Prefrontal Cortical NR2B-Containing NMDA Receptors are Essential for Spatial Working Memory Performance

doi:10.21203/rs.3.rs-5228477/v1

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Prefrontal Cortical NR2B-Containing NMDA Receptors are Essential for Spatial Working Memory Performance

https://doi.org/10.21203/rs.3.rs-5228477/v1

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NR2A and NR2B are the major GluR2 subunits of N-methyl-D-aspartate (NMDA) receptor. NR2B-containing NMDA receptor was found exclusively expressed in prefrontal cortical (PFC) layer III post-synapses of pyramidal neurons. Many studies have suggested the importance of PFC NR2B-containing NMDA receptor for working memory, especially for the persistent delay cell firing. However, direct evidence for the necessity of PFC NR2B-containing NMDA receptor on working memory is still absent, especially in non-human primates. Here, we directly evaluated the necessity of PFC synaptic NR2B in both rats and monkeys. We first examined the synaptosome expression ratio of NR2B/2A in the PFC, hippocampus and visual cortex and confirmed a relatively higher expression ratio in the PFC than in the hippocampus and visual cortex in both species. We then investigated the effect of intra-PFC blockade of NR2B on the performance of spatial working memory task and found that the spatial working memory, but not pattern discrimination, was robustly impaired in a delay length-dependent way upon NR2B blockade in both species. The present study provided behavioral and neuropharmacological evidence for the critical role of PFC NR2B-containing receptor in primate PFC.

Biological sciences/Neuroscience/Molecular neuroscience

Health sciences/Diseases/Psychiatric disorders/Schizophrenia

Biological sciences/Physiology

N-Methyl-D-Aspartate Receptor (NMDAR) is one the major excitatory ionotropic receptors that receive glutamate input in the Central Nervous System (CNS). NMDA receptor usually consists of four subunits, two NR1 and two NR2 subunits[1]. There are 4 types of NR2 subunits (A-D) that are heterogeneously distributed at different region of the brain. In the adult mammalian brain, NR2A and NR2B containing receptors are the major type of functional NMDARs. NR2B is more distributed at extra-synaptic area at the posterior brain regions such as the visual cortex and hippocampus[2–4]. Therefore, NR2B could be less contributed to synaptic transmission than NR2A in these regions. At the anterior part, i.e., prefrontal cortex, NR2B subunits are exclusively located in the postsynaptic densities of layer III neurons in adult primates[5]. Because the response kinetics of each NR2 subunit upon activation are quite different, the synaptic NR2A/2B ratio largely determines the receptor properties[6, 7]. The offset decay time constant of NR1-NR2A channel is 3–4 times faster than that NR1-NR2B channel upon glutamate stimulus[6].

Working memory is a specific memory type that reflect the ability to maintain current obtained information online and use it to instruct behavior outputs. Prefrontal cortex (PFC) is the critical brain region to execute working memory functions. In primate, dorsolateral prefrontal cortex (dlPFC) is essential for performing spatial working memory tasks. During a visual guided delayed response task, there were neurons that were able to maintain spatially tuned, persistent activity across the delay period. The delay related persistent neuronal firing was considered the underlying mechanism of working memory[8, 9]. Computational models predicted that this persistent memory related activity requires stimulation of NMDARs rather than AMPARs, and the slow kinetics of NR2B containing receptors are particularly well-suited to generate recurrent pyramidal neuron excitation in the absence of sensory inputs. Indeed, Wang et. al. demonstrated that the persistent delay firing in dlPFC pyramidal cells could be abolished by local iontophoresis of NR2B blockade[5].

While this very local and tiny amount of drug infusion only affect cells in small volume surrounding the electrode tip and would not disturb the whole neuronal network and behavior performance, large area drug application was necessary. To clearly verify the functional significance of NR2B subunit in the PFC, here we directly compared the synaptic NR2B/2A expression in PFC and other brain areas for both rodent and primate and conducted both rodent and primate version of spatial working memory tasks to observe the expected inhibition effects on the task performance by local infusing NR2B antagonist to PFC in both species.

Subjects

Sprague-Dawley rats (male, 200–250 g, 8–10 weeks old) for T-Maze task were purchased from SLACCAS (Shanghai, China). Two Sprague-Dawley rats (male, 300–350 g, 12 weeks old) for western blot were obtained from Jiangxi Province Key Laboratory of Laboratory Animal, Nanchang University. Rats were housed in plastic cages (2–3 rats per cage) at temperature controlled (24 ± 1 ºC) vivarium room on a 12/12 light/dark cycle. Food and water were available ad libitum before the behavior training period. All experimental procedures were approved by the Ethical Committee of Rat Experiments at Nanchang University (Nanchang, Jiangxi) and Fudan University (Shanghai, China).

Two adult female Rhesus monkeys (Macaca mulatta, 4.0 kg (#1) and 6.0 kg (#2), purchased from the Kunming Primate Laboratory Animal Center, Chinese Academy of Sciences, Kunming, Yunnan, China; License number: SCXK (Dian) 2008–0001 and SYXK (Dian) 2008–0001) were used in the present behavioral study. The animals were neighbor-housed individually in an established animal room with a 12 h/12 h light/dark cycle (light from 7:00 am to 7:00 pm) and a temperature-controlled (21 ± 1 ºC) environment. The animals were given free access to water and were fed with a diet of monkey chow (Kunming Gaoyun Biological Technology Co. Ltd., Kunming, Yunnan, China; License number: SCXK (Dian) 2009–0003) immediately after behavioral testing. The amounts of monkey chow were designed to keep monkey weights at 90% of the weights attained when food was freely available. Fresh skinless peanuts and raisins were used as food rewards during behavioral testing thus minimizing the need for dietary regulation. Animals were assigned to a single experimenter who trained them extensively before behavioral testing. The experimenter testing the animals was blind to treatment conditions. The experimental protocols were approved by the Laboratory Animal Supervision Committee of Yunnan, China.

All procedures were performed in accordance with the US National Institute of Health Guidelines for Use and Care of Laboratory Animals and the Use and Care of Laboratory Animals Guidelines established by China government.

Monkey brain tissues used for western blot were obtained from one male Rhesus monkey (Macaca mulatta, 7 years old) that provided by Guangdong blue-island biological technology Co. Ltd. The animal was sacrificed for other experimental purpose without affecting CNS.

Synaptosomes isolation and Western blotting

Animals were cardiac perfused with saline under anesthetization with pentobarbital sodium (40 mg/kg, i.p.). Brain tissues were obtained from rat mPFC or monkey principal sulcus, hippocampus (HIP) and visual cortex (VC) of both species, and then were used immediately or frozen under − 80°C. 50 mg tissue per sample was homogenized in a weight: volume ratio of 80:1000 using cold lysis buffer (1:50 PI and 1:100 PMSF). Homogenate was then centrifuged at 1000×g for 10 min at 4°C to separate nuclei (P1). The supernatant (S1) was then centrifuged at 10000×g for 15 min to separate the crude synaptosomes (P2) and supernatant S2. P2 was then lysed with TEVP buffer and spun at 25000×g for 20 min to get purified synaptosomes (LP1)[10].

Western blot was done with standard protocol. Proteins were separated by electrophoresis on SDS-PAGE with 200V voltage for 90 min. Proteins were then translocated to membrane with 300 mA current for 2 hr, and then incubated with blocking media (TBST containing 5% nonfat milk) for 2 hr at room temperature. The membranes were then incubated with primary NR2B (1:1000, #AB1557P, Merckmillipore), NR2A (1:1000, #AB1555P, Merckmillipore), PSD95 (1:1500) antibodies overnight at 4°C. After wash, HRP labeled secondary antibody (1:1500) was used to incubate for 2 hr at 4°C. ECL chemiluminescence substrate (Pierce) was used to detect HRP activity and developed onto x-ray films (Kodak).

Behavioral tasks

1. Delayed alteration task for rats

The T-maze paradigm was described previously[11]. In briefly, rats were subject to restrict diet to maintain at approximately 85% of normal body weight. After habituation to the T-maze until they voluntarily ate the reward food at the end of arms, the training was initiated. As shown in Fig. 3A, the animal was placed at the end of the start arm. The trial was initiated by removing the sliding door to allow the animal to explore the maze. In trial 0, both arms were baited, the animal can get reward by visiting either of them. The animal was then forced back to the start point without allowing to enter the other arm. After a certain interval (5 s delay), the formal trial started, and the animal was allowed to explore the maze again. The animal was rewarded by entering into the arm opposite to the previous trial. Error choice was followed by error-correction trials with the same inter trial interval (5 s) until the animal entered into the previous baited arm. Each session contained 10 formal trials. After the animals reached the criteria with 80% or higher correct response rate in two consecutive sessions, they were ready for the drug infusion test.

2. Delayed response task for monkeys

Behavioral testing was performed in a Wisconsin General Test Apparatus situated in a quiet room. Animals were tested at the same time of day immediately prior to feeding. Firstly, the animals were trained on the two-choice spatial delayed response task as previously described (Cai et al., 1993;Ou et al., 2001). The test board contained two food-wells (left and right) spaced 15 cm apart. An opaque screen could be lowered to separate the animal from the test board. Each trial of the delayed response task began with the experimenter putting the food reward into one of the two food-wells under the gaze of the animal (cue phase). Then, the food-wells were covered with identical plastic plaques (white squares) and the opaque screen was lowered between the animal and the test board for a specified delay (delay phase). At the end of this delay, the screen was raised, and the animal was allowed to choose one of the wells (response phase). There was an interval of 20 s between trials. Rewards were pseudo-randomly distributed between left and right over the 30 trials of a daily test session.

During the initial training phase, delays were held constantly during a daily session and were gradually increased from ‘‘0’’ s according to a stepwise procedure over the 1020 trials. After 1020 trials (34 sessions), the animals were ready for drug infusion. To observe the effect of drugs on memory capacity, the animals were trained on a variable delayed response task in which three different delays (delay a, delay b and delay c) were randomly distributed over the 30 trials. Delays were adjusted until the animal reached stable performance level of 27 trials correct out of 30 trials (90% correct) to make enough space to show the effects of Ro25-6981 or muscimol. Finally, the range of delays was ‘‘0’’ s (delay a), 20 s (delay b) and 40 s (delay c) for both monkeys. The ‘‘0’’ s delay consisted of lowering the screen and immediately raising it again.

3. Pattern discrimination task for monkeys

Once they had grasped the delayed response task, the animals were trained to learn another task, pattern discrimination task in the same apparatus. In the pattern discrimination task, the two food-wells were always covered by two opaque plaques, with an equilateral triangle or a crisscross pattern on the surface. Each trial began with the experimenter putting the food reward into one of the two food-wells and covering them with plaques (animals’ vision was blocked by the lowed screen). Then the screen was raised, and the animal was allowed to choose between the two patterns. The animals could get reward only by choosing the equilateral triangle pattern. And 20 s later (interval), a new trial began. There were 10 trials in a daily test session, and the trials (the two patterns) were quasi-randomly distributed between left and right. Once performance was demonstrated to be stable at 90% (at last 1 wrong trial) for more than 2 days, drug treatment was initiated.

4. Cannula implantation and drug infusion

4.1 Cannula implantation:

The animals were implanted with cannulae before drug infusion and behavioral test. For rats, they were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). stainless steel guide cannulae (23 G) were bilaterally implanted into the mPFC (Bregma AP: 3.5 mm, ML: ±0.75 mm, DV: 1.5 mm from skull surface). The cannulae were then affixed in place with dental cement secured with sterile stainless-steel screws. Sterile stylets (24 gauge) were inserted into each guide cannula, at a point equal to the intracranial tip of the cannula, to prevent occlusion. After one-week recovery from surgery, the animals were re-trained on the T-maze task to insure the performance level of 80%.

For primates, they were pre-anesthetized with hydrochloric ketamine (5 mg/kg, intramuscularly). Then they were deeply anesthetized with sodium pentobarbital (35 mg/kg, intraperitoneally) after pretreatment with sulfate atropine (0.2 mg/kg, intramuscularly). Chronic guide cannulae were implanted using aseptic methods as following. An incision was made along the midline of the scalp, and 23 stainless-steel guide cannulae (20 gauge, 5 cannulae in a row, total 4 rows, see Fig. 1) were implanted into the dorsolateral PFC. The stereotaxic coordinates were derived from a monkey brain atlas (Paxinos, et al., 2008). The coordinates were ranged as: AP, + 25mm ~ + 33 mm from the interaural line; ML, ± 9 mm ~ ± 14 mm from the midline. The cannulae were lowered down to touching the dura without penetration and then affixed to the skull using dental cement secured with sterile stainless-steel screws. Sterile stylets (24 gauge) were inserted into each guide cannula, at a point equal to the intracranial tip of the cannula, to prevent occlusion. All the cannulae were enclosed in a plastic chamber (fixed to the skull) to avoid grasp and infection. Great care was taken to minimize pain and post-operative infection. Surgical incisions were smeared with analgetic antiphlogistine, and the animals were treated with penicillin on seven consecutive post-operative days. Animals were given at least 7 days to recover from surgery before behavioral testing.

4.2 Drug infusion

Animals were habituated to the infusion procedure by mock treatment. For the rats, the animal was gentle restricted with hand, the stylets were removed and replaced with infusion needles (24 gauge). The needle tips were inserted beyond the guide cannulae for 2.7 mm, i.e. 4.2 mm below the skull surface. 1 µl PBS or NR2B antagonist Ro25-6981 (0.5 mg/ml) was infused at 0.1 µl/min. After infusion, the needles were kept in place for additional 3 min. Then the needles were removed, and the stylets were inserted back, and the animals were returned to home cages for 15 min before the behavior test started.

For the primates, animals were seated in a restraint chair while the stylets were removed and replaced with sterile infusion needles (24 gauge with sharp tip for dura penetration) that the tip open point extended 1mm below the guide cannulae. Animals received bilateral intracranial infusions of either 0 (saline) or 7.5 µg muscimol (Sigma Chemical, USA) or 15µg Ro25-6981 (Tocris, UK) in 3 µl saline for each cannula. Infusion was driven by a microsyringe pump (Bioanalytical System Inc., West Lafayette, IN, USA) using 5 µl microsyringes at a speed of 1 µl/min. Needles remained in place for 1 min after completion of the infusion, then the stylets were immediately reinserted. Behavioral testing was performed 60 min after the infusion.

Histology

After behavioral testing, the positions of cannulae were examined. The animals were sacrificed by overdoses of sodium pentobarbital and a stainless-steel electrode (30 gauge) was put into the same position as the infusion needle to deposit ferric ion in the infusion point with a anodal current (6V, 10 s). Cadavers were treated by transcardial infusion of 0.9% (w/v) saline followed by formaldehyde (4%, w/v) solution (containing 1% (w/v) potassium ferrocyanide for the Prussian blue reaction with deposited ferric ion). The brain surface cannulae traces were photo recorded for position re-construction. Brains were stored in 4% (w/v) formaldehyde solution for several days and were then sectioned for histological verification of cannulae positions (centers of the sites stained by Prussian blue reaction).

Single unit recordings in dlPFC

Oculomotor delayed response (ODR) task

Studies were performed on two adult male rhesus monkeys (Macaca mulatta), cared for under the guidelines of the National Institutes of Health and the Yale IACUC. The monkeys were trained in the visuospatial ODR task that was described previously (Wang et al., 2013). The subject started a trial by fixating at the central spot and maintaining fixation for 0.5 seconds (fixation period), whereupon a cue was illuminated for a period of 0.5 seconds (cue period) at one of eight peripheral targets located at an eccentricity of 13°with respect to the fixation spot. After the cue was extinguished, a 2.5-second delay period followed. The subject was required to maintain central fixation throughout both the cue presentation and the delay period. At the end of the delay, the fixation spot was extinguished which instructed the monkey to make a memory guided saccade to the location where the cue had been shown prior to the delay period. A trial was considered successful if the subject’s response was completed within 0.5 seconds of the offset of the fixation spot and was within 2°around the correct cue location. The subject was rewarded with fruit juice immediately after every successful response.

Pharmacology, physiology and data acquisition

This study used iontophoresis to apply NR2B antagonist TCN237 (Tocris) near PFC neurons. Iontophoretic electrodes were constructed with a 20-µm-pitch carbon fiber (ELSI, San Diego, CA) inserted in the central barrel of a seven-barrel non-filamented capillary glass (Friedrich and Dimmock, Millville, NJ). A Neurophore BH2 iontophoretic system (Medical Systems Corp., Greenvale, NY) was used to control of the delivery of the drugs. The drug was ejected at currents that varied from 15–25 nA. Extra-cellular voltage was amplified using a custom low-noise preamplifier (SKYLAB) and band-pass filtered (180Hz-6Khz, 20dB gain, 4-pole Butterworth; Kron-Hite, Avon, MA). Signals were digitized (15 kHz, micro 1401, Cambridge Electronics Design, Cambridge, UK) and acquired using the Spike2 software (CED, Cambridge, UK). Neural activity was analyzed using waveform sorting by a template-matching algorithm, which made it possible to isolate more than one unit at the same recording site. Post-stimulus time histograms (PSTHs) and rastergrams were constructed online to determine the relationship of unit activity to the task. Unit activity was measured in spikes per second. If the rastergrams displayed task-related activity, the units were recorded further and pharmacological testing was performed.

Data were first collected from the cell under a control condition in which at least eight trials at each of 8 cue locations were obtained. Upon establishing the stability of the cells’ activity, this control condition was followed by iontophoretic application of drug(s). Dose-dependent effects of the drug were tested in two or more consecutive conditions. Drugs were continuously applied at a relevant current throughout a given condition. Each condition had ~ 8 (6–12) trials at each location for statistical analyses of effects. The cue epoch was used for Cue cells, the delay epoch for Delay cells, and the response epoch for Response cells.

Data analysis

Western blot band intensity for NR2B and NR2A were first normalized with each PSD95 band. For each batch of experiment, the NR2B/2A ratio for HIP was set as 1, and the ratios for PFC and VC were then normalized with the HIP value.

Delayed performance data in drug treated sessions were first separated as effective and noneffective sessions with 85% criteria, i.e., more than 25 hits in a 30-trials session was labeled as noneffective session. The effective data were then evaluated against the saline sessions using a two-way analysis of variance with repeated measures. The delay and drug treatment were within-subject factors. If a significant effect was noted, data were refined with the post hoc Tukey’s test by paired t-test (T_dep) or one-way analysis of variance.

Synaptic expression of NR2B was enriched more in prefrontal cortex than other brain areas for both rats and monkeys

To evaluate the subunit components of GluR2 across PFC, hippocampus and visual cortex. Immunoblot was used to detect the protein expression of NR2B and NR2A from brain tissues of adult rodent and primate species. To restrict the protein source from synapses, synaptosomes were isolated from each brain area and used for the following Immunoblot. As shown in Fig. 1B&C, both NR2B and NR2A protein were detected in all synaptosome samples from rat mPFC, hippocampus (HIP) and visual cortex (VC). To quantify the NR2B/2A ratio in each brain area, PSD95 was used as inner synaptosome amount control for each batch of samples, then the ratio for HIP was normalized as 1. For all 3 Immunoblot runs, the NR2B/2A ratio for mPFC was all higher than HIP (1.771 ± 0.234 fold), while the one for VC was all lower (0.736 ± 0.032 fold) (Fig. 1D). The same experiments were then repeated with Rhesus macaque (Macaca mullata) brain samples. Similarly, both NR2B and NR2A were detected in synaptosome samples from the principal sulcus (PS) area of dlPFC, HIP and VC (Fig. 1F&G). Consistent with the rat data, the NR2B/2A ratio was reliably higher in PFC than HIP (1.226 ± 0.081 fold), and the one in VC was lower (0.511 ± 0.149 fold) (Fig. 1H). Therefore, our data were consistent with the hypothesis that NR2B is distributed across the brain with a descending gradient from anterior to posterior, while NR2A is with an opposite gradient.

Online blockage of NR2B subunit disrupted working memory related delay cell direction preferences in monkey dlPFC

Similar as previously reported[5], we studied the role of NR2B subunit for delay cells in monkeys performing an oculo-motor delayed response (ODR) task. Acute single-unit recordings from dlPFC were established when the monkeys were performing the task. Delay cells that maintain persistent firing through the delay period were real-time identified. After that, NR2B antagonist TCN237 was iontophoretic applied with constant current of 15–25 nA. 14 delay cells were successfully recorded and finished the drug administration. As shown in Fig. 2a & 2b, Iontophoresis of TCN237 decreased the cell firing rate overall for both preferred (p < 0.0001, paired t-test) and nonpreferred (p < 0.01, paired t-test) at a dose dependent manner. However, the decreases in the preferred direction were more dramatic. Thus, the spatial tuning index, i.e. the ratio of firing rate in preferred vs. nonpreferred directions, was significantly decreased (p < 0.001, paired t-test) (Fig. 2d).

Prefrontal cortical infusion of NR2B antagonist impaired spatial working memory in both rats and monkeys

To examine the necessity of prefrontal NR2B containing NMDA receptors for executing working memory tasks, both rodent and primate version of spatial working memory tasks were tested

Rats were trained to perform a delayed alteration T-maze (DAT) task. The animal had to remember the location where it got food reward in the previous trial and maintain the information during the delay period to instruct the following choice to the opposite direction in the next trial. The inter-trial interval was set to 5 s. After a reliable performance stage (no more than 2 errors in a 10 trials session) was established, NR2B antagonist Ro25-6981 (0.5 µg in 1 µl PBS per side) was infused to the media prefrontal cortex (PrL area) of both hemispheres. DAT test then started at 15 min after the infusion. The rats (n = 10) performed the task at the previous day with 2.00 ± 0.47 win-shift errors and only 1 of the 10 rats made 1 lose-shift error. With the NR2B antagonist infusion, the win-shift errors increased to 5.75 ± 0.43, which was not different from the 50% chance level (one sample t-test), and the lose-shift errors increased to 3.75 ± 0.43. Thus, the total errors of 9.50 ± 0.50 was matched with the chance level (expectation of total 20 trials to get 10 rewards) as well. The task performance was then recovered to the pre-infusion level at 3 days later, which excluded the possible permanent damage to the PrL area by drug infusion. However, in the control group treated with PBS, the task performance was affected neither on the test session nor in the recovery session (Fig. 3D). Therefore, inhibition of NR2B subunit in rat media prefrontal cortex totally disrupted the spatial working memory capacity.

Two Rhesus monkeys were trained to perform a delayed response task using the Wisconsin General Test Apparatus. The animals had to remember the location of a food well where she saw the experimenter put food (a piece of peanut) in. After a delay period (0, 20, or 40 s), the animals were allowed to open one of the two food wells to get the food reward (Fig. 4A). The food wells were retracted immediately if the animal made error choice, and the trial ended. Well trained monkeys maintained at a high level of performance with 90% or higher correct rate in a 30-trials (10 trials for each delay setting) session. For each drug infusion session, one pair of symmetrical cannulas that were pre-implanted to the principal sulcus area were used. Saline control sessions were conducted for all drugs (Ro25-6981 or muscimol) tested locations. The task performance remained intact in the saline sessions of both monkeys. However, both muscimol (7.5 µg in 3 µl saline for each cannulae) and Ro25-6981 (15 µg in 3 µl saline for each cannula) infusion significantly impaired the animals’ performance comparing to saline controls. Two-way ANOVA with Turkey’s repeated measure showed that, after infusion of muscimol or Ro25-6981, there were significantly delay-dependent correct rate decreases on both animals (p < 0.001 for both treatments, p < 0.001 for both treatment × delay interactions in both Monkeys). While there were no differences between muscimol and Ro25-6981 treatments in both monkeys. Paired t test analysis showed that, compared with saline infusion, the performance after Ro25-6981 or muscimol infusion was not impaired at the shortest delay (“0” s) in both monkeys, but the performances at 20 s and 40 s were significantly decreased in both monkeys (Fig. 4C & 4D).

A pattern discrimination task for long-term memory test was performed following each delayed response sessions. Infusion of muscimol or Ro25-6981 into the PFC produced no significant effect on the performance of the animals in the pattern discrimination task (All T < 1.58, p > 0.14, Fig. 4D right panel). Taken together with the 0 s delayed response data, the results further supported that the impaired performance in the delayed response task was not the outcome of impaired common ability of task execution or motivation, but impaired working memory capacity.

In the present study, we investigated the synaptic expression of NMDA receptor subunit NR2B in various brain areas. Our data confirmed the hypothesis that NR2B is enriched more in the post-synapses of adult prefrontal cortex than other areas such as hippocampus, visual cortex and cerebellum. The striking behavioral impairment of NR2B inhibition on working memory tasks in both rodent and primate emphasized the functional significance of NR2B containing NMDA receptors on the cognitive function execution in prefrontal cortex.

NR2B expression occurs prenatally and maintained at a relatively high level in adult central nervous system (CNS)[6, 12–14]. There is NR2B down-regulation from young to adult and a location shift from post-synapse to extra-synapse during CNS maturation in many brain areas such as visual cortex and hippocampus, and the synaptic NR2B is partially replaced by NR2A[15–19]. An optimal synaptic NR2A/NR2B ratio could be a good balance of plasticity and stability for information processing and storage. The ability to generate recurrent local network firing is more critical rather than long-term plasticity in the PFC for cognitive functions such as working memory. The slower kinetics of NR2B was predicted to be suitable for working memory function with computation models[20, 21]. The present data confirmed a relatively higher level of synaptic NR2B in PFC than in other areas such as hippocampus and visual cortex.

In the primate electrophysiologic parts of current study, we focused on the cells with persistent firing during the delay period. Persistent spiking is an important component, if not the whole for working memory. According to recent theories, Working memories are held between spiking by spiking-induced changes in synaptic weights[22]. The “persistent spiking” was a statistical phenomenon but not necessary to present at every single trial. As mentioned above, the ability to generate this “persistent spiking” is more important. Therefore, the impaired persistent firing during the delay period reflected the impairment of working memory in the current study.

Many brain areas could be involved in a working memory task. For example, hippocampus could be constantly required for spatial information encoding and interaction with PFC. Inhibition of Hippocampal CA1 NR2A subunit impaired DAT task performance with both 5 s and 30 s delay in rats, while inhibition of NR2B only affected the task performance with long delay [11]. Genetic depletion of NR2B specifically in hippocampus did not affect spatial learning in water maze, but impaired working memory for recently visited places[23]. Thus, NR2B is required for working memory in both PFC and hippocampus. In the present study, Ro25-6981 infusion to mPFC in rats totally disrupted the task performance to a chance level with a short delay (5 s) setting, suggesting more critical role of NR2B in mPFC for spatial working memory. Similarly, the Ro25-6981 inhibition effects on the primate delayed response task were comparable to that of the GABAa receptor agonist, muscimol, which silenced the drug affected area. On the contrast, in a long-term fear conditioning model, both the delay and the contextual fear memory acquisition was intact with NR2B inhibition in mPFC of rats, but the trace fear memory was impaired, which involved working memory components[24]. Taken together, NR2B containing NMDA receptors in PFC were essential for working memory across mammalian species.

However, it should be noted that the rodent mPFC is not equivalent to dlPFC in primates. The granular part of PFC is specific to primates but absent in rodent. Agranular parts of the PFC emerged in early mammals and were inherited by both rodent and primates[25]. The evolution of granular cells in PFC could enhance the capacity and robustness of working memory in primates.

The role of PFC in different types of working memory could be varied as well. For example, the instant tactile sensory information storage could be different from spatial information storage. Spatial information would be competitively updated by constant visual input, while tactile information could be hold in the primary sensory cortex for a while without incoming distractions[26]. Therefore, PFC could be more critical for spatial than tactile working memory holding. This could explain why in a lever-pressing based working memory model, the inactivation of NR2B in mPFC did not affect the working memory task performance of the rats. However, NMDA receptors in mPFC were still involved in the performance of that task, which relied more on NR2A subunit [27]. In the rodent version of our working memory tasks, it also included executive functions with inhibitory component. The rats needed to not only hold the location information of most recently visited, but also avoid entering this location again. This function is a kind of working memory and relies on PFC, too.

Working memory impairments are commonly seen in schizophrenia patients that associated with reduced task-related prefrontal cortical activation[28]. glutamatergic signaling deficits are one of the major neural bases of schizophrenia[29]. The significance of NR2B containing NMDA receptors for working memory in PFC has been long suggested by studies with NR2B over-expressed transgenic mice[30] and electrophysiological data from both rodent and primate[5, 11]. A previous study in monkeys with systemic administration of drugs also supported the importance of NR2B for working memory[31]. However, a direct behavioral and pharmacological evidence for the necessity of PFC NR2B on working memory was still absent to date, especially in primates. The present study provided the direct evidence showing that prefrontal cortical NR2B was crucial for working memory processing, both in rates and monkeys.

Competing Interests

The authors declare no competing interests.

Author Contributions

B.L. and J.P. conceived of the study. J.P., G.W., Y.H. and L.Q. performed the experiments. M.W. and A.F.T.A. conducted the electrophysiological recordings. J.P., G.W. and W.M. analyzed the data. J.P., G.W and B.L. wrote the manuscript.

Acknowledgements

This work was supported by grants from the STI 2030-Major Projects (2021ZD0201705 to B.L.), the National Natural Science Foundation of China (31971035, 31771182 to B.L; 32060199 and 2360197 to J.P..), Jiangxi Provincial Natural Science Foundation (20224ACB206016 to J.P.), and China Postdoctoral Science Foundation [20080440581 to G.W].

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The authors have declared there is NO conflict of interest to disclose

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Prefrontal Cortical NR2B-Containing NMDA Receptors are Essential for Spatial Working Memory Performance

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Abstract

Figures

Introduction

Material and Methods

Subjects

Synaptosomes isolation and Western blotting

1. Delayed alteration task for rats

2. Delayed response task for monkeys

3. Pattern discrimination task for monkeys

4. Cannula implantation and drug infusion

4.1 Cannula implantation:

4.2 Drug infusion

Histology

Single unit recordings in dlPFC

Data analysis

Results

Prefrontal cortical infusion of NR2B antagonist impaired spatial working memory in both rats and monkeys

Discussion

Declarations

Competing Interests

Author Contributions

Acknowledgements

References

Additional Declarations

Status:

Version 1