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Dorsal Hippocampus Not Always Necessary in a
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Radial Arm Maze Delayed Win-shift Task
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Dylan Layfield1*, Nathan Sidell1, Afnan Abdullahi1, Ehren L. Newman1
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1) Department of Psychological and Brain Sciences, 1101 E 10th St, Bloomington, IN,
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47405;
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Word count (without abstract, captions, or references) - 3025
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Number of figures - 2
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Acknowledgements:
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This work was made possible by generous funding from the Harlan Scholars Program and the
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IU-MSI STEM Initiative. We graciously thank the Indiana University Laboratory Animal
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Resources facilities for their attention and care of our animals.
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.CC-BY-NC 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint. http://dx.doi.org/10.1101/433565doi: bioRxiv preprint first posted online Oct. 2, 2018;
Abstract
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Spatial working memory is important for foraging and navigating the
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environment. However, its neural underpinnings remain poorly understood. The
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hippocampus, known for its spatial coding and involvement in spatial memory, is widely
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understood to be necessary for spatial working memory when retention intervals
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increase beyond seconds into minutes. Here, we describe new evidence that the dorsal
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hippocampus is not always necessary for spatial working memory for retention intervals
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of 8 minutes. Rats were trained to perform a delayed spatial win shift radial arm maze
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task (DSWS) with an 8-minute delay between study and test phases. We then tested
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whether bilateral inactivation of the dorsal hippocampus between the study and test
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phases impaired behavioral performance at test. Inactivation was achieved through a
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bilateral infusion of lidocaine. Performance following lidocaine was compared to control
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trials, in which, sterile phosphate buffered saline (PBS) was infused. Test performance
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did not differ between the lidocaine and PBS conditions, remaining high in each. To
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explore the possibility that this insensitivity to inactivation was a result of overtraining,
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a second cohort of animals received substantially less training prior to the infusions. In
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this second cohort, lidocaine infusions did significantly impair task performance. These
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data indicate that successful performance of a spatial win-shift task on the 8-arm maze
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need not always be hippocampally dependent.
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1. Introduction
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The ability to forage is key to survival for many animals. A critical component of
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foraging behavior is spatial working memory, wherein information regarding the spatial
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positions that food has been found or that remain to be searched is maintained (Olton &
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Samuelson, 1976). Identifying the brain structures that support spatial working memory
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remains an outstanding goal of behavioral neuroscience. Pharmacological inactivation
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and lesion studies indicate that the hippocampus is necessary when the delay between
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encoding of spatial information and use of that information increases from seconds to
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minutes (Lee & Kesner., 2003a; Lee & Kesner., 2003b; Churchwell & Kesner., 2011).
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Spatial working memory in rodents is often studied using the radial arm maze
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(Olton & Samuelson., 1976). A widely used radial arm maze task is the delayed spatial
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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win-shift task (DSWS; Packard et al., 1990; Seamans and Phillips., 1994; Seamans et al.,
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1995). In this task, rats first complete a study phase with a subset of arms available and
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baited with food. Later, rats complete a test phase where all arms are open, and the rat
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is rewarded for visiting previously un-entered arms. Though prior work has shown that
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inactivation of the ventral hippocampus/subiculum reduced performance after a 30-
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minute delay (Floresco et al., 1997), the necessity of the dorsal hippocampus for this
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task remains unknown.
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Given the existing literature showing the hippocampus is involved in spatial
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cognition (O’Keefe & Nadel., 1978; Buzsaki & Moser., 2013; Hartley et al., 2014) and
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that lesions / pharmacological inactivations of dorsal hippocampus impair performance
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in other spatial working memory tasks (McDaniel et al., 1994; Lee & Kesner., 2003a;
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Lee & Kesner, 2003b; Potvin et al., 2006; Yoon et al., 2008), we expected that the dorsal
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hippocampus would be necessary for accurate test performance the DSWS task on the
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radial arm maze with 8 min retention intervals. However, here, we describe data
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showing that bilateral infusions of lidocaine administered at the outset of an 8-minute
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retention interval between study and test did not impair performance relative to
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phosphate buffered saline infusions. To test if this insensitivity was possibly the result of
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overtraining, a second cohort of animals received substantially less training prior to the
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infusions. In this second cohort, lidocaine infusions significantly impaired task
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performance. These results challenge the traditional view that the dorsal hippocampus
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is always needed for spatial memory guided behavior at intermediate delays.
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2. Methods
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The data presented here was collected incidentally in the running of a larger study.
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In the broader study, the trials of interest were those with delays of 60 min or longer.
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Trials with 8 min retention intervals had been included to control for expectancy effects
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central to that design. We describe here the full set of methods but will focus our
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analyses on short retention interval trials, during which, the inactivation could be
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expected to be in effect.
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All animal procedures and surgery were conducted in strict accordance with National
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Institutes of Health the Indiana University Institutional Animal Care and Use
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Committee guidelines.
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2.1 Subjects
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19 Male Adult Long Evans rats were used: 10 in cohort one and nine in cohort
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two. One animal was dropped from cohort one due to inaccurate cannula placement.
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Rats were individually housed and maintained on a 12 H light/dark cycle in a
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temperature and humidity-controlled room with ad libitum access to water, and food
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restricted to maintain ~90% (85-95%) of free feeding body weight. Rats were acclimated
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to the animal facility for 5 days before being handled daily.
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2.2 Behavioral training
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Training took place on a custom automated radial maze (Maze Engineers,
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Cambridge, MA) with a 33.2 cm wide hub and pneumatic drop doors at the entrances to
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the 8 arms, each measuring 48.26 cm long, 10.79 cm wide with 20.95 cm tall walls. At
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the end of each arm were food wells in which 45 mg sucrose pellets (Bio-Serv,
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Flemington, NJ) were delivered. The maze was open on top, with clear acrylic walls,
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allowing for viewing of a variety of distal cues surround the maze. The maze was cleaned
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with chlorohexadine immediately after each trial. Figure 1A summarizes the training
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and testing phases rats in cohort one and two received.
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2.2.1 Habituation & Preliminary Training
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For 3 days prior to training, rats were handled for 10 minutes and given 20-30
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sucrose pellets to habituate them to experimenter handing and rewards. Preliminary
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training consisted of one 10 min trial daily for 4 days. During each, 4-6 pellets were
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placed along each of the 8 arms and 2 pellets in the food wells of each arm. The trial
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ended after the rat consumed all pellets or 10 minutes had elapsed.
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2.2.2 Initial Training
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Initial Training consisted of 10 sessions, 1 trial/session. During each, rats were
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trained that arms would be baited once per day with 2 pellets. Training trials began by
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placing the animal in the central hub with testing room lights off and hub doors closed.
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After 1-min the doors opened automatically, and lights turned on allowing the rat to
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freely forage. Lights were turned off during all hub placements in this and all future
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phases of training to prevent increased exposure to extra-maze cues. The rats could
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explore until all pellets were collected or 15 minutes had elapsed.
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2.2.3 Pre-task Training
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Pre-task training consisted of two phases, a study phase and a test phase. In the
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study phase, the rat was placed in the central hub, after 1 minute, a random set of four
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doors opened, and the rat was allowed to collect pellets from each. The rat was then
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removed and placed in their home cage for an 8-min retention phase. During which, the
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maze was cleaned. The test phase began by placing the rat in the hub and, after 1 min,
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all eight doors opened. The four arms not opened at study were baited with pellets. The
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test phase ended after all the pellets had been consumed or when 15 minutes elapsed.
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The trial timeline is illustrated in Figure 1B. Criterion performance, required before
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moving on in the study, was no more than three errors over four days, with any days
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having errors requiring the optimal number of arm entries (for example 1 error requires
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5 arm entries 2 errors 6 arm entries etc.).
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2.2.4 Extended task experience prior to surgery
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Rats in cohort one participated in two pilot experiments between reaching
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criterion and receiving surgery (Figure 1). These pilot experiments consisted of test runs
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through the task described below. These variants used retention intervals of 8, 60, and
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150 minutes and varied the holding location of rats during the retention interval. Rats in
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cohort two did not participate in pilot studies, reducing the total experience with the
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task prior to surgery (see Results for comparison of training time between cohorts).
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2.2.5 Task
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The larger task design, from which the current trials of interest were drawn,
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replicated the ‘retrieval practice’ design of Crystal et al. (2013). In short, rats performed
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a DSWS task on an 8-arm maze but on some trials, rats were placed back on the track at
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8 min but removed again prior to the doors opening. The full test phase was then run
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after the remainder of a 60 min delay. Our study used a fully counterbalanced 3 x 2
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design with 3 behavioral conditions {8 min retention, 60 min retention with retrieval
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practice, 60 min retention without retrieval practice} and 2 infusion types {lidocaine,
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phosphate buffered saline}.
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2.3 Surgery
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Rats were anesthetized with isoflurane (1 – 4% in oxygen) and placed in a
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stereotaxic frame (Kopf Instruments). A scalp incision was made, 2-4 screws were
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inserted into the skull and two craniotomies were drilled to target dorsal hippocampus
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with the coordinates AP: -3.8 mm, ML ±2.5 mm. Two 26-gauge guide cannulas (Plastics
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one) were lowered into the brain, DV 1.8 mm from brain surface, and secured to the
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anchor screws with dental acrylic. Dummy cannulas were inserted into the guide
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cannulas. Rats recovered for 5+ days post-surgery.
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2.4 Intracranial microinfusions
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For infusions, the dummy cannulas were removed and injector cannulas with 1
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mm projection prefilled with solution was inserted. A microinfusion pump infused a
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total volume of 0.5ul at 0.5ul per minute per hemisphere. The injector cannula
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remained in place for 1-minute post infusion to allow for liquid diffusion. The injector
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was then removed, and a sterilized dummy cannula was secured into the guide cannula.
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Inactivation was achieved by infusing Lidocaine hydrochloride (Sigma-Aldrich) diluted
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to 4% w/v in phosphate-buffered saline (PBS). The concentration and volume were
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selected to match those used by others to induce behavioral deficits in other learning
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paradigms (Lopez et al., 2012; Chang & Laing., 2017). Given the 0.5 ul infused here, a
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functional spread of slightly less than 1.0 mm would be expected (Martin, 1991; Welsh
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and Harvey, 1991). The short duration of the test phase (~ 1-3 min), run ~7 min after the
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infusion, fell within the window of lidocaine effect (Malpeli et al., 1999). The control
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condition, designed to control for the influences of the infusion process, was a volume
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matched infusion of PBS. For each animal, two lidocaine and two PBS infusions were
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performed for the 8 min retention interval trials examined here.
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2.5 Histology
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After completing all trails, rats were sacrificed via isoflurane overdose and
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perfused intracardially with saline followed by a 10% formalin solution. Brains were
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extracted and stored in formalin. 72 hours prior to slicing, brains were transferred into a
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30% sucrose solution. Brains were sectioned at 40 um along the coronal plane and
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stained with 0.5% cresyl violet. Stained slices were imaged, and cannulas tip placement
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was confirmed by reference to a rat brain atlas as shown in Figure 1C (Paxinos & Watson
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2007).
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2.6 Data Analysis & Statistics
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Memory performance at test was scored by 1) Percent correct: the percentage of
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the first four arms visited that were baited, and 2) Number of arm entries: the number
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of arms visited by the time the fourth correct arm was visited. An arm visit occurred
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when all four feet of the rat where in the arm. Statistical analysis for within group
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comparisons on both percent correct and total arm entry measures were performed with
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one-way repeated measures ANOVAs with two levels (PBS, Lidocaine), all p-values
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reported are Wilk’s Lambda test. For cross-group comparisons a 2 (cohort) x 2
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(infusion type) mixed factor ANOVA to compare percent correct and total number of
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arm entries between cohort 1 and cohort 2. Independent samples t-tests with two groups
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(cohort 1 vs cohort 2) were run to compare number of days to reach criterion first time,
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and number of days to return to criterion post-surgery prior to the start of infusions.
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Significance was defined at the α = 0.05 level. Reported values indicate mean ±
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standard deviation.
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3. Results
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Fig 1. Experiment overview. (A) Timeline of task training. Rats in cohort one and two
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received the same training up to reaching behavioral criterion. Only cohort one
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participated in two pilot studies prior to surgery. Both cohorts received the same
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training following surgery. The mean number of trials required at each step is shown.
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(B) Timeline of task trials. Rats were placed in the hub for 1 minute before 4 doors open
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in the study phase. Immediately following study phase rats were given infusions and
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placed back in their home cage (in the maze room) for remainder of the delay. After the
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8-minute delay, rats were placed back in the hub for 1 minute before all 8 doors open for
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test phase. (C) Coronal plates showing cannula termination spots. Each circle represents
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a cannula termination spot for a rat. (Red circles cohort 1, Blue circles cohort 2).
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Numbers next to each plate indicate distance from bregma in mm. trls = trials.
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Fig 2. Dorsal hippocampal inactivation only impaired DSWS performance in cohort two.
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(A) Percentage of first four arm entries that were correct for both cohorts. No difference
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was observed in cohort one. In cohort two, the accuracy was significantly lower
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following lidocaine infusions. (B) Total arm entries to visit all four target arms for both
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cohorts. No difference was observed in cohort one. In cohort two, the number increased
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significantly following lidocaine infusions. Bar height reflects means, error bars indicate
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±SEM. * - p < 0.05; n.s. – not significant.
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To test whether the dorsal hippocampus is necessary for accurate test
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performance in a delayed win-shift spatial working memory task, we compared the
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percent accuracy and number of arm entries at test in animals that received lidocaine or
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PBS infusions between the study and test phases.
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Performance did not significantly differ between the lidocaine and PBS
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conditions in cohort one whether it was measured as the percentage of the first four arm
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entries that were correct (81 ± 14% vs. 83 ± 17%, Wilk’s λ =0.98, F(1,17) = 0.39 p = 0.54;
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Figure 2A) or as the total number of arm entries needed to collect all four rewards (5.2 ±
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1.1 arms vs. 4.8 ± 0.9 arms, Wilk’s λ = .89, F(1,17) = 2.07, p = 0.17; Figure 2B). That is,
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rats in cohort one performed well whether or not their dorsal hippocampus was
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inactivated prior to test. The lack of difference between these conditions suggests that
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the accurate performance of the radial arm maze DSWS task does not always necessitate
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the dorsal hippocampus.
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Prior studies strongly suggest that the dorsal hippocampus should be required for
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this task. We hypothesized that the lack of hippocampal dependence observed in the
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first cohort was due to over training. Between reaching behavioral criterion for the first
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time and receiving the cannulation surgery, rats in the first cohort participated in two
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pilot experiments. This extending the amount of task training they received by an
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average of 28.3 ± 8.1 days. To test whether less training would uncover a sensitivity to
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the hippocampal inactivation, we ran a second cohort of rats. These received an average
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of only 7.6 ± 4.5 days of training between reaching criterion and undergoing surgery,
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differing significantly from the first cohort (F(1,16) = 4.49, p =< 0.0001). Consistent
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with the idea that the first cohort had a better grasp of the task prior to surgery, they
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returned to criterion significantly faster than the second cohort following surgery (6.4 ±
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3.2 days vs. 17.3 ± 11.0 days, F(1.16) = 3.68, p = 0.011). Importantly, however, both
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cohorts reached the initial criterion (before surgery) in a similar number of days (17.44
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± 9.54 days vs. 23.44 ± 15.6 days, F(1,16) = 3.383, p = .340), demonstrating that there
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was not a systematic difference in capability between the cohorts. It is also important to
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note that in performing the two pilot experiments, the first cohort also received
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experience (12.55 trials on average) with retention intervals of between 60 and 150
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minutes whereas the second cohort remained naïve to these long delays.
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Consistent with the hypothesis that the additional experience the first cohort
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received reduced the necessity of the hippocampus for accurate performance at test, the
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second cohort, which did not receive this experience, was sensitive to hippocampal
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inactivation. Test performance in the second cohort was significantly lower following
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lidocaine infusions relative to PBS infusions whether measured by percent correct (81 ±
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16% vs. 64 ± 25%, Wilk’s λ = 0.667, F(1,17) = 8.50, p = 0.010; Figure 2A) or by arm
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entries (4.9 ± 0.8 arms vs. 5.8 ± 1.3 arms, Wilk’s λ = .704, F(1,17) = 7.15, p = 0.016;
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Figure 2B). The sensitivity of the second cohort to hippocampal inactivation
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demonstrates that the dorsal hippocampus is involved performance of the delayed
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spatial win-shift task, at least initially. When considered together with the data from the
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first cohort, the present findings suggest that, with sufficient training, performance of
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this task become independent of the dorsal hippocampus.
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4. Discussion
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Understanding the brain structures that play a necessary role in spatial working
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memory remains an outstanding challenge. The traditional view holds that, when
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retention intervals span minutes in a delayed spatial win shift (DSWS) task and the
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arms shown at study are randomized over trials, the hippocampus is always required. In
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this brief report, we describe data showing that radial arm maze DSWS task
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performance was not impaired when lidocaine was infused into the dorsal hippocampus
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prior to the test phase in a cohort of rats. The same infusions did impair performance,
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however, in a second cohort of rats that had received significantly less task training.
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These results indicate that the dorsal hippocampus is important for DSWS task
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performance at test early in training but, with additional experience, becomes less
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important.
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Our finding that lidocaine infusions did not impair performance when done prior
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to the 8 min tests was surprising given previous work showing the importance of the
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hippocampus for working memory at similar delays (Lee & Kesner., 2003a; Lee &
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Kesner., 2003b; Yoon et al., 2008; Churchwell & Kesner., 2011). Lee & Kesner (2003a),
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for example, showed that temporary inactivation/lesion of dorsal hippocampus/mPFC
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during a radial arm DSWS task impaired performance with a 5-minute retention
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interval. Importantly, the deficit could not be compensated for by an intact mPFC
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indicating the specific importance of the dorsal hippocampus at such delays.
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The discrepancy between prior reports and our findings may be attributable to
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methodological differences. The most significant difference between prior studies and
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ours is that in prior work the inactivation/lesions had effect on both the study and test
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phases. Here, the inactivation was performed between the study and test phases leaving
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the dorsal hippocampus fully functional during the study phase. As such, unlike prior
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work, the manipulation used here asked whether the dorsal hippocampus is necessary
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specifically at test. Unfortunately, we were not able to test this hypothesis directly by
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performing the inactivation prior to the study phase in the first cohort because this
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striking pattern of results was identified incidentally in analysis of data collected as a
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control for a larger study after the rats had already been sacrificed. Future research will
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be needed to test this hypothesis directly.
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Another key difference may be the amount of training the animals received. The
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cohort of rats that was insensitive to the inactivation had received considerably more
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training on the task prior to the cannulation surgery. With this training, they also
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received experience with delays of 60 minutes or longer. Given the cohort that did not
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receive the extra training or experience with long delays remained sensitive to the
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inactivation, it is likely that this experience contributed to the reduction in hippocampal
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dependence. Whether it was the extra trials of experience or specifically the experience
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with the long delays that led to this shift remains unclear. The experimental design used
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here was poorly suited to sorting between these possibilities because it was initially
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designed for a separate study.
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In summary, we describe here the incidental finding that pharmacological
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inactivation of the dorsal hippocampus prior to the test phase of a DSWS task did not
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impair spatial working memory performance in a cohort of rats. While in a second
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cohort, the task was sensitive to dorsal hippocampus inactivation. This finding is
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striking in that it contrasts with what prior work has shown on hippocampal
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contributions to spatial working memory would have predicted. These results do not
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directly contradict prior findings however, as there are important procedural differences
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between what was done here and what has been done previously. Perhaps most
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importantly, this is the first study to test the effect of dorsal hippocampal inactivation
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selectively during the DSWS test phase. The present results were found during analysis
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of non-essential trials of a larger study and additional work is needed, using purpose
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designed experiments, to better understand the boundary conditions of the current
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findings.
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.CC-BY-NC 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint. http://dx.doi.org/10.1101/433565doi: bioRxiv preprint first posted online Oct. 2, 2018;
Captions
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Fig 1. Experiment overview. (A) Timeline of task training. Rats in cohort one and two
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received the same training up to reaching behavioral criterion. Only cohort one
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participated in two pilot studies prior to surgery. Both cohorts received the same
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training following surgery. The mean number of trials required at each step is shown.
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(B) Timeline of task trials. Rats were placed in the hub for 1 minute before 4 doors open
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in the study phase. Immediately following study phase rats were given infusions and
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placed back in their home cage (in the maze room) for remainder of the delay. After the
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8-minute delay, rats were placed back in the hub for 1 minute before all 8 doors open for
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test phase. (C) Coronal plates showing cannula termination spots. Each circle represents
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a cannula termination spot for a rat. (Red circles cohort 1, Blue circles cohort 2).
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Numbers next to each plate indicate distance from bregma in mm. trls = trials.
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Fig 2. Dorsal hippocampal inactivation only impaired DSWS performance in cohort two.
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(A) Percentage of first four arm entries that were correct for both cohorts. No difference
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was observed in cohort one. In cohort two, the accuracy was significantly lower
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following lidocaine infusions. (B) Total arm entries to visit all four target arms for both
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cohorts. No difference was observed in cohort one. In cohort two, the number increased
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significantly following lidocaine infusions. Bar height reflects means, error bars indicate
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±SEM. * - p < 0.05; n.s. – not significant.
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.CC-BY-NC 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint. http://dx.doi.org/10.1101/433565doi: bioRxiv preprint first posted online Oct. 2, 2018;