 5-sec interval, the reward place was changed to a different location and reactivated. In the RRPST, the mouse learned to navigate randomly in the open field in order to obtain rewards. The trials for the above spatial tasks were terminated when the mouse received 50 ICSS rewards or 10 min had passed

 

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4.4.4 Manipulations in the circular open field (familiar environment) Place-cell activity was monitored in a circular cylindrical chamber over several 10-min sessions, during which mice randomly sought out ICSS rewards. Neurons were recorded in sequential sessions in order to determine the stability of the place fields between baseline sessions and in order to assess the changes in firing rate maps in response to extra-maze (distal) and intra-maze (proximal) rotation. Figure 2C shows a diagram of the sequential testing sessions. In the standard session (pre-rotation, baseline session 1), neuronal activities were monitored while mice foraged in the circular open field with the speaker continuously emitting white noise at a 9 o’clock position and an incandescent electric bulb was on at a 3 o’clock position. Neurons were then recorded in distal cue rotation and proximal cue rotation sessions. In the distal cue rotation session, the position of the distal cues was rotated 180° while the chamber was kept constant. In the proximal cue rotation, the chamber was rotated 180° while the distal cues remained unchanged. After each manipulation of the distal or proximal sessions, an additional session was recorded with the distal and proximal cues being returned to the standard conditions. Because multiple sessions were recorded sequentially, the mouse was usually not disconnected from the recording cable between sessions. We did not conduct any manipulations that interfered with the spatial orientations of the animals. Before and after each recording session, the mouse rested on a box that was placed on a pedestal outside of the recording chamber for 5 min.

4.4.5 Manipulations in the square open field (novel environment) Place cells were then recorded in a new open field that the mouse was exposed to for the first time (Fig. 2D). The new open field was a square chamber (sized 55 × 55 cm, 25-cm height) that replaced the familiar open field. Two identical square chambers were used alternatively. The sequences of the

 

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manipulations in the new environment were similar to those that were used in the familiar environment. Before each session in either a familiar or a new environment, the floor was cleaned with a 0.5% Hibitane solution (Sumitomo Corporation, Osaka, Japan).

4.5 Data analysis 4.5.1 Unit isolation and recording The recording electrode assembly was advanced into the HF at ~20 µm per day. Neural activities were recorded with a conventional recording procedure when the mice performed foraging. Complexspike cells were determined with the criteria that have been described in previous studies (Foster and Wilson, 2006; Ranck, 1973). Data collection started when the signal-to-noise ratio exceeded ~4 times in one of the electrodes. Signal amplification, filtering, and digitizing of the spike waveforms was accomplished with a platform with a Plexon system. The recorded signals were amplified 10,000 times, filtered between 0.6 and 3 kHz, digitized at a 40-kHz sampling rate, and stored on a computer hard disk in order to allow for off-line spike sorting. The digitized neuronal activities were isolated into single units by their waveform components with an off-line sorter program (OfflineSorter, Plexon Inc.). The waveforms of the isolated units were superimposed in order to check for invariability throughout the recording sessions. Each cluster was then checked manually in order to ensure that the cluster boundaries were well separated and that the waveform shapes were consistent with action potentials. For each isolated cluster, an interspike interval histogram was constructed, and an absolute refractory period of at least 1.0 ms was used to exclude suspected multiple units. Examples of tetrode recordings are shown in Fig. 5.

 

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4.5.2 Place field delineation Dividing the total number of spikes by the cumulative dwell time in each pixel for the entire session yielded a firing rate map. The distribution map of the pixel firing rate was represented by a color scale with a pixel size of 2.4 × 2.4 cm. Pixels that the mouse had not visited in the open field were shown in dark blue, and those that the mouse visited but that the cell never fired were denoted by white pixels. A firing rate that was greater than zero was rated on an ascending scale, with the color scales being gray, cyan, green, yellow, and red. Pixels with firing rates greater than twice the mean were shown as red pixels. Place fields were delineated as clusters of pixels with firing rates that exceeded twice the average of the session firing rates. A place field could be continued through any edge that was shared by 2 pixels the met the criterion, but not through the corners. If 1 or more neighboring pixels satisfied the criterion, the field was expanded to include the pixels. Each added pixel was then tested for the presence of a neighboring pixel that met the criterion. When no neighboring pixels satisfied the criterion, the limit of the field was identified. The minimum field size of a place-related cell was set at 9 pixels. A cell that showed place field(s) in at least one of the sessions in the familiar or the novel environment was defined as a cell with place-related activity. Noncontiguous patches of adjacent pixels that contained significantly increased firing rates were defined as subfields if they satisfied the above criterion of place fields.

4.5.3 Baseline session analysis For each place cell, the firing rate plot that was determined during baseline session 1 was used to calculate the following: (1) the place field size, (2) the mean overall firing rate, (3) the mean intra-field firing rate, (4) the mean extra-field firing rate, (5) the maximum intra-field firing rate, (6) sparsity, (7)

 

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spatial tuning, (8) spatial coherence, and (9) the spatial information content (bits/spike). These analyses were conducted with previously described methods (Hetherington and Shapiro, 1997; Jung et al., 1994; Shapiro et al., 1997; Skaggs et al., 1993; Wiener et al., 1989). The values of these parameters were compared between the 2 groups of mice with Student’s t-tests, Mann-Whitney U-tests, or repeated measures 2-way ANOVAs with Tukey’s tests, as appropriate. Briefly, the size of the place field was estimated as the percentage of the place field over the visited arena. The mean overall firing rate was calculated as the total number of spikes that were fired within a session divided by the session time. The mean intra-field and mean extra-field firing rates were determined as the averaged firing rate of the cell within and outside the place field. The maximum intra-field firing rate was the highest firing rate of all the pixels within the place field. The spatial tuning of the cell was determined as the ratio of the mean firing rate of the place field to the mean extra-field firing rate (Wiener et al., 1989). The spatial coherence was calculated by performing a z-transformation of the correlation between the rate in a pixel and the mean rate in the adjacent pixels (Cacucci et al., 2007). The spatial information (Inf) that was signaled by each unit (Skaggs et al., 1993) was calculated with the following equation: Inf =

∑

Pi × (ri/R) log2 (ri/R)

i

where R is the session average firing rate, ri is the rate in pixel i, and Pi is the probability that the mouse was detected in pixel i.

4.5.4 Distal rotation, proximal rotation, and remapping analyses In order to quantify the rotation of the place fields between different sessions with environmental manipulations (rotation of the distal or proximal cues), a rotation correlation score was measured for each place cell. The bins were smoothed by recalculating the firing rate of each bin as the average of  

20

itself and the adjacent bins. For each cell, (1) the Pearson product-moment correlation between the firing rate array in the original session and that in the second session with environmental manipulation was measured, and, then, (2) the amount of angular rotation in the firing rate maps was quantified between the pair of sessions. This latter value was determined by rotating the firing rate map of the second session in 5° rotational increments in order to determine the position at which the rotated firing map was maximally correlated with the firing rate map of the original session. The rotation angle that produced the highest correlation was set as the amount that the place field had rotated between the 2 sessions, and a cell was justified as following the cues if its place fields shifted more than 50% of the angle that was rotated for the given cue rotation session compared to the preceding baseline session. The measures of the divergence of the firing fields (remapping) in the 2 chambers were also computed; firing rate maps in baseline session 1 were compared between the familiar and novel environments. A cell was considered for remapping if it met 1 of the following conditions: (1) the cell stopped firing after being exposed to the novel chamber, (2) the field moved to a location that did not overlap in position and direction with the previous location in the familiar chamber, and (3) the cell became more robustly active (more than 2 times) in the novel chamber than in the familiar chamber.

4.5.5 Assessment of place field stability In order to assess place field stability, firing rate maps in baseline session 1 were compared with those in baseline sessions 2 and 3 with Pearson product-moment correlations. Correlation coefficients that were less than 0.4 were considered firing rate map changes. When the correlation coefficient between the 2 firing rate maps was larger than 0.4, we analyzed the intra-field firing changes of whether the cell became more robustly active (more than 2 times) in baseline sessions 2 and 3 compared to

 

21

baseline session 1 (intra-field firing change). We further analyzed the place cells that had correlation coefficients less than 0.4 in order to assess the appearance and/or disappearance of place fields among the baseline sessions. New place fields were identified in baseline sessions 2 and 3 as activity that met the criteria of a place field but that was not localized within the constraints in baseline session 1 [new field(s)]. If no field in baseline sessions 2 and 3 could be found in the original location in baseline session 1, the place field was considered to have disappeared [field(s) loss].

4.5.6 Statistical analysis Collected data in visual acuity test had been compared using hypothesis tests (a Chi square test for numbers of responses or a two tailed Student’s T-test for the time of response). Those tests were also applied in analyzing differences in place-cell characteristics, including basic properties (Table 2), number of HF place cells (Table 3), and changes in baseline sessions (Tables 4, 5). Behavioral data in open-field between WT and D2R-KO mice over 18 days of training were compared using the two-tailed Student’s T-test and repeated two-way ANOVA (Supplementary Fig. 1). Statistical tests with significant differences were all set at p < 0.05.

4.6 Histology After the recording electrodes were estimated to have been advanced below the pyramidal cell layer of CA1 of the HF, the locations of the recording electrodes were verified histologically. Mice were deeply anesthetized with pentobarbital sodium (40 mg/kg i.p.). An electrolytic lesion (30 µA negative current for 15 s) was applied through the recording electrodes. The mouse was perfused on the following day with 0.9% saline, which was followed by 10% buffered formal-saline. The brain was removed and

 

22

fixed in 30% formal-saline for a week. The brains were sectioned coronally (50 µm) on a freezing microtome and stained with cresyl violet.

Acknowledgments This work was supported partly by the Japan Society for the Promotion of Science Asian Core Program and the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B) (25290005). The founders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Special thanks should be given to Dr. Motoya Katsuki and his colleagues at Okazaki National Research Institute for preparing a number of animals.

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Figure legends

Fig. 1 - Verification of electrode placement. Locations of the recording electrode tips (black-filled circles) for WT mice (A) and D2R-KO mice (B) that were used for the unit recording experiment. Only the electrode tip locations of the neurons with place-related activity were shown. Electrode positions were plotted on the atlas by Hof et al. (2000). The numbers beside each session correspond to millimeters from the bregma.

Fig. 2 - Experimental set up, spatial tasks, and experimental manipulations. A: Experimental apparatus. An open field containing a mouse was viewed from the top center by a charge-coupled device camera that signaled the mouse’s position. Incandescent electric light bulbs and white-noise speakers at the 4 peripheral positions of the ceiling served as distal cues. A computer plotted the trail of the mouse and controlled reward delivery from a stimulator. B: Random reward place search task (RRPST). A computer program randomly delineated a circular reward place (small thick red circle). The mouse was rewarded when it entered the reward place, which was then made inactive (small thin red circles). START: location of the mouse at the start of the session. Yellow dots: locations of reward delivery. C: Manipulations in the familiar open field. In the standard session (baseline 1), a light bulb was turned on at the 3 o’clock position and a speaker continuously emitted white noise at the 9 o’clock position. In the distal cue rotation session, the position of the distal cues (the light and noise) was rotated 180° with the chamber constant. In the proximal rotation session, the open field chamber was rotated

 

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180° while the distal cues remain unchanged. Note that the proximal cues indicate cues within the openfields, including open-field’s wall, olfactory and tactile cues in the fields, and so on. D: Manipulations in the novel open field. A square chamber replaced the circular open field. All manipulation tests were similar to those that were used in the familiar open field. A time scale shows the durations for the recording sessions and intersession intervals.

Fig. 3 - The responses of hippocampal place cells to manipulations in familiar and novel environments in the wild-type mice. A: In the familiar environment, a place cell had a place field around the 8 o’clock position (1) in the standard session, and it shifted to the 2 o’clock position (2) in the distal rotation session. It returned to the same position (3) as in the baseline 2 session. However, the place field did not shift (4) in neither the proximal rotation session nor (5) in the baseline 3 session in which the recording chamber was returned to the normal position. In the novel environment, the location-specific firing of this cell also followed the distal cues (6–7) but not the proximal cues (8–9). B: A place cell had a place field that did not follow the rotation of the distal cues (1–2) but that followed the proximal cues (3–4) in the familiar environment. In the novel environment, the place field still followed the rotation of the distal cues (6–7). C: A place cell had a place field that did not follow either of the changes of the distal (1–2) or proximal (3–4) cues in the familiar environment. In the novel environment, the place field followed only the distal cues (6–7) and not the proximal cues. Symbols of the light bulbs and speakers beside the firing maps indicate their arrangements under the spatial manipulations. The curved arrows indicate the rotation of the recording chamber in the proximal cue rotation session. Color scale tables to the right of

 

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the firing maps indicate the calibration for the firing rate. The numbers in bold and in parentheses to the right of the firing maps indicate the intra-field and extra-field firing rates, respectively. The green open squares enclose the firing maps in order to emphasize the sessions in which the place field of the cell had rotated.

Fig. 4 - The responses of hippocampal place cells to manipulations in familiar and novel environments in the D2R-KO mice. A: A place cell had a place field that followed the rotation of the distal cues (1–2), but it did not follow the rotation of the proximal cues (3–4) in the familiar environment. In the novel environment, the place field of this cell was not changed by either the distal or the proximal cue manipulations. B: A place cell whose place-related activity did not follow the rotation of the distal cues (1–2) but followed the rotation of the proximal cues (3–4) in the familiar environment. In the novel environment, the place field of this cell was not changed by either the distal or the proximal cue manipulations. C: A place cell had a place field that did not follow either of the changes of the distal (1–2) or proximal (3–4) cues in the familiar environment. In the novel environment, the place field of this cell followed only the distal cues (6–7) and not the proximal cues. The other descriptions are the same as those for Fig. 3.

Fig. 5 - An example of a multiple-unit recording with a tetrode isolated by an off-line sorter. A: Superimposed waveforms of 4 neurons (a, b, c, d) recorded by each electrode (EL1–4) from a tetrode that corresponded to the cluster analysis in (B).

 

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B: Cluster cutting analysis based on a principal component analysis. Each dot represents 1 neuronal spike that exceeded the defined threshold. Three clusters (a, b, c, d) were identified. Peak – valley EL2 and peak – valley EL3: principle components. Calibration: 500 µs, 200 µV. C: Correlations among three clusters (a, b, c). Each cluster had been chosen as reference once; and auto-correlations were correlations of each of these clusters to itself, while cross-correlations were correlations of these clusters to others.

Highlights   •

Place cells of Dopamine D2 receptor knock‐out (D2R‐KO) mice were recorded. 

•

D2R‐KO mice showed deficits in place‐cell properties (e.g., spatial tuning). 

•

Place fields were less stable in the D2R‐KO mice in the familiar environment. 

•

The place cell properties of D2R‐KO mice were similar to aged animals.   

 

 

38

Figure 1

WT

B

1

CA1

CA

DG

CA1

CA1

-2.10

DG

3

3

DG

CA

-1.60

DG

CA

A

D2R-KO

Figure 2

A. Experimental setup TV monitor

B. Task (RRPST) CCD camera

40W house light

Speaker

Computer

Multi-channel acquisition processor (MAP)

ICSS to MFB

Stimulator

C. Experimental manipulations in the familiar environment Distal cue rotation experiment

9

Proximal cue rotation experiment

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D. Experimental manipulations in the novel environment Distal cue rotation experiment 12 9

Proximal cue rotation experiment 12

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Figure 3

Figure 4

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Table 1

Table 1. Results of the visual cliff test for the WT and D2R-KO mice.

Group

WT

D2R-KO

p-value

Positive

78

71

0.88

Negative

22

19

Latency (s)

63.0 ± 51.0

52.0 ± 54.3

0.14

The numbers of positive and negative responses were compared between the 2 groups of mice with a 2 test, and the latencies in those responses were compared by a Student’s t-test. The values of the response latencies are presented as mean ± standard deviation (SD).

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Table 2

Table 2. Comparison of HF place-cell firing properties of the WT and D2R-KO mice in the familiar environment.

Group

Basic properties

WT

D2R-KO

p-value

Number of place cells/total recorded neurons (%)

79/144 (54.9)

39/117 (33.3)

0.01

Place-field size (% of total visited area)

13.65 ± 6.39

14.47 ± 8.40

0.59

Place-field number

1.52 ± 0.78

1.62 ± 0.78

0.31

Mean overall firing rate (spikes/s)

1.28 ± 0.98

1.25 ± 0.90

0.73

Mean firing intra-field rate (spikes/s)

4.73 ± 3.27

3.61± 2.29

0.03

Mean extra-field firing rate (spikes/s)

0.74 ± 0.70

0.70 ± 0.56

0.54

Maximum intra-field firing rate (spikes/s)

27.91 ± 15.48

23.82 ± 12.53

0.12

Sparsity

0.47 ± 0.20

0.47 ± 0.18

0.94

Spatial tuning

12.04 ± 10.82

6.63 ± 4.40

0.02

Spatial coherence

0.64 ± 0.24

0.55 ± 0.23

0.05

Spatial information content (bits/spike)

2.24 ± 0.93

2.26 ± 1.17

0.91

Values are means ± SD. Comparisons between the 2 groups of mice were performed with Student’s ttests or Mann-Whitney U-tests. See Materials and Methods for the details of these parameters.

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Table 3

Table 3. Numbers of HF place cells in the WT and D2R-KO mice in response to rotation of the distal and proximal cues in both the familiar and novel environments.

Number of place cells (%) Group

Chamber

Total tested

Distal

Proximal

Both

Neither

WT

Familiar

66 (100)

10 (15.2)

30 (45.5)

0 (0.0)

26 (39.4)

Novel

46 (100)

24 (52.2)

2 (4.4)

1 (2.2)

19 (41.3)

Familiar

33 (100)

2 (6.1)

17 (51.5)

1 (3.0)

13 (39.4)

Novel

23 (100)

10 (43.5)

2 (8.7)

0 (0.0)

11 (47.8)

D2R-KO

Only place cells that had at least one place field in each of the 5 sessions in each environment were analyzed. In the WT mice, the majority of place cells followed changes of the proximal cues in the familiar environment, and they switched preference cues to the distal cue in the novel environment. The D2R-KO mice showed a similar tendency of predominant spatial cues as those in the WT group. There were no significant differences in the percentages of place cells that responded to the distal and proximal cues between the WT and D2R-KO mice in both the familiar and novel environments (2 test).

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Table 4

Table 4. Number of HF place cells in the WT and D2R-KO mice showing changes in place responses

between the baseline sessions in both the familiar and novel environments.

Chamber

Group

Intra-field firing

Firing rate map

Firing rate map

change(Corr > 0.4)

change(Corr < 0.4)

change(Corr < 0.4)

(BL1 vs. BL2)

(BL1 vs. BL 2)

(BL1 vs. BL3)

(BL1 vs. BL3)

Changed/Total (%)

Changed/Total (%)

Changed/Total (%)

Familiar

Novel

WT

6/66 (9.1)

7/66 (10.6)

15/66 (22.7)

D2R-KO

5/33 (15.2)

15/33 (45.5)*

21/33 (63.6)*

WT

7/50 (14.0)

19/50 (38.0)

20/48 (41.7)

D2RKO

2/24 (8.3)

13/24 (54.2)

13/24 (54.2)

The firing rate maps of baseline sessions 2 (BL2) and baseline session 3 (BL3) were compared with that in baseline session 1 (BL1). The number of neurons with correlation coefficients less than 0.4 (Changed) and the total number of neurons tested (Total) are indicated in each comparison in each group. An intrafield firing rate change was defined as such if the cell with a correlation coefficient greater than 0.4 became more robustly active (more than 2 times) in baseline sessions 2 and/or 3. Corr, correlation coefficient. *, significant difference compared to WT mice (P < 0.05).

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Table 5

Table 5. Numbers of HF place cells in the WT and D2R-KO mice showing the appearance (new field) and disappearance (field loss) of place field(s) between the baseline sessions in the familiar and novel environments.

Chamber

Familiar

Novel

Group

New field(s)(Corr < 0.4)

Field(s) loss(Corr < 0.4)

(BL1 vs. BL2, BL3)

(BL1 vs. BL2, BL3)

Appeared/Total (%)

Disappeared/Total (%)

WT

4/66 (6.1)

4/66 (6.1)

D2R-KO

7/33 (21.2)*

9/33 (27.3)**

WT

6/50 (12.0)

8/50 (16.0)

D2RKO

4/24 (16.7)

6/24 (25.0)

Firing rate maps in baseline session 1 (BL1) were compared with those in baseline sessions 2 and 3 in the place cells with correlation coefficients less than 0.4 (Table 4). New place fields were identified in baseline sessions 2 and 3 (BL2, BL3) as firing that met the criteria of a place field but that was not localized within the constraints in baseline session 1 [new field(s)]. If no field in baseline sessions 2 and 3 could be found in the original location in baseline session 1, the place field was considered to have disappeared [field(s) loss]. Corr, correlation coefficient. *, **, significant difference compared to the WT mice (P < 0.05 and P < 0.01, respectively, 2 tests).

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