This work was supported by the University of Strasbourg and Neurex&#8212;Neuroscience Upper Rhine Network (post-doc fellowship to RP) and by BrainLinks-BrainTools Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086). We thank Nadja Martini for expert technical assistance.

1. General Considerations
This protocol is approved by the Animal Care and Use Committee of University Hospital Freiburg (same for Strasbourg). Visual acuity is necessary for performance in tests of spatial learning. Rodents with impaired visual systems are thus not suitable. Also, lighting must be sufficient in order for the rats to see the different cues located on the surrounding walls. It is useful to utilize basic-shaped (square, circle, triangle) but well-contrasted cues (e.g., black-painted cues on a white-painted background). Likewise, severe motoric deficits are exclusion criteria because swimming is required for this test and drowning may occur. Finally, hyper-anxious rodents can display a strongly biased search behavior, which impacts on performance.
2. Apparatus Set Up
<ol>
	<li>Construct a double-H maze consisting of a 160 cm central alleyway, which is intersected at both ends and in the center by three 160 cm parallel run arms (see Figure 1). Ensure that the central alleyway and its perpendicular arms are 20 cm wide, and are surrounded by 35 cm high transparent Plexiglas walls. Thickness of the Plexiglas is 6 mm for all parts.</li>
	<li>Secure the walls in place using glue and screws, and waterproof the maze using silicone joints at all internal angles. These joints can be replaced easily should they lose their waterproofness. Place a drain outlet at the extremity of one of the corner arms (or in the middle of the maze) for emptying.</li>
	<li>Place the maze on an 80 cm high table, with adequate space around it for a) walking around the maze, and b) placement of well-contrasted cues. Designate each arm in the maze by its position, i.e., northwest (NW), north (N), northeast (NE), southwest (SW), south (S), and southeast (SE).</li>
	<li>Place a ceiling-mounted camera above the maze for post-test analysis of animal behavior, using either manual or automatic (video-tracking) methods (see &#167;5).</li>
</ol>
3. General Comments
<ol>
	<li>Prior to use, fill the maze with water to an approximate height of 18 cm (200 L). This is high enough to prevent rats from touching the bottom of the maze with their feet, but shallow enough to prevent escape.</li>
	<li>After pre-training, render the water opaque by mixing 250 g skim milk powder. Change the water on a daily basis to prevent the milk-water from becoming rancid. Maintain the water temperature between 21 - 23 &#176;C to provide incentive for the rats to seek the escape platform. 
	NOTE: As rats become familiar with the position of the platform during pre-training, its position is moved to a different arm during training.</li>
	<li>Prior to use, immerse a 17 cm high, 10 cm diameter platform at the extremity of one of the corner arms (NE, NW, SE or SW). Ensure the height to be submerged is 1 cm below the surface of the water surface. Train the rats to reach the target platform using either an allocentric or egocentric learning strategy, which is dependent on the type of paradigm used (see &#167;4).</li>
</ol>
4. Basic Training Protocols
NOTE: Rats are typically provided with an initial day of pre-training, which allows them to become familiar with the maze.
<ol>
	<li>For pre-training release the rat from the extremity of one of the center arms (e.g., S arm) and place the goal platform at the extremity of one of the corner arms (e.g., NE), then give the rats 4 consecutive 60 sec trials in which to reach the target platform.</li>
	<li>Upon reaching the escape platform, allow the rat to wait there for 15 sec, so that they can rest and observe their surroundings. Regardless of the start position, always block the opposite arm with a transparent guillotine door, which prevents entry. 
	NOTE: During pre-training, leave the water transparent, and adjust the platform height such that it protrudes 1 cm above the water&#8217;s surface, thus making it visible to the rat.</li>
	<li>Perform daily training session consisting of up to 4 consecutive trials, separated by a 10 sec gap at least (discrete training, i.e., with intervals of several minutes between trials, is an alternative).</li>
	<li>For training, relocate the platform from its pre-training position to the chosen arm (e.g., NW), and submerge it at its extremity 1 cm below the water surface. Now render the water opaque by addition of milk power and perform the following training (section 4).</li>
	<li>For rats that do not reach the target platform within 60 sec, return them to the starting position, and gently guide them to the platform by the experimenter.</li>
	<li>Measure several variables during the training and probe sessions, such as, distance swam, latency to goal arm / platform, time spent in each arm, as well as the number of initial/repetitive errors (see Figure 2). Keep in mind that latencies may be influenced by motor difficulties. Should the case arise, distance and errors appear as more reliable variables regarding cognitive performance.</li>
	<li>Allocentric Strategy Training:
	<ol>
		<li>Day&#160;1 - Pre-Training:
		<ol>
			<li>Do not add milk powder for this step. Place escape platform protruding 1 cm above the water&#8217;s surface in a fixed location. Train rats in 4 consecutive trials to reach the platform.</li>
		</ol>
		</li>
		<li>Days 2 - 5 &#8211; Training:
		<ol>
			<li>Add 250 g of skim milk powder to the water to render it opaque. Move the platform to a different arm (e.g., from NE to NW) and add water so that the platform is 1 cm below the water surface. Release rats from either the N or S arm in unpredictable sequences for each session, such that both arms are used twice as trial start in a single session (4 trials/day, e.g., SNNS, NSNS; see Figure 3).</li>
		</ol>
		</li>
		<li>Day 6 &#8211; Probe Session:
		<ol>
			<li>Remove platform for the probe trial. Release rats from a different arm to those used during training (e.g., SW), and allow them to swim for 60 sec. See Figure 4 for representative swim tracks. Analysis of the time spent in the target arm (former location of the platform) gives an indication about whether rats use a spatial strategy, another type of strategy, a sequential combination of different strategies (see below), or a disorganized search pattern.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Egocentric Strategy Training:
	<ol>
		<li>Day 1 &#8211; Pre-Training:
		<ol>
			<li>This is the same as first step in the allocentric strategy training (step 4.1). Do not add milk powder for this step. Place escape platform protruding 1 cm above the water&#8217;s surface in a fixed location. Train rats in 4 consecutive trials to reach the platform.</li>
		</ol>
		</li>
		<li>Days 2 - 5 &#8211; Training:
		<ol>
			<li>Add 250 g of skim milk powder to the water to render it opaque. Move the platform to a different arm and add water so that the platform is 1 cm below the water surface. Release rats from the same start arm (S or N; see Figure 5) for every trial (4 trials/day).</li>
		</ol>
		</li>
		<li>Day 6 &#8211; Probe Session:
		<ol>
			<li>Remove platform for the probe trial. Rats are released from an arm different from where they were released during the training. Allow the rats to swim for 60 sec. Block the opposite arm with a transparent guillotine door. 
			NOTE: Modify the above training strategies for according to the requirements of the particular experiment, e.g., Testing Drug Effects etc.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>
	Dry the rat off with absorbent towels after each session in the water.
	</li>
</ol>
5. Analysis
<ol>
	<li>Perform measurements of latencies, initial and repetitive errors, first choices and response type manually by recording these variables from the videos taken from the overhead camera.</li>
	<li>Alternatively if available, utilize commercially available video-tracking software and configured to record these variables automatically.</li>
	<li>Statistical Analysis: 
	NOTE: The specific implementation of statistical analysis depends on the study, which is taking place.
	<ol>
		<li>Perform a one, two or three-way ANOVAs regarding the initial/repetitive errors, latency to goal arm/platform, and time spent in the target arm; with factors that include the test day, and treatment group(s).</li>
		<li>Where necessary, follow these ANOVAs using post-hoc Newman-Keuls multiple comparisons tests. To compare performance, use a reference value (e.g., the time spent in the target arm during a probe trial vs. the chance level), and perform t-test.</li>
		<li>If required, use non parametric statistics in addition (e.g., &#967;&#178;, see below) or instead when conditions for parametric ones are not fulfilled.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Izquierdo, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+connection+between+the+hippocampal+and+the+striatal+memory+systems+of+the+brain:+a+review+of+recent+findings.">The connection between the hippocampal and the striatal memory systems of the brain: a review of recent findings.</a> Neurotoxicity Research. 10 (2), 113-121 (2006).</li><li>Albouy, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+the+hippocampus+and+striatum+are+involved+in+consolidation+of+motor+sequence+memory.">Both the hippocampus and striatum are involved in consolidation of motor sequence memory.</a> Neuron. 58 (2), 261-272 (2008).</li><li>Albouy, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Hippocampal+and+Striatal+Systems+Predicts+Subsequent+Consolidation+of+Motor+Sequence+Memory.">Interaction between Hippocampal and Striatal Systems Predicts Subsequent Consolidation of Motor Sequence Memory.</a> Plos One. 8 (3), (2013).</li><li>Chang, Q., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-hippocampal+lidocaine+injections+impair+acquisition+of+a+place+task+and+facilitate+acquisition+of+a+response+task+in+rats.">Intra-hippocampal lidocaine injections impair acquisition of a place task and facilitate acquisition of a response task in rats.</a> Behav Brain Res. 144 (1-2), 19-24 (2003).</li><li>McDonald, R. J., Hong, N. S., Devan, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+understanding+mammalian+cognition+and+memory-based+behaviours:+an+interactive+learning+and+memory+systems+approach.">The challenges of understanding mammalian cognition and memory-based behaviours: an interactive learning and memory systems approach.</a> Neurosci Biobehav Rev. 28 (7), 719-745 (2004).</li><li>Packard, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anxiety+cognition,+and+habit:+a+multiple+memory+systems+perspective.">Anxiety cognition, and habit: a multiple memory systems perspective.</a> Brain Res. 1293, 121-128 (2009).</li><li>Packard, M. G., McGaugh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+hippocampus+or+caudate+nucleus+with+lidocaine+differentially+affects+expression+of+place+and+response+learning.">Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning.</a> Neurobiol Learn Mem. 65 (1), 65-72 (1996).</li><li>Lawrence, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+and+mnemonic+functions+in+early+Huntington's+disease.">Executive and mnemonic functions in early Huntington's disease.</a> Brain. 119 (Pt 5), 1633-1645 (1996).</li><li>Lawrence, A. D., Watkins, L. H., Sahakian, B. J., Hodges, J. R., Robbins, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+object+and+visuospatial+cognition+in+Huntington's+disease:+implications+for+information+processing+in+corticostriatal+circuits.">Visual object and visuospatial cognition in Huntington's disease: implications for information processing in corticostriatal circuits.</a> Brain. 123 (Pt 7), 1349-1364 (2000).</li><li>Walker, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease.">Huntington's disease.</a> Lancet. 369 (9557), 218-228 (2007).</li><li>Cushman, L. A., Stein, K., Duffy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+navigational+deficits+in+cognitive+aging+and+Alzheimer+disease+using+virtual+reality).">Detecting navigational deficits in cognitive aging and Alzheimer disease using virtual reality).</a> Neurology. 71 (12), 888-895 (2008).</li><li>Liu, L., Gauthier, L., Gauthier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+disorientation+in+persons+with+early+senile+dementia+of+the+Alzheimer+type.">Spatial disorientation in persons with early senile dementia of the Alzheimer type.</a> A`m J Occup Ther. 45 (1), 67-74 (1991).</li><li>Mapstone, M., Steffenella, T. M., Duffy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visuospatial+variant+of+mild+cognitive+impairment:+getting+lost+between+aging+and+AD.">A visuospatial variant of mild cognitive impairment: getting lost between aging and AD.</a> Neurology. 60 (5), 802-808 (2003).</li><li>Vliet, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropsychological+profiles+of+mild+Alzheimer's+disease+and+questionable+dementia+as+compared+to+age-related+cognitive+decline.">The neuropsychological profiles of mild Alzheimer's disease and questionable dementia as compared to age-related cognitive decline.</a> J Int Neuropsychol Soc. 9 (5), 720-732 (2003).</li><li>Hanagasi, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+impairment+in+amyotrophic+lateral+sclerosis:+evidence+from+neuropsychological+investigation+and+event-related+potentials.">Cognitive impairment in amyotrophic lateral sclerosis: evidence from neuropsychological investigation and event-related potentials.</a> Brain Res Cogn Brain Res. 14 (2), 234-244 (2002).</li><li>Robbins, T. W., Murphy, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+pharmacology:+40++years+of+progress,+with+a+focus+on+glutamate+receptors+and+cognition.">Behavioural pharmacology: 40+ years of progress, with a focus on glutamate receptors and cognition.</a> Trends Pharmacol Sci. 27 (3), 141-148 (2006).</li><li>Paul, C. M., Magda, G., Abel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+memory:+Theoretical+basis+and+comparative+review+on+experimental+methods+in+rodents.">Spatial memory: Theoretical basis and comparative review on experimental methods in rodents.</a> Behav Brain Res. 203 (2), 151-164 (2009).</li><li>Hodges, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+procedures:+the+radial-arm+and+water+maze+compared.">Maze procedures: the radial-arm and water maze compared.</a> Brain Res Cogn Brain Res. 3 (3-4), 167-181 (1996).</li><li>Kesner, R. P., Farnsworth, G., Kametani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+parietal+cortex+and+hippocampus+in+representing+spatial+information.">Role of parietal cortex and hippocampus in representing spatial information.</a> Cereb Cortex. 1 (5), 367-373 (1991).</li><li>Barnes, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+deficits+associated+with+senescence:+a+neurophysiological+and+behavioral+study+in+the+rat.">Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat.</a> J Comp Physiol Psychol. 93 (1), 74-104 (1979).</li><li>Whishaw, I. Q., Cassel, J. C., Jarrad, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats+with+fimbria-fornix+lesions+display+a+place+response+in+a+swimming+pool:+a+dissociation+between+getting+there+and+knowing+where.">Rats with fimbria-fornix lesions display a place response in a swimming pool: a dissociation between getting there and knowing where.</a> J Neurosci. 15 (8), 5779-5788 (1995).</li><li>Staay, F. J., Raaijmakers, W. G., Lammers, A. J., Tonnaer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+fimbria+lesions+impair+acquisition+of+working+and+reference+memory+of+rats+in+a+complex+spatial+discrimination+task.">Selective fimbria lesions impair acquisition of working and reference memory of rats in a complex spatial discrimination task.</a> Behav Brain Res. 32 (2), 151-161 (1989).</li><li>Olton, R. S., Samuelson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remembrance+of+places+past:+Spatial+memory+in+rats.">Remembrance of places past: Spatial memory in rats.</a> J Exp Psych. 2 (2), 97-116 (1976).</li><li>Pol-Bodetto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+double-H+maze+test,+a+novel,+simple,+water-escape+memory+task:+acquisition,+recall+of+recent+and+remote+memory,+and+effects+of+systemic+muscarinic+or+NMDA+receptor+blockade+during+training.">The double-H maze test, a novel, simple, water-escape memory task: acquisition, recall of recent and remote memory, and effects of systemic muscarinic or NMDA receptor blockade during training.</a> Behav Brain Res. 218 (1), 138-151 (2011).</li><li>Cassel, R., Kelche, C., Lecourtier, L., Cassel, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+match/mismatch+of+visuo-spatial+cues+between+acquisition+and+retrieval+contexts+influences+the+expression+of+response+vs.+place+memory+in+rats.">The match/mismatch of visuo-spatial cues between acquisition and retrieval contexts influences the expression of response vs. place memory in rats.</a> Behavioural Brain Research. 230 (2), 333-342 (2012).</li><li>Schumacher, A., de Vasconcelos, A. P., Lecourtier, L., Moser, A., Cassel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+high+frequency+stimulation+in+the+dorsal+striatum:+Effects+on+response+learning+and+on+GABA+levels+in+rats.">Electrical high frequency stimulation in the dorsal striatum: Effects on response learning and on GABA levels in rats.</a> Behavioural Brain Research. 222 (2), 368-374 (2011).</li><li>Lecourtier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+neurobehavioral+development+and+dramatic+impairments+of+procedural-like+memory+following+neonatal+ventral+hippocampal+lesion+in+rats.">Intact neurobehavioral development and dramatic impairments of procedural-like memory following neonatal ventral hippocampal lesion in rats.</a> Neuroscience. 207, 110-123 (2012).</li><li>Kirch, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+deficits+in+declarative+and+procedural+memory+dependent+behavioral+function+in+a+transgenic+rat+model+of+Huntington's+disease.">Early deficits in declarative and procedural memory dependent behavioral function in a transgenic rat model of Huntington's disease.</a> Behav Brain Res. 239, 15-26 (2013).</li><li>Cholvin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventral+midline+thalamus+contributes+to+strategy+shifting+in+a+memory+task+requiring+both+prefrontal+cortical+and+hippocampal+functions.">The ventral midline thalamus contributes to strategy shifting in a memory task requiring both prefrontal cortical and hippocampal functions.</a> J Neurosci. 33 (20), 8772-8783 (2013).</li><li>Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Brain Res Brain Res Rev. 36 (1), 60-90 (2001).</li></ol>

The authors have nothing to disclose.

Comments on Study Design and Analysis
Since its conception, the double-H maze has been utilized in a number of behavioral experiments in rats, which collectively were designed to study egocentric and/or allocentric responses in rats under normal24,25 and altered26-29 brain states. The latter studies include striatal deep-brain stimulation (DBS)26, animal models of neurological disorders27,28, as well as bilateral deactivations of various cortico-hippoca...

In animals, learning is principally mediated by the hippocampal- and striatal-based memory systems1,2, which play central roles regarding place- and procedural-memory, respectively. The relationship between these two systems is complex, and they are known to interact with each other in cooperative or competitive manners1,3. In addition, studies have shown that the influence of either of these memory systems on animal behavior can increase following the absence or damage of the other system4-7. Both of these systems are connected to the prefrontal cortex via the thalamus.
Numerous neurological disorders and neurodegenerative diseases can affect spatial cognition in humans, which rely on the interplay between procedural and declarative memory systems. Examples include Parkinson&#8217;s disease (PD), Huntington&#8217;s disease (HD)8-10, Alzheimer&#8217;s disease (AD)11-14, as well as amyotrophic lateral sclerosis (ALS)15. Animal models, which are relevant to these disorders can be induced through various drug treatments which block certain receptors16, as well as through targeted lesions. When such animals are used with spatial memory tasks, a valuable insight can be gained into the underlying mechanisms related to these disorders, as well as to various treatment options.
There are many different types of spatial memory tasks in rodents, which collectively are designed to assess specific aspects of learning and memory, as well as the effects of potential treatments for various disorders17,18. These tasks can be distinguished by the number of goals and pathways, the degree of behavioral flexibility in solving the task, the memory duration or delay, as well as the choice of strategy used in solving the task. A good performance may be acquired based on external cues or landmarks which are used to orientate the animal towards the goal (an allocentric or place strategy). Alternatively, a rodent may develop a strategy which is based on bodily direction and cues with regards to the direction to move in (an egocentric or procedural strategy), e.g., if a rat knows that the goal is one left turn followed by one right turn, then there is little need for an allocentric or place strategy. Maze tasks often differ based on the degree of flexibility offered to the rodent in solving them. For instance, in the Morris Water Maze, a dry version of the latter (e.g., 19) or the Barnes maze (e.g., 20), there are potentially infinite routes the rat can take to reach the goal. In the Morris Water Maze, for example, the location of the goal may be learned based on external landmarks or cues (allocentric strategy), or by simply swimming in circles towards the center until the platform is found (egocentric strategy)21. Certain tasks have multiple goals and a high degree of flexibility, such as the cone-field task22 or Olton&#8217;s radial maze23. At the other end of the scale are tasks, which offer limited flexibility in reaching the goal, e.g., the Stone maze, or the alternating version of the T-maze. These tasks provide only one correct way of reaching the goal and facilitate the emergence of cognitive routines that are principally governed by the striatal-based procedural memory system.
The double-H maze is a novel spatial memory testing device, which was designed to allow the experimenter to direct the type of strategy that is learned by rodents in solving the task24. Consisting of three parallel run arms intersected by a perpendicular central alleyway, the double-H maze is a water-escape task in which rodents learn to reach an escape platform that is immersed in one of the maze locations. During training, a procedural strategy can be developed by maintaining the same start and goal locations throughout. Alternatively, an allocentric strategy may be developed by alternating the starting location in a random order, thus requiring the rat to learn the location of the hidden platform based on environmental cues as it has to do in a water maze. This overcomes an obstacle present in many different maze tasks, in which the experimenter otherwise has little control over the type of strategy that rodents utilize. This is important when considering that the effects of certain cognition-enhancing drug candidates rely on the hippocampal-based place-memory system, thus the emergence of cognitive routines or procedures may confound the interpretation of the behavioral observations when animals, for example switch from allocentric to procedural memory during the course of training. Similarly, it may be desirable to assess the effects of drugs and treatments on procedural memory, without the influence of allocentric place-based memory. Finally, this device can be utilized to study the cooperative or competitive interactions between these memory systems, and the conditions under which rodents may switch from one system to another.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Rats or Mice</td>
			<td></td>
			<td>Charles River</td>
			<td></td>
		</tr>
		<tr>
			<td>Towels for drying</td>
			<td></td>
			<td>University Hospital</td>
			<td>1 / animal</td>
		</tr>
		<tr>
			<td>Water</td>
			<td></td>
			<td></td>
			<td>~200 L / day</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td></td>
			<td>Grocery store</td>
			<td>250 g / 200 L water</td>
		</tr>
		<tr>
			<td>Garden Hose</td>
			<td></td>
			<td>Hardware store</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying rack for towels</td>
			<td></td>
			<td>Hardware store</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinect camera</td>
			<td></td>
			<td>Kinect</td>
			<td></td>
		</tr>
		<tr>
			<td>PC computer</td>
			<td></td>
			<td>any</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Double H Maze, (plexiglass) (Custom-Built)</td>
		</tr>
		<tr>
			<td>External lateral walls, 1600 &#215; 350 &#215; 6 mm</td>
			<td></td>
			<td align="right">2</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal lateral walls, 706 &#215; 350 &#215; 6 mm</td>
			<td></td>
			<td align="right">8</td>
			<td></td>
		</tr>
		<tr>
			<td>Central corridor panels, 500 &#215; 350 &#215; 6 mm</td>
			<td></td>
			<td align="right">4</td>
			<td></td>
		</tr>
		<tr>
			<td>Arm extremities, 188 &#215; 350 &#215; 6 mm</td>
			<td></td>
			<td align="right">6</td>
			<td></td>
		</tr>
		<tr>
			<td>Guillotine doors, 187 &#215; 350 &#215; 6 mm</td>
			<td></td>
			<td align="right">3</td>
			<td></td>
		</tr>
		<tr>
			<td>Extremity covers, 200 &#215; 250 mm</td>
			<td></td>
			<td align="right">6</td>
			<td></td>
		</tr>
		<tr>
			<td>Crossbars, 200 &#215; 40 mm</td>
			<td></td>
			<td align="right">6</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Double-H Maze Platform (to be ballasted)</td>
			<td></td>
			<td></td>
			<td>Double-H Maze Platform (to be ballasted) (Custom-Built)</td>
		</tr>
		<tr>
			<td>Metal platform, 100 mm diameter &#215; 150 mm</td>
			<td></td>
			<td align="right">2</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform cover, 100 mm diameter &#215; 6mm</td>
			<td></td>
			<td align="right">2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the double-h maze: a robust behavioral test for learning and memory in rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

Egocentric Learning Strategy
A study was carried out to determine whether the chosen memory strategy in rats changes based on alterations of their perspective of external environmental cues, following an egocentric-learning paradigm25. Rats were trained over 4 days (4 trials/day) to reach a goal arm located at NE, and were subsequently tested on the fifth day using a misleading probe trial, in which the start arm was either moved 60 cm to the left (i.e., NE start for animals r...

Watch this Scientific Journal Video about The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents at JoVE.com

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

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12.4K Views.  University Hospital Freiburg. The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Video: The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.

This video shows the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression, defensive behavior and violence in a semi-natural setting. The use of the paradigm for social stress experiments is explained as well.

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

A Real-world What-Where-When Memory Test

Episodic memory is a complex memory system which allows recall and mental re-experience of previous episodes from one&#39;s own life. Real-life episodic memories are about events in their spatiotemporal context and are typically visuospatial, rather than verbal. Yet often, tests of episodic memory use verbal material to be recalled (word lists, stories). The Real-World What-Where-When memory test requires participants to hide a total of 16 different objects in 16 different locations over two temporal occasions, 2 h apart. Another two hours later, they are then asked to recall which objects (What) they had hidden in which locations (Where) and on which of the two occasions (When). In addition to counting the number of correctly recalled complete what-where-when combinations, this task can also be used to test real-world spatial memory and object memory. This task is sensitive to normal cognitive aging, and correlates well with performance on other episodic memory tasks, while at the same time providing more ecological validity and being cheap and easy to run.

A Real-world What-Where-When Memory Test

The Real-World What-Where-When memory test is a novel episodic memory test, in which participants need to recall which objects have been hidden in which locations on which of two distinct occasions. It is easy to run and is sensitive to normal cognitive aging.

Episodic memory is a complex memory system which allows recall and mental re-experience of previous episodes from one&#39;s own life. Real-life episodic ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in  Free Moving Rodents

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper understanding of the neurobiological causes underlying the decision-making problems. A cost-benefit based T-maze task is used for measuring decision making in which rodents choose between a high reward arm (HRA) and a low reward arm (LRA). There are two paradigms of the T-maze decision-making task, one in which the cost is a time delay and the other in which it is physical effort. Both paradigms require a tedious and labor-intensive management of experimental animals, multiple doors, pellet reward, and arm choice recordings. In the current work, we invented an apparatus based on traditional T-maze with full automation for pellet delivery, door management and choice recordings. This automated setup can be used for the evaluation of both delay- and effort-based decision making in rodents. With the protocol described here, our lab investigated the decision-making phenotypes of multiple genetically modified mice. In the representative data, we showed that the mice with ablated medial habenular showed aversions of both delay and effort and tended to choose the immediate and effortless reward. This protocol helps to decrease the variability caused by experimenter intervention and to enhance experiment efficiency. In addition, chronic silicon probe or microelectrode recording, fiber-optic imaging and/or manipulation of neural activity can be easily applied during the decision-making task using the setup described here.

This article introduces an automated T-maze apparatus that we invented, and a protocol based on this apparatus for analyzing delay-based decision making and effort-based decision making in free moving rodents.

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...