Name: shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish
Uploaded: 2021-07-12T13:00:00
Description: Watch this Scientific Journal Video about Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish at JoVE.com

The authors would like to thank the Hyde lab members for their thoughtful discussions and the Freimann Life Sciences Center technicians for zebrafish care and husbandry. This work was supported by the Center for Zebrafish Research at the University of Notre Dame, the Center for Stem Cells and Regenerative Medicine at the University of Notre Dame, and grants from National Eye Institute of NIH R01-EY018417 (DRH), the National Science Foundation Graduate Research Fellowship Program (JTH), LTC Neil Hyland Fellowship of Notre Dame (JTH), Sentinels of Freedom Fellowship (JTH), and the Pat Tillman Scholarship (JTH). Figure 1 made with BioRender.com.

Zebrafish were raised and maintained in the Notre Dame Zebrafish facility in the Freimann Life Sciences Center. The methods described in this manuscript were approved by the University of Notre Dame Animal Care and Use Committee (Animal Welfare Assurance Number A3093-01).
1. Shuttle box learning paradigm (Figure 1A)
NOTE: The learning paradigm provides a rapid assessment of cognition regarding associative learning.
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	<li>Deutsch, M., Mendez, M., Teng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+traumatic+brain+injury+and+frontotemporal+degeneration.">Interactions between traumatic brain injury and frontotemporal degeneration.</a> Dementia and Geriatric Cognitive Disorders. 39, 143-153 (2015).</li><li>Gardner, R., 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=Traumatic+brain+injury+in+later+life+increases+risk+for+Parkinson+disease.">Traumatic brain injury in later life increases risk for Parkinson disease.</a> Annals in Neurology. 77, 987 (2015).</li><li>Fleminger, S., Oliver, D., Lovestone, S., Rabe-Hesketh, S., Giora, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head+injury+as+a+risk+factor+for+Alzheimer's+disease:+the+evidence+10+years+on;+a+partial+replication.">Head injury as a risk factor for Alzheimer's disease: the evidence 10 years on; a partial replication.</a> Journal of Neurology, Neurosurgery and Psychiatry. 74, 857-886 (2003).</li><li>Johnson, V., Stewart, W., Smith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury+and+amyloid-&#946;+pathology:+a+link+to+Alzheimer's+disease.">Traumatic brain injury and amyloid-&#946; pathology: a link to Alzheimer's disease.</a> Nature Reviews Neurosciences. 11, 361-370 (2010).</li><li>Korley, F. K., Kelen, G. D., Jones, C. M., Diaz-Arrastia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergency+department+evaluation+of+traumatic+brain+injury+in+the+United+States,+2009-2010.">Emergency department evaluation of traumatic brain injury in the United States, 2009-2010.</a> The Journal of Head Trauma Rehabilitation. 31, 379-387 (2016).</li><li>Corrigan, J. D., Selassie, A. W., Orman, J. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+traumatic+brain+injury.">The epidemiology of traumatic brain injury.</a> The Journal of Head Trauma Rehabilitation. 25, 72-80 (2010).</li><li>Levin, H., Arrastia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+prognosis,+and+clinical+management+of+mild+traumatic+brain+injury.">Diagnosis, prognosis, and clinical management of mild traumatic brain injury.</a> The Lancet Neurology. 14, 506-517 (2015).</li><li>GBD 2016 Traumatic Brain Injury and Spinal Cord Injury Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+and+national+burden+of+traumatic+brain+injury+and+spinal+cord+injury,+1990-2016:+A+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2016.">Global, regional, and national burden of traumatic brain injury and spinal cord injury, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016.</a> The Lancet, Neurology. 18 (1), 56-87 (2019).</li><li>Campbell, L. J., 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=Notch3+and+DeltaB+maintain+M&#252;ller+glia+quiescence+and+act+as+negative+regulators+of+regeneration+in+the+light-damaged+zebrafish+retina.">Notch3 and DeltaB maintain M&#252;ller glia quiescence and act as negative regulators of regeneration in the light-damaged zebrafish retina.</a> Glia. 69 (3), 546-566 (2021).</li><li>Green, L. A., Nebiolo, J. C., Smith, 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=Microglia+exit+the+CNS+in+spinal+root+avulsion.">Microglia exit the CNS in spinal root avulsion.</a> PLoS Biology. 17 (2), 3000159 (2019).</li><li>Hentig, J., Byrd-Jacobs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+zinc+sulfate+results+in+differential+effects+on+olfactory+sensory+neuron+subtypes+in+the+adult+zebrafish.">Exposure to zinc sulfate results in differential effects on olfactory sensory neuron subtypes in the adult zebrafish.</a> International Journal of Molecular Sciences. 17 (9), 1445 (2016).</li><li>Ito, Y., Tanaka, H., Okamoto, H., Oshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neural+stem+cells+and+their+progeny+in+the+adult+zebrafish+optic+tectum.">Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum.</a> Developmental Biology. 342, 26-38 (2010).</li><li>Lahne, M., Nagashima, M., Hyde, D. R., Hitchcock, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+Muller+glia+to+regenerate+retinal+neurons.">Reprogramming Muller glia to regenerate retinal neurons.</a> Annual Reviews of Vision Sciences. 6, 171-193 (2020).</li><li>Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+adult+zebrafish+brain+from+neurogenic+radial+glia-type+progenitors.">Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.</a> Development. 138 (22), 4831-4841 (2011).</li><li>Kishimoto, N., Shimizu, K., Sawamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regeneration+in+a+zebrafish+model+of+adult+brain+injury.">Neuronal regeneration in a zebrafish model of adult brain injury.</a> Disease Models &amp; Mechanisms. 5 (2), 200-209 (2012).</li><li>Bhattarai, P., 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=Neuron-glia+interaction+through+Serotonin-BDNF-NGFR+axis+enables+regenerative+neurogenesis+in+Alzheimer's+model+of+adult+zebrafish+brain.">Neuron-glia interaction through Serotonin-BDNF-NGFR axis enables regenerative neurogenesis in Alzheimer's model of adult zebrafish brain.</a> PLoS Biology. 18 (1), 3000585 (2020).</li><li>Kalueff, 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=Towards+a+comprehensive+catalog+of+zebrafish+behavior+1.0+and+beyond.">Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.</a> Zebrafish. 10 (1), 70-86 (2013).</li><li>Chanin, 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=Assessing+startle+responses+and+their+habituation+in+adult+zebrafish.">Assessing startle responses and their habituation in adult zebrafish.</a> Zebrafish Protocols for Neurobehavioral Research. 66, (2012).</li><li>L&#243;pez-Schier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroplasticity+in+the+acoustic+startle+reflex+in+larval+zebrafish.">Neuroplasticity in the acoustic startle reflex in larval zebrafish.</a> Current Opinion in Neurobiology. 54, 134-139 (2019).</li><li>Maheras, A. 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=Genetic+pathways+of+neuroregeneration+in+a+novel+mild+traumatic+brain+injury+model+in+adult+zebrafish.">Genetic pathways of neuroregeneration in a novel mild traumatic brain injury model in adult zebrafish.</a> eNeuro. 5 (1), (2018).</li><li>Gaspary, K. V., Reolon, G. K., Gusso, D., Bonan, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+object+recognition+and+object+location+tasks+in+zebrafish:+Influence+of+habituation+and+NMDA+receptor+antagonism.">Novel object recognition and object location tasks in zebrafish: Influence of habituation and NMDA receptor antagonism.</a> Neurobiology of Learning and Memory. 155, 249-260 (2018).</li><li>Hentig, J., Cloghessy, K., Dunseath, C., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scalable+model+to+study+the+effects+of+blunt-force+injury+in+adult+zebrafish.">A scalable model to study the effects of blunt-force injury in adult zebrafish.</a> Journal of Visualized Experiments. , (2021).</li><li>Wu, Y. J., 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=Fragile+X+mental+retardation-1+knockout+zebrafish+shows+precocious+development+in+social+behavior.">Fragile X mental retardation-1 knockout zebrafish shows precocious development in social behavior.</a> Zebrafish. 14 (5), 438-443 (2017).</li><li>Rea, V., Van Raay, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+zebrafish+to+model+autism+spectrum+disorder:+A+Comparison+of+ASD+risk+genes+between+zebrafish+and+their+mammalian+counterparts.">Using zebrafish to model autism spectrum disorder: A Comparison of ASD risk genes between zebrafish and their mammalian counterparts.</a> Frontiers in Molecular Neuroscience. 13, 575575 (2020).</li><li>Zhdanova, I. V., 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=Aging+of+the+circadian+system+in+zebrafish+and+the+effects+of+melatonin+on+sleep+and+cognitive+performance.">Aging of the circadian system in zebrafish and the effects of melatonin on sleep and cognitive performance.</a> Brain Research Bulletin. 75 (2-4), 433-441 (2008).</li><li>Yu, L., Tucci, V., Kishi, S., Zhdanova, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+aging+in+zebrafish.">Cognitive aging in zebrafish.</a> PloS One. 1 (1), 14 (2006).</li><li>Bahl, A., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+for+evidence+accumulation+and+decision+making+in+larval+zebrafish.">Neural circuits for evidence accumulation and decision making in larval zebrafish.</a> Nature Neuroscience. 23 (1), 94-102 (2020).</li><li>Ngoc Hieu, B. 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=Development+of+a+modified+three-day+t-maze+protocol+for+evaluating+learning+and+memory+capacity+of+adult+zebrafish.">Development of a modified three-day t-maze protocol for evaluating learning and memory capacity of adult zebrafish.</a> International Journal of Molecular Sciences. 21 (4), 1464 (2020).</li><li>Williams, F. E., White, D., Messer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+spatial+alternation+task+for+assessing+memory+function+in+zebrafish.">A simple spatial alternation task for assessing memory function in zebrafish.</a> Behavioural Processes. 58 (3), 125-132 (2002).</li><li>Zohar, O., 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=Closed-head+minimal+traumatic+brain+injury+produces+long-term+cognitive+deficits+in+mice.">Closed-head minimal traumatic brain injury produces long-term cognitive deficits in mice.</a> Neuroscience. 118 (4), 949-955 (2003).</li><li>Becker, C., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+as+a+model+for+successful+central+nervous+system+regeneration.">Adult zebrafish as a model for successful central nervous system regeneration.</a> Restorative Neurology and Neuroscience. 26 (2-3), 71-80 (2008).</li><li>Grandel, H., Kaslin, J., Ganz, J., Wenzel, I., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurogenesis+in+the+adult+zebrafish+brain:+origin,+proliferation+dynamics,+migration,+and+cell+fate.">Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration, and cell fate.</a> Developmental Biology. 295 (1), 263-277 (2006).</li></ol>

Cognitive impairment can significantly and negatively impact the quality of life. Because of the increased visibility and occurrence of concussions and traumatic brain injuries throughout the population, it is important to understand how they cause cognitive impairment and how the damage can be minimized or reversed. For these reasons, model organisms that can be tested for cognitive decline play a critical role in these studies. Rodents have long been the primary model to investigate neurobehavior and cognition, however...

Learning and memory are routinely used as metrics of cognitive impairment, which happens due to aging, neurodegenerative disease, or injury. Traumatic brain injuries (TBIs) are the most common injury that results in cognitive deficits. TBIs are of growing concern because of their association with several neurodegenerative disorders, such as frontotemporal dementia and Parkinson&#39;s disease1,2. In addition, the increased beta-amyloid aggregations observed in some TBI patients suggest that it may also be associated with the development of Alzheimer&#39;s disease3,4. TBIs are often the result of blunt-force trauma and span a range of severities5, with mild brain injuries (miTBI) being the most common. However, miTBIs are often unreported and misdiagnosed because they result in minor cognitive impairments for only a short period, and the injured individuals usually recover fully6. In contrast, repeated miTBI events have been a growing concern because it is highly prevalent in young and middle-aged adults, can accumulate over time7, can impair cognitive development, and exacerbate neurodegenerative diseases1,2,3,4,5, similar to individuals who experience either a moderate or severe TBI8.
Zebrafish (Danio rerio) is a useful model for exploring a variety of topics in neuroscience, including the ability to regenerate lost or damaged neurons throughout the central nervous system9,10,11,12,13. Neural regeneration was also demonstrated in the telencephalon, which contains the archipallium in the dorsal-inner region. This neuroanatomical region is analogous to the hippocampus and is likely required for cognition in fish and for the short-time memory in humans14,15,16. Furthermore, zebrafish behavior has been extensively characterized and cataloged17. Learning has been studied through various techniques, including habituation to the startle response18, which can represent a rapid form of non-associative learning when performed in short blocks and with attention to the rapid decay time19. More complex tests of associative learning, such as T-boxes, plus-mazes, and visual discrimination20,21 are used but often are time-consuming, require days or weeks of preparation, and rely on shoaling or positive reinforcement. Here, we describe a rapid paradigm to assess both associative learning and either immediate or delayed memory. This shuttle box assay uses an aversive stimulus and negative reinforcement conditioning to assess cognitive deficits and recovery following blunt-force TBI. We demonstrate that undamaged control adult zebrafish (8-24 months) reproducibly learn to avoid the red light within 20 trials (&#60;20 min of assessment) in the shuttle box, with a high degree of consistency across observers. Additionally, using the shuttle box we demonstrate that learning and memory abilities across adult (8-24 months old) are consistent and are useful for assaying cognition with significant impairments between either different TBI severities or repeated TBI. Furthermore, this method could be rapidly employed as a metric to track a wide range of disease progressions or efficacy of drug interventions impacting maintenance or recovery of cognition in adult zebrafish.
Here, we provide an instructional overview of a rapid cognitive assessment that can examine both complex associative learning (section 1) and memory in terms of both immediate and delayed memory.This paradigm provides an assessment of the short and long-term memory of a learned associative cognitive task (section 2).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Flashlight</td>
			<td>Ultrafire</td>
			<td>9145</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Large DNA Gel Box</td>
			<td>Fisher Scientific</td>
			<td>FB-SB-1316</td>
			<td>Shuttle Box</td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>Fisher Scientific</td>
			<td>FB-105</td>
			<td></td>
		</tr>
	</tbody>
</table>

shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

The learning paradigm, outlined in the protocol and schematic (Figure 1), provides a rapid assessment of cognition with respect to associative learning. In addition, this paradigm has a high level of stringency, by defining learning as a repeated and consistent display of 5 consecutive positive trials. This paradigm is also applicable to a range of ages and injuries. Undamaged fish at 8 months (young adult), 18 months (middle-aged adult), and 24 months (elderly adult) required a similar numb...

Watch this Scientific Journal Video about Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish at JoVE.com

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

The Shuttle Box Assay reproducibly tracks the progression and recovery of cognitive impairment following a blunt force trauma or any other type of brain injury in zebrafish. The Shuttle Box Assay allows for relatively simple, rapid and robust measurement of associative learning in both short and long-term memory in zebrafish. To begin, prepare the Shuttle Box by modifying a 30.5 by 19 by 7.5 centimeter gel box with a 5 by 19 centimeter piece of aquarium-grade plexiglass added to each side at a 45 degree angle.Then make a line marking the halfway point of the tank to assess when fish have crossed the middle of the tank. After adding 800 milliliters of system water to the Shuttle Box, place two to three fish into a holding tank containing system water. Leave the tank in the dark room where the Shuttle Box Assay will be performed.Next, in the dark room, place one fish in the center of the Shuttle Box. Secure the lid and attach the electrodes to a power supply. The fish should be acclimated in the dark.Turn off all lights and acclimate the fish to the Shuttle Box for 15 minutes. Successful acclimation can be considered when the fish freely explores the tank. After successful acclimation of the fish, manually shine an 800 lumen red lens flashlight approximately two centimeters from the gel box wall on the side occupied by the fish.Do not start a trial if the fish is resting next to the platinum wire. Shine the light stimulus directly on the fish and manually follow any lateral movement of the fish with the light to ensure continual visualization of the stimulus. Continue to provide the light stimulus until either of the following conditions for a successful or failed trial are met.Consider the trial successful if the fish crosses over the halfway point of the tank within 15 seconds of light exposure. Once the fish crosses the halfway point, stop the light stimulus immediately. If the trial failed, use an electrophoresis power supply to apply a negative shock stimulus of 20 millivolts to one ampere, alternating two seconds on, two seconds off for 15 seconds with a maximum of four shocks or until the fish passes the halfway point of the box.Then terminate both the light and negative stimulus. Prior to the training period, ensure a successful acclimation to the Shuttle Box by leaving the fish under dark conditions for 15 minutes. Then manually shine an 800 lumen red lens flashlight approximately two centimeters from the gel box wall on the side occupied by the fish.Shine the light stimulus directly on the fish and follow any lateral movement of the fish with the light to ensure continual visualization of the stimulus. While the light is shining on the fish, simultaneously apply the adverse shock stimulus of 20 millivolts to one ampere, alternating two seconds on, two seconds off for 15 seconds with a maximum of four shocks or until the fish passes the halfway point of the box. Once this is achieved, terminate the light and adverse stimulus.Repeat for 25 iterations. Upon completion, allow the fish another acclimation period of 15 minutes under dark conditions. During the initial testing period, perform a successful acclimatization in the Shuttle Box for 15 minutes, then apply only the light stimulus for up to 15 seconds and record the responses.Consider the trial successful if the fish crosses over the halfway point of the Shuttle Box within 15 seconds after starting the light stimulus and stop the light stimulus immediately when the fish crosses the halfway point. Consider the trial as failed if the fish does not cross over the halfway point of the Shuttle Box 15 seconds after starting the light stimulus and stop the light stimulus after 15 seconds. During the initial testing, do not apply an adverse stimulus following a failed attempt.Perform short-term memory testing immediately following the initial testing period. Induce dramatic brain injury and wait for a period of four hours before testing. Then acclimate the fish in the Shuttle Box for 15 minutes.Assess short-term memory by applying only the light stimulus for up to 15 seconds and record if the fish crosses over the halfway point of the box before the light is turned off, considered as passed trial, or fails to cross the halfway point within the 15 seconds considered as failed trial. Repeat the above step 25 times with a 30-second rest period between each trial and record the number of successful and failed trials. Perform long-term memory testing four days after initial testing.Induce traumatic brain injury and wait for a period of four hours before testing, then acclimate the fish in the Shuttle Box for 15 minutes. Assess long-term memory by applying only the light stimulus for up to 15 seconds and record if the fish crosses over the halfway point of the box before the light is turned off, considered as passed trial, or fails to cross the halfway point within the 15 seconds considered as failed trial. Repeat the above 25 times with a 30-second rest period between each trial and record the number of successful and failed trials.The instructional overview of the learning and memory paradigms for cognitive assessment is shown here. The undamaged fish at eight months, young adult, 18 months, middle-aged adult, and 24 months, elderly adult, required a similar number of trials to learn the behavior of avoiding the red light. After utilizing the severe blunt force traumatic brain injury model, or STBI, the fish at different ages required a similar number of trials to master the assay across one to five days post-injury.On day one following STBI, fish of all ages required a similar number of trials to learn the behavior, which was significantly greater than the undamaged controls. The undamaged fish rapidly mastered the Shuttle Box, achieving five consecutive positive trials in approximately 17 trials whereas one day following a mild brain injury, or a mild TBI, fish display a significant increase in the number of trials to learn the behavior. This deficit increased after two mild TBI and was further elevated after three mild TBI injuries.Undamaged fish exhibit a slight increase in the percent difference of successful trials in immediate memory and delayed memory relative to the initial testing period. Following a single mild TBI, fish displayed significant and immediate memory deficits compared to undamaged fish. This trend continued with repeated injury with increasing deficits following both two mild TBI and three mild TBI.The timing of applied adverse stimuli is critical. Pairing the adverse stimuli with the light and their simultaneous removal solidifies their association and is crucial for the paradigm. The assay allows for rapid assessment or complex associative learning that can be used to investigate developmental, aging, and environmental impacts on cognitive impairment.

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Research

JoVE Journal

Neuroscience

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the concurrent brain activity of two or more interactive individuals with a novel index called inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is presented here. Further, a pipeline of analyzing IBS of oxygenated hemoglobin (Oxy-Hb) signal is explained. Specifically, the experimental design, the process of NIRS data recording, data analysis methods, and future directions are all discussed. Overall, implementing a standardized fNIRS hyperscanning pipeline is a fundamental part of second-person neuroscience. Also, this is in line with the call for open-science to aid the reproducibility of research.

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.