Introduction

One cognitive task frequently utilized to evaluate learning and memory performance in rodents is the hippocampus-dependent Morris water maze (MWM) test of spatial navigation [1], [2]. In this test the subject is placed in a large tank of water and is required to learn to swim to a small escape platform that is submerged below the surface of the water in a fixed spatial location. Placing the subject at different start locations around the tank ensures that it must navigate to the platform by the flexible use of distal visual cues placed about the room (e.g. cabinets, posters etc.).

Spatial navigation performance exhibits age-dependent decline in many species, including in humans [3]–[5], monkeys [6], [7], rats [8], [9] and mice [10] and hippocampal dysfunction has been reported as one of the earlier hallmarks of Alzheimer’s disease [11]. Thus, standard intercross linkage analysis aiming to identify typical genetic variations underlying strain differences in spatial navigation could help to establish a paradigm for investigation in humans.

We have recently reported the first genome-wide scan for quantitative trait loci (QTLs) affecting spatial learning and memory in Dahl rats [12]. We detected nine spatial navigation (Nav) QTLs on chromosomes 1, 2, 3, 8, 9, 11, 17, 20 and X affecting spatial learning and memory with various levels of significance [12]. The Nav-5 QTL region on chromosome 17 (58–80 Mbp) was detected with the highest significant linkage (LOD 5.3) explaining 13% of total trait variance [12]. Thus, the present study was undertaking to 1) confirm the presence of one Nav QTL in this chromosome 17 region, and 2) delimit more precisely the chromosomal region harboring this Nav-5 QTL.

Results

To substantiate the existence of Nav-5 QTL in the chromosome 17 58–80 Mbp region, we transferred two Dahl R chromosomal segments spanning the Nav-5 QTL onto the Dahl S genetic background (shown in Figure 1). We implemented a “speed congenic” strategy towards the development of highly inbred S.R17A and S.R17B (Figure 1) congenic lines. At back-cross six we established homozygous congenic lines for assessment of navigational performance on the Morris water maze (MWM) task. At BC6 S.R17A was >99.85% of Dahl S genetic background and S.R17B >99.70% of Dahl S genetic background.

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Figure 1. Congenic analysis of Nav-5 QTL region on chromosome 17.

On the left of the figure is shown the relevant region of the physical map of rat chromosome 17. Values in parenthesis next to the marker names denote physical locations in base pairs. The mapped Nav-5 QTL region of approximately 9.64 Mbp is noted (to the right). Congenic strains are shown as solid bars (representing the Dahl R introgressed fragments) flanked by open bars (representing the putative regions of recombination).

https://doi.org/10.1371/journal.pone.0058280.g001

Measurements of spatial learning in the MWM task revealed equivalent acquisition performance among all three groups (Figure 2A, F_2,360 = 1.39, P>0.24). In the probe trial for spatial memory all three groups exhibited target selectivity showing enhanced preference for the target quadrant over other quadrants (Figure 2B, Dahl S rats: P<0.001; Figure 2C, S.R17A rats: P<0.001; Figure 2D, S.R17B rats: P<0.001). However, direct comparison of selectivity for the target quadrant in the probe trial shows better performance of Dahl S controls compared with S.R17B subjects (Figure 2E, P = 0.02) and equivalent performance when compared with S.R17A subjects (Figure 2E, P = 0.79). Consistently, Dahl S rats showed increased spatial accuracy performance when compared with S.R17B subjects (Figure 2F, P = 0.02), corroborating their superior search accuracy for the hidden platform. Navigational performance in S.R17A congenic rats did not differ from Dahl S controls (Figure 2F, P = 0.96) demonstrating absence of genes affecting spatial learning and memory in this chromosomal segment, thus delimiting the chromosomal region to <10 Mbp (65.02–74.66 Mbp) that contains the gene underlying Nav-5.

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Figure 2. Testing of spatial learning and memory in Dhal S, S.R17A and S.R17B congenic male rats

(A) Acquisition performance measured as mean distance in cm during the 24 trials (•, Dahl S; ◊, S.R17A; ○, S.R17B). Percentage distance traveled in quadrants (B, C, D, E) and spatial accuracy performance (F) during the probe trial after completion of Morris water maze training. Quadrants are: target (T), opposite (O), adjacent right (A_R) and adjacent left (A_L). *P = 0.02, ***P<0.001. Data represent means ± s.e.m. (one-way ANOVA followed by Holm-Sidak’s test for quadrant occupancy in the probe trial of the Morris water maze task and for spatial accuracy performance).

https://doi.org/10.1371/journal.pone.0058280.g002

Discussion

Our results obtained in the present study confirmed the existence of Nav-5 localizing to a single chromosome 17 congenic segment. Our congenic analysis maps Nav-5 QTL between 65.02–74.66 Mbp (Figure 1) on chromosome 17. Importantly, the observed effect on navigational performance upon introgression of a Dahl R congenic fragment is consistent with expected directionality of spatial navigation effects; worsening performance in spatial learning and memory in S.R17B congenic rats [12].

Inspection of the chromosome 17 65–75 Mbp region encompassing Nav-5 QTL shows 77 annotated genes. One annotated gene, LOC689258 [similar to Serine/threonine-protein kinase MARK1 (MAP/microtubule affinity-regulating kinase 1)] at 67.94 Mbp, could be a candidate for the observed effects of Nav-5 QTL on spatial navigation. MARK1 has been shown to regulate microtubule assembly, neuronal differentiation and tau toxicity [13] an event implicated in late-onset Alzheimer’s disease. More recently, the MARK1-tau axis has been shown to play a critical role in mediating the toxic effects of Aβ on synapses and dendritic spines [14], neurodegenerative processes broadly associated with Alzheimer’s disease.

Our study demonstrates the existence of a QTL on chromosome 17 65–75 Mbp region that affects spatial learning and memory in Dahl S rats. The successful genetic isolation of the chromosome 17 Nav-5 QTL present in S.R17B congenic line will form the basis to further fine map this QTL region to <0.1 Mbp via sub-strain construction and eventually identify the specific gene variant underlying this QTL. Identification of genes that influence spatial navigation in rats could help to establish a paradigm for investigation of similar pathways pertinent to age-dependent cognitive decline and Alzheimer’s disease in humans.

Materials and Methods