Intracortical Injection of Okadaic Acid Increases Locomotor Activity and Decreases Anxiety-like Behaviour in Adult Male Rats: Potential Involvement of NMDA Receptor

Abstract

Background

Neuronal transmission through the N-methyl-d-aspartate receptor (NMDAR) is a key event in synaptic plasticity and excitotoxicity. The channel properties and biochemical signalling activities of the receptor are regulated by phosphatases such as protein phosphatase 1. While the immediate consequences of NMDAR activation have been reported previously, the long-term behavioural changes remain unclear.

Purpose

We attempted to investigate the long-term behavioural effects of N-methyl-d-aspartic acid (NMDA) injection and the role of phosphatases during NMDAR signalling.

Methods

NMDAR was activated by stereotaxic injection of NMDA into the prefrontal cortex of adult rats. To elucidate the role of phosphatases in mediating NMDAR signalling and associated animal behaviour, okadaic acid (OA), a phosphatase inhibitor, was administered before NMDA injection. The animals were tested for their general locomotion and cognitive function using behavioural assays.

Results

A single injection of NMDA impaired cognition in the long term. Interestingly, intracortical OA injection resulted in increased locomotor activity and decreased anxiety-like behaviour in animals without major cognitive effects.

Conclusion

We demonstrate that the inhibition of phosphatases during NMDAR signalling can affect locomotion and anxiety-like behaviour in adult male rats. Our study underscores the potential of modulating phosphatases as a pharmacological target for anxiety disorders.

Keywords

phosphatases okadaic acid anxiety hyperlocomotion

Introduction

The N-methyl-d-aspartate receptor (NMDAR) is an important ionotropic glutamate receptor that is permeable to calcium (Ca²⁺) ions. NMDAR dysfunction has been linked to several neurological diseases, such as stroke, epilepsy, traumatic brain injury (TBI), Alzheimer’s disease (AD), Huntington’s disease and schizophrenia.^{1, 2} Various serine/threonine (Ser/Thr) kinases like Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII), death-associated protein kinase 1 (DAPK1), and phosphatases like protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B) have been reported to regulate NMDAR channel properties.³ Both kinases and phosphatases are involved, either directly or indirectly, in excitotoxicity,^{4, 5} a condition in which excessive activation of NMDAR causes neuronal damage, to the extent of being proposed as potential targets in combating neurodegeneration.^{5, 6}

Previously, we had reported a CaM kinase-mediated increase in NMDAR phosphorylation in an acute in vivo model of excitotoxicity induced by intracerebroventricular (ICV) injection of N-methyl-d-aspartic acid (NMDA).⁷ Pre treatment with okadaic acid (OA), a phosphatase inhibitor, also caused an increase in NMDAR-GluN2B-Ser¹³⁰³ phosphorylation⁷; however, the role of phosphatases during excitotoxicity was not addressed. Building on this earlier report, we investigated the chronic effects induced by NMDAR overactivation in vivo by studying alterations in behaviour. We further focused on the long-term behavioural effects of inhibiting phosphatases during NMDAR activation by OA pre treatment. OA and its derivatives are potent inhibitors of PP2A (IC₅₀ = 0.2 nM) and PP1 (IC₅₀ = 20 nM). It can readily enter cells and induce neurotoxicity in vitro and in vivo by blocking protein dephosphorylation.⁸ Stereotaxic intrahippocampal injection of OA enhances Ser phosphorylation of the NMDAR-GluN2B subunit and induces neuronal hyperexcitability and epilepsy in vivo.⁹ In this study, a rodent model of excitotoxicity was created by stereotaxic intracortical injection of NMDA. OA was administered before NMDA injection to elucidate the role of phosphatases in modulating animal behaviour. Our work aims to explore the behavioural changes triggered by neurotoxic events in the cortex, a region known to be susceptible to damage following conditions like ischemic stroke or trauma.^{10, 11}

Methods

Animals

Male Wistar rats (2–3 months, 200–300 g) were used for stereotaxic surgery and behavioural tests. Animals were obtained from the Animal Research Facility (ARF) of Rajiv Gandhi Centre for Biotechnology (RGCB). The animals were housed individually in a facility maintaining a 14:10 h light-dark cycle. Individual housing for the male rats was followed to avoid intermale aggression. Food pellets and water were provided ad libitum to the animals. All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) of RGCB and followed the rules set by the Committee for Control and Supervision of Experiments on Animals, Government of India.

Drugs

NMDA (M3262, Sigma-Aldrich, USA) was dissolved in sterile 0.9% NaCl solution (saline). OA (O8010, Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich) and was used at a final concentration of 10% DMSO in saline. The pH was adjusted to 7–7.4 for all the solutions.

Stereotaxic Surgery

Stereotaxic surgeries were performed using methods similar to previous reports.^{7, 12} The male rats were assigned to the treatment groups at random. The animals were deeply anaesthetised by 2–5% isoflurane (Troikaa Pharmaceuticals, India) inhalation. The head was positioned in a stereotaxic frame (Leica Microsystems, Germany). The skull was exposed using a sagittal incision. Holes were drilled into the skull over the prefrontal cortex (PFC) bilaterally, using the stereotaxic coordinates, anterior–posterior (AP) = +3.2 mm, medial–lateral (ML) = ±0.7 mm, dorsal–ventral (DV) = −3 mm with respect to the bregma, adapted from Paxinos and Watson.¹³ The saline pre treated animals were first bilaterally injected with 0.5–0.75 µL saline, followed by either a second injection of 0.5–0.75 µL saline (Saline + Saline group) or 33.98 mM NMDA (Saline + NMDA group) 30 min later. Similarly, the OA and DMSO pre treated groups of animals received bilateral injections of 0.5–0.75 µL of 45.6 µM OA or 10% DMSO, respectively, followed by either saline (OA + Saline and DMSO + Saline groups) or NMDA (OA + NMDA or DMSO + NMDA groups) injections after 30 min. DMSO-treated animals were used as the vehicle control for OA-injected animals. The drugs were manually injected using a needle attached to a Hamilton syringe (25 µL). The solutions were administered slowly, and the needle was left in place for 5 min before its withdrawal. After injection, the holes were sealed, and the incision was sutured. Antibiotics and analgesics were administered subcutaneously for 3 days post-surgery. The animals were given 7–9 days to recover before the commencement of behavioural tests.

Behavioural Tests

The behavioural tests were performed for the animals as described previously.¹⁴ Open field test (OFT), novel object recognition test (NORT), object location test (OLT) and the Morris water maze (MWM) test were employed to assess the general locomotion, anxiety and spatial cognition of the animals. OFT, NORT and OLT were performed ~7–9 days after surgery, whereas the MWM test was conducted ~4–5 weeks after the surgical procedure. The time interval between the dry maze and water maze tests was allowed for the complete healing of the surgical wound. We observed that some animals had a tendency to disrupt the sutures while grooming themselves; consequently, proper healing of the wound was ensured prior to conducting the MWM test. Animals were transported to the behavioural testing facility no less than an hour before the start of each test. All behavioural tests were carried out between 14:00 and 18:00 h. The arena and objects used for behavioural tests were wiped with 70% ethanol before and after each animal was tested.

OFT

OFT assesses the spontaneous locomotor activity and anxiety-like behaviour of the animals. While locomotion is assessed by the total distance travelled, velocity and the time spent moving, the anxiety-like behaviour of the animals can be determined from the time spent in the peripheral zone (PZ) and central zone (CZ) of the test arena. In OFT, the rats were allowed to freely explore an open field arena (70 × 70 × 40 cm L × B × H) for 10 min. The videos were captured using a Panasonic CCTV camera (WV-CP500) or Microsoft LifeCam Studio webcam (Q2F-00013), and analyses for various OFT parameters were carried out using EthoVision XT software (Noldus Information Technology, Wageningen, The Netherlands).

NORT and OLT

In NORT, the animals were habituated to an arena for 10 min. Habituation was also used as OFT by video recording the movements of the animals. Twenty-four hours later, the rats were trained by allowing them to explore two familiar objects placed in the arena for 5 min. A day after training, during the test phase, one of the familiar objects was replaced with a novel object, and the time spent by the animals exploring each object was recorded for 5 min. The familiar/novel object(s) used, and the position of the objects were interchanged among the animals in a group to rule out any preference towards an object or position in the box. The first 3 min of object exploration during testing¹⁵ were used to calculate the discrimination index (DI) and recognition index (RI).

Discrimination index = \frac{T_{N} - T_{F}}{T_{N} + T_{F}}

Recognition index = \frac{T_{N}}{T_{N} + T_{F}}

where

T_N: Time spent exploring the novel object.

T_F: Time spent exploring the familiar object.

In OLT, one of the objects was relocated, and the animals were tested 1 h after training for the time spent exploring the displaced and non-displaced objects for 5 min. The first 3 min of object exploration during testing¹⁵ were used to calculate DI and RI.

MWM Test

Approximately 4–5 weeks after stereotaxic surgery, the animals were subjected to the MWM test. The procedure adopted has been described earlier.^{14, 16} The animals were trained to locate the hidden platform using a regimen that involved 5–6 trials, each 60 s long, with an intertrial interval of 25–30 s, carried out over 5 consecutive days. The mean escape latency for five trials per day was calculated for each animal. The distance travelled, percentage of moving time, and velocity of the animals were determined by a 60 s probe trial performed after the last trial on day 5 of training. The animals which exhibited thigmotaxis behaviour (n = 0–3 per group) during the 5 days of training were excluded from analysis.

Statistical Analysis

GraphPad Prism version 10 (10.3.1) was used for data analysis, and the results are represented as mean ± standard deviation (SD). Outliers, if any, in all the data sets were identified using the Robust Regression and Outlier Removal (ROUT) method, set at Q = 1%. The data after the outlier test were used for subsequent statistical tests. Unpaired parametric Student’s t-test was used to compare the data of saline and NMDA-treated animals within the saline, DMSO and OA pre treatment groups. One-way analysis of variance (ANOVA) followed by Šidák’s multiple comparisons test was used to compare the data for the saline pre treatment group with OA and DMSO pre treatment groups. Two-way repeated measures ANOVA, followed by either Šidák’s (for comparison between the saline and NMDA treatment groups within the saline, DMSO and OA pre treatment groups) or Tukey’s multiple comparisons test (for comparison between OA and DMSO pre treatment groups with the saline pre treatment group), was used to analyse the data from the MWM test. The OFT variables were also tested for correlation using Pearson’s correlation method. Pearson’s correlation coefficients were assessed as followed earlier.¹⁷ Statistical significance was defined as p < .05.

Results

Effect of Intracortical NMDA Injection on Behaviour

OFT

Intracortical NMDA injection did not affect the locomotor activity (t₍₂₇₎ = 0.2515, p = .8033 for total distance travelled, t₍₂₇₎ = 0.1977, p = .8448 for velocity and t₍₂₇₎ = 0.3842, p = .7038 for the percentage of time spent moving) (Figure 1A) and anxiety-like behaviour of the animals (t₍₂₅₎ = 1.403, p = .1730 for the percentage of time in CZ and PZ) (Figure 1B). Animals with higher anxiety will spend less time in the CZ of the open field. The saline- and NMDA-injected animals did not show a significant difference in the time spent in the CZ or PZ in OFT (Figure 1B).

Figure 1.

Notes: Rats were subjected to intracortical injection of saline followed by NMDA or saline.

NORT and OLT

The NMDA-treated animals did not show a difference in DI (t₍₂₃₎ = 0.07510, p = .9408) and RI (t₍₂₃₎ = 0.07449, p = .9413) in NORT (Figure 1C) and in OLT (t₍₂₂₎ = 0.1761, p = .8618 for DI and t₍₂₂₎ = 0.1752, p = .8625 for RI) (Figure 1D). Moreover, the total time spent exploring the objects did not show a significant difference between the two groups (t₍₂₃₎ = 0.7460, p = .4632 for NORT and t₍₂₂₎ = 0.06969, p = .9451 for OLT) (Figure 1C and D). This indicates that NMDA did not affect short-term and long-term memory.¹⁸

MWM Test

In the MWM test, there was an effect in the interaction between the treatment groups and time (F_(4,84) = 5.213, p = .0008) on escape latency. Šidák’s post hoc test indicated that the NMDA-treated animals had a significantly higher escape latency on day 5 of trials compared to the saline group (Figure 1E). This suggests that NMDA injection induced cognitive impairment, although mild. Student’s t-test revealed no significant difference between the saline- and NMDA-injected animals in the distance travelled (t₍₂₀₎ = 0.5941, p = .5591), velocity (t₍₂₁₎ = 0.06518, p = .9486) and percentage of time spent moving (t₍₂₀₎ = 0.1594, p = .8749) in MWM trials (Figure 1F).

Effects of Intracortical OA Injection on Behaviour

OFT

In OFT, pre treatment with OA increased the spontaneous locomotor activity as observed by a significant increase in the total distance travelled (F_(3,47) = 4.314, p = .0091) (Figure 2A), velocity (F_(3,47) = 4.224, p = .0100) (Figure 2B) and percentage of time spent moving (F_(3,47) = 5.560, p = .0024) when compared to the saline pre treated group (Figure 2C). Šidák’s multiple comparisons test attributed this significant effect to the OA + NMDA-treated group of animals. Comparison of the OA pre treated group with the DMSO pre treated group also revealed an increase in the locomotor activity of the animals (F_(3,42) = 2.861, p = .0481 for total distance travelled, F_(3,42) = 2.729, p = .0559 for velocity and F_(3,42) = 4.425, p = .0086 for the percentage of time spent moving) with OA pre treatment (Figure 2A–C). No differences in the above variables were observed with DMSO pre treatment when compared to saline pre treatment (F_(3,49) = 0.7853, p = .5079 for total distance travelled, F_(3,49) = 0.7602, p = .5218 for velocity, F_(3,49) = 1.377, p = .2608 for the percentage of time spent moving). Since the trend observed in the OA + NMDA group was an increase when compared to the OA + Saline-treated group (t₍₂₀₎ = 2.364, p = .0283) (Figure 2C), a synergistic effect of OA and NMDA on the general locomotor activity levels of the animal is likely.

Figure 2.

Notes: Rats were subjected to intracortical injection of saline/DMSO/OA, followed by NMDA or saline. After recovering from stereotaxic surgery (7–9 days), the locomotory and anxiety-like behaviour of the animals were studied using OFT.

Further analysis of the OFT data revealed a significant difference in the anxiety-like behaviour of the OA pre treated animals compared to saline pre treated animals (F_(3,45) = 10.32, p < .0001 for the percentage of CZ and PZ time). The OA-treated animals spent a considerable amount of time in the CZ, indicating an anxiolytic effect (Figure 2D and E). This effect was significant, particularly for the OA + NMDA-treated animals, when compared to the DMSO-treated group also (F_(3,41) = 3.586, p = .0216 for both CZ and PZ time) (Figure 2D and E). This indicates that OA pre treatment can alter the exploratory activity and cause a decrease in anxiety-like behaviour in the OFT.

Correlation analysis, by and large, revealed very weak interactions between the CZ/PZ time and locomotion parameters for the OA pre treated group, except for the time spent moving for the OA + NMDA-treated group (Pearson’s correlation coefficient: for OA + Saline treatment, r = 0.05468, p = .8731 for total distance, r = 0.03253, p = .9244 for velocity, r = 0.2371, p = .4828 for time spent moving; for OA + NMDA treatment, r = 0.01093, p = .9745 for total distance, r = 0.02265, p = .9473 for velocity, r = 0.6181, p = .0427 for time spent moving). Nevertheless, this suggests that the increased exploratory behaviour observed with OA pre treated animals is independent of their increased locomotion. Therefore, intracortical OA treatment can alter exploratory activity and lead to a decrease in anxiety-like behaviour in OFT.

NORT and OLT

Pre treatment with OA did not induce any significant alterations in DI and RI in NORT (F_(3,41) = 0.3690, p = .7758 for DI and F_(3,41) = 0.3682, p = .7763 for RI) (Figure 3A and B) and OLT (F_(3,42) = 2.239, p = .0977 for DI and F_(3,42) = 2.241, p = .0976 for RI) (Figure 3D and E) when compared to saline-treated animals. No significant differences were observed between DMSO and saline pre treated animals (F_(3,43) = 0.9087, p = .4448 for DI and F_(3,43) = 0.9061, p = .4460 for RI in NORT; F_(3,44) = 0.3366, p = .7989 for DI and F_(3,44) = 0.3367, p = .7988 for RI in OLT) and between DMSO and OA treatments (F_(3,38) = 0.2369, p = .8701 for DI and F_(3,38) = 0.2361, p = .8706 for RI in NORT; F_(3,42) = 0.8831, p = .4576 for DI and F_(3,42) = 0.8840, p = .4572 for RI in OLT) (Figure 3A, B, D and E).

Figure 3.

Notes: Rats were subjected to intracortical injection of saline/DMSO/OA, followed by NMDA or saline. After recovering from stereotaxic surgery (7–9 days), the long-term recognition memory and short-term spatial memory of the animals were studied using NORT (A–C) and OLT (D–F), respectively.

Additionally, no significant difference in the total time spent exploring the objects was observed for any of the groups studied (Saline versus OA: F_(3,41) = 2.151, p = .1085, Saline versus DMSO: F_(3,43) = 0.3221, p = .8093, DMSO versus OA: F_(3,38) = 1.116, p = .3545 for NORT and Saline versus OA: F_(3,42) = 0.3050, p = .8216, Saline versus DMSO: F_(3,44) = 0.1225, p = .9463, DMSO versus OA: F_(3,42) = 0.6077, p = .6137 for OLT) (Figure 3C and F). This indicates that transient disruption of OA-sensitive phosphatases did not affect the long-term recognition memory and short-term spatial memory processes.

MWM

Data from the MWM test were analysed by two-way repeated measures ANOVA. Statistical tests revealed a significant interaction effect between the treatment groups with time (Saline versus OA: F_(12,160) = 2.712, p = .0023, Saline versus DMSO: F_(12,156) = 1.880, p = .0407) on escape latency in the MWM test (Figure 4A). With DMSO versus OA treatment comparison, no significant effect could be observed in the time course of learning (F_(12,148) = 0.6816, p = .7672). Tukey’s multiple comparisons test detected appreciable differences in the escape latencies only between the saline and NMDA treatments within the saline pre treatment group on day 4 (p = .0565) and on day 5 (p = .0314) (Figure 4A). This indicates that intracortical OA or vehicle pre treatment did not cause any significant effect on hippocampal-dependent spatial learning and memory.

Figure 4.

Notes: Rats were subjected to intracortical injection of saline/DMSO/OA, followed by NMDA or saline. After recovering from stereotaxic surgery (4–5 weeks), the spatial cognition of the animals was studied using the MWM test.

During the MWM trials, we observed that the OA pre treated animals displayed an increased tendency to stay afloat, starting from day 3 of trials. Therefore, we analysed the percentage of time spent moving in the probe trial and found that the OA + Saline-treated animals exhibited a decrease in time spent moving (F_(3,37) = 6.589, p = .0011) with no significant difference in distance travelled (F_(3,37) = 0.6854, p = .5667) and velocity (F_(3,38) = 0.5296, p = .6647) compared to the saline group of animals (Figure 4D). One-way ANOVA followed by Šidák’s comparison between DMSO and OA pre treated animals did not detect a significant difference in distance travelled (F_(3,34) = 1.295, p = .2918) and velocity (F_(3,34) = 1.209, p = .3214), but it could yield a difference in the percentage of time spent moving (F_(3,33) = 5.489, p = .0036) (Figure 4D). Although DMSO pre treated animals did not show impairment in memory performance, the statistical test revealed a significant decrease in the distance travelled (F_(3,37) = 4.340, p = .0102) and velocity (F_(3,38) = 3.924, p = .0156) of DMSO + Saline-treated animals compared to Saline animals, with no change in the time spent moving (F_(3,36) = 0.2277, p = .8765) (Figure 4B and C). Interestingly, the OA-induced decrease in moving time and the DMSO-induced decrease in distance and velocity were negated with NMDA treatment (OA + Saline versus OA + NMDA: t₍₁₇₎ = 2.304, p = .0341, DMSO + Saline versus DMSO + NMDA: t₍₁₇₎ = 2.239, p = .0388 for distance travelled and t₍₁₇₎ = 2.380, p = .0293 for velocity) (Figure 4B–D).

Discussion

During neurotoxic conditions such as stroke or TBI, the activity of phosphatases decreases temporarily,¹⁹ and overactivation of NMDARs ensues, resulting in neuronal death.²⁰ In this study, we evaluated the long-term behavioural consequences of transient fluctuations in phosphatases during excessive activation of NMDAR in vivo. Our study reveals significant changes in locomotion and anxiety-like behaviour in adult rats when phosphatases are inhibited during NMDAR activation.

Alterations in Behaviour with NMDA/OA Injection

The rodent PFC plays an essential role in higher brain functions such as attention, cognition, motivation, reward, decision making and emotional control.^21–23 It acts as the hub for the integration of signals projected from multiple regions of the brain to guide adaptive behavioural responses.^{21, 23} The neuronal population in the PFC consists of 80–90% glutamatergic pyramidal neurons and 10–20% GABA (γ-aminobutyric acid)-ergic inhibitory interneurons.²⁴ Several subcortical autonomic, motor and limbic brain structures, such as the hypothalamus and thalamus, nucleus accumbens, ventral tegmental area, hippocampus and amygdala receive excitatory efferents from the PFC.^{23, 24}

OFT

Interestingly and unexpectedly though, we observed that the animals treated with OA exhibited increased locomotion and decreased anxiety-like behaviour. It can be argued that the increased time in the CZ is due to the hyperlocomotor activity of the OA-treated animals. Although the increased locomotory effect was specific only to the OA + NMDA-treated animals, the changes in anxiety were exhibited by the animals in the OA pre treatment group, irrespective of subsequent saline or NMDA treatment. This indicates an OA-induced effect on anxiety levels. This conclusion is supported by the significant increase in CZ time of OA + Saline-treated animals compared to the Saline + Saline group. Furthermore, we observed a weak correlation between the exploratory and locomotory parameters.

The alterations in locomotion and anxiety-like behaviour could happen due to modulation in the levels of neurotransmitters like dopamine,²⁵ glutamate,²⁶ adenosine²⁷ or GABA.²⁸ Animals treated with OA display some symptoms of overexcitation of their excitatory synapses.²⁹ In our study, hyperlocomotion was exhibited by OA + NMDA-treated animals only. We speculate that the action of OA on NMDAR activity⁹ and/or dopamine transporters³⁰ may have been further enhanced by NMDA injection in these animals. OA-mediated hyperphosphorylation of NMDARs enhances their sensitivity to the endogenous transmitter and increases NMDA receptor-mediated currents,⁹ which in turn can selectively upregulate dopamine D1 receptors.³¹ OA can also inhibit the dephosphorylation of dopamine transporters and/or reduce dopamine uptake³⁰ and thereby induce hyperactivity. A similar behavioural pattern was also reported by Coelho et al. in transgenic mice overexpressing adenosine A_2A receptors in the forebrain, presumed to be due to increased levels of phosphorylated DARPP-32 (dopamine and cyclic AMP-regulated-phosphoprotein of 32 kDa), an endogenous inhibitor of PP1,²⁷ thereby probably causing downregulation of PP1 activity. On the other hand, OA + Saline-injected animals failed to show any changes in locomotion, consistent with previous reports.³²

The amygdala plays a central role in modulating fear and anxiety responses. Reciprocal communication between the PFC and amygdala can regulate stress-induced behavioural alterations.^{23, 33} The anxiolytic action of OA, observed in our study, can be attributed to its ability to regulate phosphorylation and endocytosis of GABA_A receptors^34–36 and/or NMDARs,^37–39 thereby affecting the neuronal firing pattern in the amygdala.^{33, 40} OA can also regulate the phosphorylation of presynaptic transporter proteins, which plays a role in the clearance of biogenic amine neurotransmitters.⁴¹ To the best of our knowledge, the injection of OA into the cortex has not been reported so far. This could explain the absence of effects on locomotion and anxiety-like behaviour in earlier studies on intrahippocampal⁴² or ICV injection of OA.⁴³

NORT and OLT

Neither the NMDA-injected nor OA-treated group of animals displayed an impairment in discriminating between familiar and novel/displaced objects in NORT and OLT. Multiple studies have reported that bilateral lesions induced by NMDA in the medial prefrontal cortex (mPFC) of rodents did not cause impairment in NORT and OLT.^44–46 Spanswick and Dyck demonstrated that in the absence of contextual information, mPFC-lesioned mice were able to successfully differentiate between the novel and familiar object, thereby indicating the role of mPFC in integrating different features of recognition memory and not when the behavioural task varies by a single item,^{47, 48} as in the present study.

MWM Test

Although both NORT and MWM tests provide similar information in terms of hippocampal integrity and PFC connections,⁴⁹ we observed defects in spatial cognition, assessed by the MWM test, in the NMDA-treated group, as opposed to no impairment in NORT and OLT tests. The involvement of different brain circuits for each behavioural task⁴⁹ and/or a reduction in long-term potentiation induction observed after 2 weeks, following an initial increase upon intracerebral injection of an excitotoxin⁵⁰ might explain the variation in our results. Intracortical NMDA injection induced a significant increase in escape latency compared to saline-injected animals on day 5 of trials.

In contrast to the published evidence on the AD model induced by OA injection,^{42, 51} we did not observe a significant cognitive impairment with intracortical OA treatment. The site of OA injection (intracortical/intrahippocampal/ICV) can exert different effects on memory processes. Also, we observed a ‘floating’ behaviour in the OA-treated animals, particularly from day 3 of trials, as evident in the decreased amount of time spent moving. This ‘non-search’ behaviour can be an indication of accelerated ageing and AD,⁵² since the alternate possibility of behavioural despair^{53, 54} in these less anxious animals is highly, although not completely, improbable. In the probe trial of the MWM test, the DMSO-treated animals surprisingly displayed a decrease in distance travelled and swimming velocity compared to their saline-treated counterparts, despite the absence of any observable memory impairment. Previous research has noted a similar decrease in swim speed in DMSO-treated wild-type mice⁵⁵ or when DMSO was administered intraperitoneally after TBI.⁵⁶ This effect of vehicle injection has been linked to disruptions in myelin function and a reduction in peripheral nerve conduction.^{56, 57}

Altogether, our results from behavioural tests indicate that a transient dysregulation in the activation of the NMDA receptor and its downstream phosphatases can cause long-term defects in locomotion and anxiety-like behaviour. Antagonism to phosphatases can either directly modulate the protein expression levels or indirectly alter the activity of regulators of NMDAR signalling proteins to cause alterations in behavioural patterns.^{58, 59} Intracortical phosphatase inhibition by OA injection can affect the anxiety-like behaviour of the animal.

Conclusion

We investigated the long-term effects of intracortical NMDA injection and the role of phosphatases during NMDAR signalling in modulating animal behaviour. NMDA injection resulted in cognitive decline in spatial learning tasks. Intracortical OA treatment increased locomotor activity, while decreasing anxiety-like behaviour. Our findings underscore the significance of maintaining a delicate balance between the activities of phosphatases and kinases in the cortex in regulating psychological states. Future investigations into the role of kinases and phosphatases in modulating the behavioural phenotype associated with neuropsychiatric disorders like autism spectrum disorders, mania, schizophrenia, bipolar disorder and attention deficit hyperactivity disorder are called upon.

Abbreviations

AD: Alzheimer’s disease; ANOVA: Analysis of variance; AP: Anterior–posterior; Ca²⁺: Calcium ions; CaMKII: Calcium/calmodulin-dependent protein kinase II; CZ: Central zone; DI: Discrimination index; DMSO: Dimethyl sulfoxide; DV: Dorsal–ventral; GABA: γ-Aminobutyric acid; ICV: Intracerebroventricular; ML: Medial–lateral; mPFC: Medial prefrontal cortex; MWM: Morris water maze; NMDA: N-Methyl-d-aspartic acid; NMDAR: N-Methyl-d-aspartate receptor; NORT: Novel object recognition test; OA: Okadaic acid; OFT: Open field test; OLT: Object location test; PFC: Prefrontal cortex; PP1: Protein phosphatase 1; PZ: Peripheral zone; RI: Recognition index; SD: Standard deviation.

Footnotes

Acknowledgements

The authors thank Dr Mayadevi, Dr Vishnu Sunil Jaikumar, Dr Remya Chandran and Mr Devaraj T P for their support, valuable suggestions and advice. We are grateful to Dr Jackson James and their team for sharing their stereotaxic instrument.

Authors’ Contribution

Jacob RS and Omkumar RV contributed to the conception and experimental design of the work.

Jacob RS, Gunasekaran S and Kumar M performed the experiments.

Jacob RS and Gunasekaran S were involved in the investigation and formal analysis of scientific data.

Jacob RS prepared the data figures and wrote the original draft of the manuscript.

Omkumar RV acquired all necessary funds and resources and supervised the work.

All authors contributed to the manuscript revision and approved the submitted version.

Data Availability Statement

Data supporting the results is provided in the manuscript and is also available with the authors.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by Rajiv Gandhi Centre for Biotechnology, Department of Biotechnology, Ministry of Science and Technology, India [Grant No: RGCB-LRF] and Department of Science and Technology, Ministry of Science and Technology, India [Grant Nos: DST-INSPIRE Fellowships IF150638 and IF150643].

Patient Consent

Not applicable.

Statement of Ethics

Protocols for animal experiments were approved by the Institutional Animal Ethics Committee of Rajiv Gandhi Centre for Biotechnology (IAEC/768/OMK/2019 and IAEC/860/OMK/2021) approved on 20 December 2019 and 31 January 2022, respectively, and followed the rules and regulations prescribed by the Committee for Control and Supervision of Experiments on Animals, Government of India.

ORCID iD

Reena Sarah Jacob

Ramakrishnapillai Vyomakesannair Omkumar

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