The Lashley III maze used for these experiments was built by Tim Bowmaster, Paul Corman, Barry Dutrow, Ryan Jabco, and Tim Treaster from the Pennsylvania State University Physics Department Machine Shop. The authors would like to thank Ms. Shoba Belegundu, Ms. Tara Chrzanowski, and Ms. Alexandra Lewis for assistance in performing the experiments presented in Figures 2-4. Mr. Walter Jackson provided valuable feedback in the preparation of this protocol. This work was supported by funding from the National Institute of Mental Health (MH064756 and MH077978 to AMA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institutes of Health.

1. Preparation:
<ol>
	<li>The Lashley III maze is constructed of Plexiglas and is shown in Figure 1. The start box, maze arms, and goal box are modular to allow for cleaning and modifiability of the maze. The walls of the start box, maze arms, and goal box are constructed of black Plexiglas to eliminate visual cues from outside and inside the maze. Maze arms are 45 cm long, 7 cm high, and 5 cm wide. Doors leading to consecutive arms of the maze are 4 cm x 4 cm and are situated 11 cm from the outer walls of the maze. The goal box is 19.5 cm long x 7 cm high x 5 cm wide. The start box is 8 cm x 9.5 cm x 7 cm. Separate clear Plexiglas pieces are placed on top of the start box, maze, and goal box to allow visualization of each mouse as it progresses through the maze while preventing mice from escaping. The maze rests on a base constructed of red Plexiglas that is large enough to hold the three pieces of the maze plus a pseudo-home cage that is the same size as the animal's standard home cage.</li>
	<li>To prepare pseudo-home cages prior to the first maze trial, fresh bedding material of the same kind as that used in the animals' home cages is added to individual cage bottoms having doorways (2 cm x 2 cm) cut into the sides. Place wire tops on these cage bottoms and individually number them, one for each animal in a testing cohort. These cages will serve as individual pseudo-home cages for each animal for the duration of the testing timeframe. Because we are investigating the roles of stress and/or anxiety-related behaviors in learning and memory, we use pseudo-home cages to avoid individually housing mice in their home cages. However, if these factors are not of primary concern, animals can be singly housed in home cages modified with doorways and these cages can serve as the home cages for successful maze navigation.</li>
</ol>
2. Testing:
<ol>
	<li>Each day, animals are moved to the behavior testing room a minimum of one hour before testing to allow them to acclimate to the testing environment. </li>
	<li>Thirty min before the testing start time, food and water is removed from all home cages. Keep the food and water in the behavior testing room but out of reach of the mice. Food and water is typically removed 30 min after the change from the light phase to the dark phase, after peak feeding has occurred. This ensures that animals are mostly food- and water -satiated prior to testing; however, testing during this period provides a small additional motivating force to navigate the maze even though there is no overt food or water reward upon task completion.</li>
	<li>After the 30 min pretest period has elapsed, clean the maze with 70% ethanol. Leave the door leading from the start box into the maze arms closed. The doors at both ends of the goal box are left open.</li>
	<li>Place the pseudo-home cage corresponding to the first animal at the end of the goal box.</li>
	<li>Carry the first mouse to the maze, cupping it in your hand. Put the mouse in the start box and replace the lid.</li>
	<li>When in position to observe the maze without obstruction, open the start box door and immediately start three stopwatches.</li>
	<li>When all four paws of the mouse have left the start box, close the start box door and stop the first timer.</li>
	<li>Record by hand the pathway the mouse travels as it navigates the maze. A mouse enters a new zone or arm of the maze when all four paws have crossed into that area (See Fig. 1).</li>
	<li>When all four paws enter the goal box, close the door leading between the maze and the goal box and stop the second stopwatch.</li>
	<li>When the mouse has completely entered the pseudo-home cage, close the door between the goal box and the cage. Stop the third stopwatch.</li>
	<li>Record the times from the three stopwatches. Count the number of errors the mouse has made. An error is defined as an entry into a dead-end cul-de-sac zone (e.g., going from arm L to zone K; Fig. 1) or travelling back through an already-travelled arm of the maze (e.g., going from arm F to arm C; Fig. 1). While we score mouse performance as it is occurring, it is possible to videotape or otherwise record maze trials to allow for subsequent scoring either by eye or by automated software. Experimenter discretion should be used to choose an appropriate method.</li>
	<li>Leave the animal in the pseudo-home cage for 1 min before returning it to its home cage.</li>
	<li>Repeat steps 2.3 to 2.12 for all mice in the testing cohort.</li>
	<li>When the last mouse in the cohort has been returned to its home cage, start the 30 min timer. At the end of this 30 min period, return food and water to all mice in the cohort and then return the animals to their colony room.</li>
	<li>Repeat this procedure on consecutive nights, with one trial per testing day per animal, until all mice in the cohort have reached a defined learning criteria or until a predetermined number of test trials has occurred. A mouse is classified as having learned the maze when it can perform the task with 0 or 1 error(s) on two consecutive trials. Mice are tested in the same order on successive trials.</li>
</ol>
3. Representative Results:
Days to learn (reach criterion) can be analyzed and compared between different groups in a particular study (Figs. 2A and 3A). Additionally, a learning index (learning ratio), which characterizes maze acquisition, can be evaluated over the first four days of the testing period (Figs. 2B and 3B). The learning index is the ratio of the number of correct path segments travelled to the total number of path segments travelled for each trial. The average learning index should be approximately 0.5 for trial 1, when mice are first exposed to the maze and navigating by chance. The learning index increases and approaches 1 between trials 2 and 4 indicating that learning is occurring. In Figure 2, young (2 mo) and aging (24 mo) male C57Bl/6NCr mice were trained on the Lashley maze for up to 15 trials. The young mice learned the maze in 7.2 (&plusmn; 1.5) d. By contrast, the aging animals took 11.7 (&plusmn; 2.1) d to reach criterion. There was a strong trend for aging C57Bl/6NCr mice to take longer to learn the maze ([t(23)=1.59, P=0.056]; one-tailed t-test, a priori hypothesis that days to learn is longer in aging mice). When the learning index is calculated, both age groups show a steady increase in the learning index from trials 2 through 4 (significant main effect of time P&lt;0.001). However, there were no statistically significant differences in the learning index between young C57 mice and old C57 mice (main effect of age P=0.58). 
Figure 3 is representative of studies examining the effects of background strain on learning behavior. Data from the young male C57Bl/6NCr shown in Fig. 2 are now compared to data from young Crl:CD-1(ICR)BR mice [CD-1, 4-5 mo]. CD-1 mice learned the Lashley III maze in 4.5 (&plusmn; 1.4) d; however, there was no significant difference between the strains in days to learn (Fig. 3A; [t(18)=1.328, P=0.20]; two-tailed t-test). By contrast, examination of the learning index shows that, while all animals were navigating the maze on the first trial by chance, CD-1 mice learned the task more quickly (main effect of strain P&lt;0.001). This is illustrated by the statistically significant increase in the learning index in trials 2 through 4 in CD-1 mice compared to C57Bl/6NCr mice. Interestingly, if CD-1 mice continue to be trained in the maze beyond the point of reaching criterion, they exhibit behavior indicative of overtraining (Fig. 4) which is discussed in more detail below.
<img src="/files/ftp_upload/1786/1786fig1.jpg" alt="Figure 1" /> Figure 1: The Lashley III maze. The dead-end cul-de-sac zones of the maze are colored red in this diagram for illustrative purposes only. The base and arms of the actual maze are solid colors (inset). The maze is drawn to scale (dimensions in text).
<img src="/files/ftp_upload/1786/1786fig2.jpg" alt="Figure 2" /> Figure 2: Lashley III maze performance in 2 and 24 month-old male C57Bl/6NCr mice. The Lashley III maze was used to assess route learning in young (2 mo) and aging (24 mo) male C57Bl/6NCr mice. The parameters measured were (a) days to criterion, the number of days required for a mouse to run the maze for two consecutive nights with 0 errors or 1 error; and (b) learning index, the number of correct four-paw arm entries made versus the total number of arm entries made on days 1-4 of Lashley III maze testing. Numbers of animals per group were n=10 for 2 month-old and n=15 for 24 month-old mice. 
<img src="/files/ftp_upload/1786/1786fig3.jpg" alt="Figure 3" /> Figure 3: Lashley III maze performance in young male C57Bl/6NCr and CD-1 mice. The Lashley III maze was used to assess route learning in 2 month-old C57Bl/6NCr and 4-5 month-old CD-1 mice . The parameters measured were (a) days to criterion, the number of days required for a mouse to run the maze for two consecutive nights with 0 errors or 1 error; and (b) learning index, the number of correct four-paw arm entries made versus the total number of arm entries made on days 1-4 of Lashley III maze testing. Numbers of animals per group were n=10 for C57Bl/6NCr and n=10 for CD-1 mice. Statistical significances: *P&lt;0.05, **P&lt;0.01 versus C57Bl/6NCr mice.
<img src="/files/ftp_upload/1786/1786fig4.jpg" alt="Figure 4" /> Figure 4: Lashley III maze performance in 4-5 month-old male CD-1 mice. The Lashley III maze was used to assess route learning in ten 4-5 month-old male CD-1 mice on 15 consecutive days. The parameters measured were (a) number of errors made per day, with an error defined as a four-paw entry into a dead-end cul-de-sac zone or travelling an already-travelled arm of the maze; and (b) time spent in the maze per day. The day at which criterion was reached is indicated with a red arrow.

<ol>
	<li>Blizard, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Return+to+home+cage'+as+a+reward+for+maze+learning+in+young+and+old+genetically+heterogeneous+mice.">Return to home cage' as a reward for maze learning in young and old genetically heterogeneous mice.</a> Comparative Med. 56, 196-201 (2006).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's wrong with my mouse?: Behavioral phenotyping of transgenic and knockout mice. , (2007).</li><li>Blizard, D. A., Klein, L. C., Cohen, R., McClearn, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse-friendly+cognitive+task+suitable+for+use+in+aging+studies.">A novel mouse-friendly cognitive task suitable for use in aging studies.</a> Behav. Genet. 33, 181-189 (2003).</li><li>Warren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetitive+learning+by+old+mice.">Appetitive learning by old mice.</a> Exp. Aging Res. 12, 99-105 (1986).</li><li>Lashley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+mechanisms+and+intelligence:+A+quantitative+study+of+injuries+to+the+brain.">Brain mechanisms and intelligence: A quantitative study of injuries to the brain.</a> , (1929).</li><li>Barnes, C. <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. Psych. 93, 74-104 (1979).</li><li>Matzel, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+differences+in+the+expression+of+a+general+learning+ability+in+mice.">Individual differences in the expression of a general learning ability in mice.</a> J. Neurosci. 23, 6423-6433 (2003).</li><li>Brandeis, R., Brandys, Y., Yehuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Morris+water+maze+in+the+study+of+memory+and+learning.">The use of the Morris water maze in the study of memory and learning.</a> Int. J. Neurosci. 48, 29-69 (1989).</li><li>Hepler, D. J., Wenk, G. L., Cribbs, B. L., Olton, D. S., Coyle, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+impairments+following+basal+forebrain+lesions.">Memory impairments following basal forebrain lesions.</a> Brain Res. 346, 8-14 (1985).</li><li>Sci, B. e. h. a. v. B. r. a. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2, 313-313 (1979).</li></ol>

All behavior protocols were approved by the Pennsylvania State University Institutional Animal Care and Use Committee. There are no financial disclosures to report.

The most commonly used rodent behavioral tests to assess learning and memory are the Morris water maze and variants of fear conditioning2. These tests rely on visual or auditory cues for learning and aversive stimuli (e.g., water, footshock) to motivate animals to complete the tasks3. In aging rodents, as in aging humans, sensory modalities decline over time. Several background strains of mice lose their hearing or develop cataracts as they age, and mortality rates after 23.5 h of water deprivation ...

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<td>Lashley III maze</td>
<td>&nbsp;</td>
<td>PSU Physics Machine Shop</td>
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<td> Maze must be custom-built, not commercially available</td>
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low-stress route learning using the lashley iii maze in mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

Watch this Scientific Journal Video about Low-stress Route Learning Using the Lashley III Maze in Mice at JoVE.com

Low-stress Route Learning Using the Lashley III Maze in Mice

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

low-stress-route-learning-using-the-lashley-iii-maze-in-mice

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Neuroscience

17.8K Views.  Pennsylvania State University. Il labirinto Lashley III è un percorso di apprendimento-lavoro che non si basa su stimoli avversi o segnali visivi. E 'quindi una scelta molto attraente per la valutazione dell'apprendimento e della memoria, soprattutto nei topi l'invecchiamento o in altro modo in cui lo stress è una considerazione.

Video: Low-stress Route Learning Using the Lashley III Maze in Mice

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

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 ...

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 ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

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.

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 ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural synchronization (INS) during social interactions, functional near-infrared spectroscopy (fNIRS) is one of the most popular techniques for hyperscanning naturalistic social interactions because of its relatively high spatial resolution, sound anatomical localization, and exceptionally high tolerance of motion artifacts. Previous fNIRS-based hyperscanning studies usually calculate a time-lagged INS using wavelet transform coherence (WTC) to describe the direction and temporal pattern of information flow between individuals. However, the results of this method might be confounded by the autocorrelation effect of the fNIRS signal of each individual. For addressing this issue, a method termed partial wavelet transform coherence (pWTC) was introduced, which aimed to remove the autocorrelation effect and maintain the high temporal-spectrum resolution of the fNIRS signal. In this study, a simulation experiment was performed first to show the effectiveness of the pWTC in removing the impact of autocorrelation on INS. Then, step-by-step guidance was offered on the operation of the pWTC based on the fNIRS dataset from a social interaction experiment. Additionally, a comparison between the pWTC method and the traditional WTC method and that between the pWTC method and the Granger causality (GC) method was drawn. The results showed that pWTC could be used to determine the INS difference between different experimental conditions and INS's directional and temporal pattern between individuals during naturalistic social interactions. Moreover, it provides better temporal and frequency resolution than the traditional WTC and better flexibility than the GC method. Thus, pWTC is a strong candidate for inferring the direction and temporal pattern of information flow between individuals during naturalistic social interactions.

This protocol describes partial wavelet transform coherence (pWTC) for calculating the time-lagged pattern of interpersonal neural synchronization (INS) to infer the direction and temporal pattern of information flow during social interaction. The effectiveness of pWTC in removing the confounds of signal autocorrelation on INS was proved by two experiments.

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...