Morris Water Maze Model

Brief Description

Assessment of the effect of an investigational compound on spatial learning and memory, a measure of cognitive function, in rats.

Introduction

One of the comorbidities associated with epilepsy is cognitive dysfunction and/or impairment [1-4], which can also arise as a side effect of some ASDs [5-8]. In light of these observations, ASDs that have proven effective in multiple acute behavioral seizure screens are further differentiated by their effect on learning and memory processes using the MWM [9, 10]. The MWM is a spatial navigation task that has been used extensively in the study of learning and memory [11, 12]. Based on the principle that rodents are highly motivated to escape from water by the quickest, most direct route [13], learning can be assessed in this task by the time it takes to find the escape platform (latency) over a given number of trials. Performance on this behavioral task can also be disrupted by pharmacological manipulations, including treatment with cholinergic and glutamatergic receptor antagonists [14, 15]. The ETSP assesses the effect of a candidate drug on MWM performance by evaluating whether a single dose of an investigational compound, when administered 30 min after the onset of pilocarpine-induced SE, can ultimately prevent the cognitive decline associated with pilocarpine-induced SE. Systemic administration of a single dose of pilocarpine, a muscarinic agonist, will induce SE, followed by the development of spontaneous recurrent seizures after a latent period, neurodegeneration and loss of cognitive abilities [16]. Pilocarpine-treated SE animals are normally impaired in their ability to perform the MWM task [17-19]. Thus, the ETSP uses the MWM studies to identify compounds that may protect against SE-induced cognitive impairments.

Methods

The cognitive assessment tasks and staining for hippocampal cell loss are performed as a blinded study to avoid any bias towards treatment conditions. All animal experiments are conducted in accordance with guidelines set by National Institutes of Health and the University of Utah Institutional Animal Care and Use Committee.

Pilocarpine treatment protocol : Twenty-four hours prior to pilocarpine administration, male Sprague-Dawley rats (100-120 g) are pretreated with lithium chloride (127 mg/kg; i.p.). On the following day, the rats receive pilocarpine hydrochloride (50 mg/kg; i.p.). The animals are then monitored carefully for convulsive seizure activity. Administration of pilocarpine induces behavioral seizures within 5-20 min. Any animal not showing convulsive seizure activity within 45 min of pilocarpine administration is removed from the study. For the experimental groups, rats are given either vehicle (0.5% methyl cellulose) or the test compound (at a dose effective in Test 72/Test 75 or as suggested by the supplier) 30 minutes after the first observed stage 3 seizure [20]. All animals are observed and scored for seizure severity for an additional 90 min. Before being returned to their home cages, 3 mL of Lactated Ringer’s solution is administered (s.c.) to each rat to compensate for the SE-induced fluid loss. All animals are given access free access to food and water, with a supplemental diet of rat chow moistened with electrolyte solution, water melon and/or orange slices, provided during the first week of recovery. Animals are allowed to recover from pilocarpine-induced SE before testing in the MWM study.

Morris water maze : Two weeks after pilocarpine treatment, rats are tested for SE-induced memory deficits in the MWM task. This test uses a round pool of water (1.8 m in diameter, 0.3 m deep) maintained at 25 ± 1oC, in which an escape platform is submerged 1.5 cm beneath the surface. The task tests the rat’s ability to find the hidden platform using only extra maze visual cues. Learning can be assessed by the time it takes to find the platform (latency) over a number of trials. In our spatial learning protocol rats receive 4 training trials per day, wherein the outcome measure evaluated is their ability to find the hidden platform. On each of the trials, rats are placed in water facing the wall at one of the 4 randomly determined starting locations (north, west, east or south) and allowed 120 sec to find the platform. The trial ends when the rats either climb onto the platform or after the 120 sec interval has elapsed. Once the rat has found the platform, it is permitted a 10 second rest period on the platform. If it does not find the platform within 120 sec, it is guided to the platform and given 10 sec to re-orient to the spatial cues. After each trial the rats are placed in a warm holding chamber for an inter-trial interval of at least 5 min. Five successive training days are conducted (days 1-5); with the last trial of day 5 being a probe test, wherein the platform is removed. After two days, the rats are re-tested using a visible non-cued platform trial session (4 trials per day) for two additional days (days 8-9). Visible/raised platform trials (1.5 cm above the water level) assess any potential impairment in visual acuity associated with the treatment protocol. The escape latency and distance traveled for each animal are recorded using a HVS image tracking system and compared across treatment groups.

FluoroJade staining for assessing in vivo neuroprotection : At the completion of the behavioral testing, all rats are anaesthetized with Nembutal (60 mg/kg; i.p.) and transcardially perfused with 1X PBS followed by 4% paraformaldehyde (PFA). The brains are removed and fixed overnight in 4% PFA. 40 µm thick coronal brain slices are cut and stained with FluoroJade-B to assess neuronal loss in the dentate gyrus (DG), CA1 and CA3 cell layers of the hippocampus.

Results

For MWM studies, animals in all treatment groups are tested in the MWM for changes in escape latency over the course of the five training sessions. Additionally, animals are observed for distance traveled, number of platform crossings and swim speed, if necessary, in both the hidden and visible trials. The dependent measures are analyzed by using one- or two-way ANOVA with Bonferroni multiple comparison tests; p < 0.05 is considered statistically significant. Additionally, the ability of an investigational compound to prevent SE-induced cell death is qualitatively assessed by FluoroJade-B staining in the dentate gyrus (DG), CA1, and CA3 cell layers, with 3 sections per region for each rat analyzed. Results obtained from rats treated with an investigational drug (n= 8) are compared to those obtained with pilocarpine-treated (n=8) and age-matched vehicle-treated naïve rats (n= 8).

Discussion

The MWM is a well-validated behavioral assay that can readily reveal impairments in learning and memory processes [12-14]. As epilepsy can often be associated with cognitive deficits, which can arise due to biochemical and physiological remodeling during the epileptogenic process [16], understanding whether investigational compounds may also disrupt normal learning and memory processes is of critical value to the ETSP. Prototypical ASDs, such as phenytoin and carbamazepine, can also disrupt discrete aspects of performance on the MWM [21]. Pilocarpine-induced SE leads to long-term deficits in cognitive performance on the MWM [10, 18]. As such, the ETSP can assess the neuroprotective capabilities of investigational compounds by examining behavioral performance on the MWM two weeks after pilocarpine-induced SE. Compounds found to mitigate such SE-induced learning and memory deficits, as detected in the MWM, may ultimately confer neuroprotection in the context of clinical status epilepticus. The ETSP differentiates novel investigational compounds on many levels during the screening process. Those compounds found to mitigate the cognitive decline associated with pilocarpine-induced SE and to be neuroprotective may afford a unique benefit to some of the currently available AEDs and could be suitable candidates for future preclinical and clinical investigation.