Our research program involving the biological effects of ALAN on birds has received funding from the FWO Flanders (to M.E. and R.P., project ID: G.0A36.15N), the University of Antwerp and the European Commission (to M.L.G, Marie Sk&#322;odowska-Curie fellowship ID: 799667). We acknowledge the intellectual and technical support of members of the Behavioral Ecology and Ecophysiology Research group at the University of Antwerp, especially Peter Scheys and Thomas Raap.

All applications of this system to animal experiments were approved by the University of Antwerp&#39;s ethical committee and conducted in accordance with Belgian and Flemish laws. Methodology adhered to the ASAB/ABS guidelines for the use of animals in behavioral research. The Belgian Royal Institute for Natural Sciences (Koninklijk Belgisch Instituut voor Natuurwetenschappen; KBIN) provided licenses for all researchers and personnel.
1. Creating the experimental system
<ol>
	<li>Obtain broad-spectrum LED(s) to use in creating ALAN. Take LED light(s) from a LED headlight. Use either a single LED light or multiple (e.g., 4) broad-spectrum LED lights for more diffuse lighting (Figure 1). 
	NOTE: As a modification, LEDs with different spectral properties (e.g., red versus blue) could be used but would have to be obtained from a different source (see the Supplementary material of Grunst et al. 201915 for the spectral properties of the LEDs used in past studies using this system).</li>
	<li>Design a system to mount the LEDs along with an IR camera to allow for behavioral monitoring. Researchers can accomplish this end in a number of ways.
	<ol>
		<li>Option 1. Insert a single broad-spectrum LED into the nest box separately in a plastic tube adjacent to an IR camera mounted with adhesive on a plastic or metal plate that fits within the nest box (Figure 1A, B).</li>
		<li>Option 2. Mount an IR camera in a central position on a plastic or metal plate and then mount LED lights in fixed positions on the plate surrounding the IR camera (Fig. 1C).</li>
	</ol>
	</li>
	<li>Design a means to connect the system to a power source (battery) and timer.
	<ol>
		<li>Use a knife or drill to make groves in the side of the nest box through which wire connectors can extend to connect the system to a Fe-battery (12 V; 120 Wh) and homemade timer (12 V).</li>
		<li>Design a dark green wooden enclosure that matches the nest box in coloration, length, and width (e.g., nest boxes used in past studies had the dimensions: 120 mm x 155 mm x 250 mm ), and with one side opening via a hinge to house the battery, recorder for the video, and timer system for the LEDs (Figure 2; Supplementary Figure 1 and Supplementary Figure 2).</li>
	</ol>
	</li>
	<li>Design a means through which to adjust ALAN intensity.
	<ol>
		<li>Obtain a resistor (value contingent on battery voltage and illumination) and connect it in series with the LED(s).</li>
	</ol>
	</li>
	<li>Design &#34;dummy&#34; boxes with the same dimensions as the enclosures that house the timer and battery for use in habituating birds to the system (i.e., as in Figure 2A, but without the internal electronics). 
	NOTE: Section 2 and section 3 discuss the step-by-step methods used to study the effects of ALAN on the focal organism.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63381/63381fig01.jpg" /> 
Figure 1: Two systems consisting of IR cameras and LED light(s) used to manipulate ALAN inside nest boxes. (A) Top view of the nest box with plate holding the older system in place. (B) Older system with 1 broad-spectrum LED to manipulate ALAN and central camera with 10 IR LEDs (c) Newer system with 4 broad-spectrum LEDs and central IR camera with 4 IR LEDs. <a href="https://www.jove.com/files/ftp_upload/63381/63381fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63381/63381fig02.jpg" /> 
Figure 2: The homemade battery and timer unit used to manipulate ALAN and video-record behavior. (A) The unit is enclosed within a wooden box that is mounted on top of the nest box. (B) View of the electronics inside the unit. Connectors extend from inside the nest box up into the wooden enclosure to connect the electronics to the IR camera and broad-spectrum LEDs. <a href="https://www.jove.com/files/ftp_upload/63381/63381fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Planning the experiment and adjusting ALAN intensity and timing
<ol>
	<li>Determine the desired light intensity to which to expose animals.
	<ol>
		<li>Carefully consider which experimental light intensity to use so as to produce meaningful results that answer the research question. In general, this will mean selecting an ecologically relevant light intensity, which free-ranging animals are likely to encounter (see Table 1 for guidance).</li>
	</ol>
	</li>
	<li>Adjust the LED lights to the desired light intensity (e.g., 1-3 lux, as used in past studies; Table 1 and Table 2).
	<ol>
		<li>Prior to placement in the field, place the system on a nest box taken into the laboratory to calibrate the light intensity. Connect the LEDs to the power source, as described further below (Protocol section 3).</li>
		<li>Adjust the light emitted by the LEDs to the desired intensity (lux) by placing a light meter at the level of the bird within the nest box (~8 cm from the bottom) and simultaneously adjusting the resistor in series with the LEDs. 
		NOTE: It is possible to achieve very low light intensities (e.g., rural sky glow levels; 0.01 lux).</li>
	</ol>
	</li>
	<li>Determine the timeframe over which to expose animals to ALAN.
	<ol>
		<li>Determine the length and timing of exposure across the night. For instance, one can expose animals to ALAN across the entire night, for only part of the night, or leave a period of darkness in the middle of the night to reduce the degree of perturbation.</li>
		<li>In cases that an animal must enter the nest box (or a specific area) to be exposed to the ALAN, also consider whether the light should be turned on before or after the entry event is likely to occur.</li>
	</ol>
	</li>
	<li>Set the timer to control the period of light exposure during the night.
	<ol>
		<li>Set the timer connected to the broad spectrum LEDs so that the light turns on and off at specified periods (e.g., on at least 2 h before sunset; off 2 h after sunrise). 
		NOTE: The IR camera allows the behavior of the animal to be recorded simultaneously for the duration of the light exposure and will be on as long as it is connected to a charged battery.</li>
	</ol>
	</li>
	<li>Determine the appropriate experimental design to use for the target research question(s). 
	NOTE: For some questions, a repeated measures experimental design will be the most powerful option (e.g., How does exposure to ALAN affect sleep behavior?). For others, paired control and experimental groups will be needed (e.g., How does exposure to ALAN affect telomere loss in developing nestlings?).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Source/exposure level</td>
			<td>Intensity (lux)</td>
		</tr>
		<tr>
			<td>Full sunlight</td>
			<td>103000</td>
		</tr>
		<tr>
			<td>Full moonlight</td>
			<td>0.05&#8211;1</td>
		</tr>
		<tr>
			<td>Urban Sky glow</td>
			<td>0.2&#8211;0.5</td>
		</tr>
		<tr>
			<td>Exposure of free-living European blackbirds</td>
			<td>0.2 (0.07&#8211;2.2)</td>
		</tr>
		<tr>
			<td>Past experimental studies using the system</td>
			<td>1&#8211;3</td>
		</tr>
		<tr>
			<td>LED street lights</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>Low pressure sodium street lights</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>High pressure sodium</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>Florescent lighting</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Metal halide</td>
			<td>400&#8211;2000</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristic light intensities in the environment3,9, exposure levels of free-ranging birds41, and intensities used in past studies using this system (references in Table 2).
3. Implementing the exposure to ALAN
<ol>
	<li>Habituate the animals to the experimental setup.
	<ol>
		<li>If possible within the context of the experiment, habituate animals to the setup by placing dummy boxes on the top of the nest boxes at least 1 day prior to the experiment to minimize the effects of novelty aversion.</li>
	</ol>
	</li>
	<li>Survey the focal individuals.
	<ol>
		<li>Fit animals in the study population with&#160;passive integrative transponder (PIT) tags to allow for identification within nest boxes without disturbing the birds.</li>
		<li>In experiments involving the effect of ALAN on sleep behavior, visit the nest boxes on the night before the experiment and scan the boxes with a radio-frequency identification (RFID) reader to determine which birds are roosting inside.</li>
		<li>In experiments during the breeding season involving exposure of developing nestlings to ALAN, consistently monitor (e.g., every other day) nest boxes, and check for nest contents and adult identity. Carefully select nest boxes containing broods with certain characteristics (i.e., modal brood size, both parents present&#160;and feeding) for use in the experiment.</li>
	</ol>
	</li>
	<li>Select and implement the experiment.
	<ol>
		<li>For experiments involving sleep behavior, implement a repeated measures design by first recording individuals sleeping under conditions of darkness for at least one night to record undisturbed sleep in the absence of ALAN (control treatment) following steps 3.3.2-3.3.21.</li>
		<li>To this end, make sure to synchronize the time on the IR cameras with the local time prior to taking them into the field.</li>
		<li>Insert an SD card into the SD slot in&#160;the mini DVR recorder adjacent to the battery (Figure 2B; Supplementary Figure 2). Check to make sure that the SD card is empty, and if not, erase the data it contains.</li>
		<li>At least 2 h prior to the onset of darkness, remove the dummy box from on top of the nest box.</li>
		<li>Open the nest box lid.</li>
		<li>Place the plate containing the IR camera inside the nest box with the camera objective oriented downward.</li>
		<li>Extend the electronic connectors out of the grove in the nest box.</li>
		<li>Close the nest box lid.</li>
		<li>Place the enclosure containing the battery, recorder, and timer on top of the nest box.</li>
		<li>Connect the battery power connectors. Connect the red connector from the recorder to the white connector from the camera (audio), the yellow connector from the recorder to the yellow connector from the camera (video), and the black connector from the battery to the red connector from the camera (power) (Supplementary Figure 1 and Supplementary Figure 2).</li>
		<li>Push the record button to initiate the camera recording. 
		NOTE: The timer will not be set and/or the power will not be connected to the timer controlling the LEDs so that no ALAN will be produced on control nights.</li>
		<li>Check with a small tft screen to ensure the recording has started and that the image is correct. A port to connect the tft screen is located below the recorder (Supplementary Figure 2).</li>
		<li>Approximately 1 h after dark, return to the nest box and check the identity of the bird sleeping inside by moving a RFID transponder reader around the bottom and sides of the nest box and recording the unique identification number communicated from the PIT tag.</li>
		<li>On the morning following the control recording, at least 2 h after sunrise, return to the nest box and collect the battery system and IR camera.</li>
		<li>Again, place a dummy box on top of the nest box.</li>
		<li>In the laboratory or office, charge the battery and remove and download the SD card from the recorder to collect the behavioral data. 
		NOTE: Batteries have a life span of ~30 h in cold conditions to enable recording for the entire night, but need to be fully recharged between consecutive nights of recording.</li>
		<li>After successfully downloading the data, erase the data from the SD card and then reinsert it into the mini DVR recorder.</li>
		<li>On the subsequent night, implement the light exposure treatment (e.g., 1-3 lux, as used in past experiments using the system; Table 1 and Table 2).</li>
		<li>Set the timer system for the desired time period of light exposure.</li>
		<li>Follow the same steps (3.3.2-3.3.17) described above for the control recording, but also connect the timer to the power and the LEDs to the timer (Supplementary Figure 1 and Supplementary Figure 2).</li>
		<li>If desired, repeat the control recording (of sleep behavior under conditions of darkness, i.e., absence of ALAN) on night three.</li>
		<li>For experiments involving exposure of nestlings to ALAN, use control and experimental broods as described in steps 3.3.23-3.3.25.</li>
		<li>Place dummy boxes (lacking electronics) on top of the nest boxes of control broods and handle both control and experimental nestlings in equivalent ways.</li>
		<li>Implement the experimental ALAN exposure for experimental boxes. During the experimental period, mount the LED system and IR camera within the nest box, as described above, and set the timer to control the desired period of light exposure.</li>
		<li>Recharge the batteries. For experiments involving multiple nights of light exposure and video recording, collect the systems each morning to recharge the batteries during the day and then replace the system in the evening.</li>
	</ol>
	</li>
	<li>Collect data on the response variable(s) of interest.
	<ol>
		<li>If behavior within the nest box is the variable of interest, the IR camera will allow simultaneously documenting behavior (e.g., sleep behavior; Figure 3).</li>
		<li>Collect any other data of interest via additional monitoring methods, with sampling occurring at variable points in time (e.g., blood samples taken before and after light exposure15).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63381/63381fig03.jpg" /> 
Figure 3: Infrared image of a great tit inside a nest box exposed to ALAN. (A) Sleeping and (B) Alert great tit <a href="https://www.jove.com/files/ftp_upload/63381/63381fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors declare that they have no conflicts of interest.

This nest box-based system of LED lights and a paired IR camera has allowed researchers to assess a range of intriguing questions regarding the biological effects of ALAN. Moreover, there are many more research directions that can be pursued with the system. In addition, expanding the use of the system to other species could help foster an understanding of interspecific differences in sensitivity to ALAN. Below some non-exhaustive possibilities for future research are presented in the hope that this paper will help motiv...

Animals have evolved with the natural patterns of light and darkness that define day and night. Thus, circadian rhythms in hormonal systems orchestrate rest and activity patterns and allow animals to maximize fitness1,2,3. For instance, the circadian rhythm in glucocorticoid hormones, with a peak at the onset of daily activity, primes vertebrates to behave appropriately across the 24-h period via effects on glucose metabolism and responsiveness to environmental stressors4. Similarly, the pineal hormone melatonin, which is released in response to darkness, is integrally involved in governing patterns of circadian rhythmicity and also has antioxidant properties5,6. Entrainment of many aspects of circadian rhythmicity, such as melatonin release, is affected by the photoreception of levels of light in the environment. Thus, the introduction of artificial light into the environment to support human activity, recreation, and infrastructure has the potential to have wide-reaching effects on the behavior, physiology and fitness of free-ranging animals7,8. Indeed, diverse effects of exposure to artificial light at night (ALAN) have been documented9,10, and ALAN has been highlighted as a priority for global change research in the 21st century10.
Measuring the effects of ALAN on free-ranging animals poses non-trivial challenges for a number of reasons. First, mobile animals moving through the environment constantly experience different levels of light. Thus, how does one quantify the level of light that individual animals are exposed to? Even if levels of light on the territory of the animal can be quantified, the animal may employ avoidance strategies that affect patterns of exposure, thus demanding simultaneous tracking of animal location and light levels. Indeed, in most field studies, the mean and variation in light exposure levels are unknown11. Second, exposure to ALAN is often correlated with exposure to other anthropogenic disturbance factors, such as noise pollution, chemical exposure, and habitat degradation. For instance, animals occupying habitats along the margins of roadways will be exposed to light from street lamps, noise from vehicular traffic, and air pollution from vehicular emissions. How then does one effectively isolate the effects of ALAN from the effects of confounding variables? Rigorous field experiments that enable good measurements of both light exposure levels and response variables are essential to evaluating the severity of the biological effects of ALAN, and to developing effective mitigation strategies11.
This article describes an experimental approach that, although not without its limitations (see discussion section), helps assuage, if not eliminate the difficulties identified above. The approach entails experimentally manipulating ALAN levels inside the nest boxes of a free-living, diurnal bird species, the great tit (Parus major), using a system of light-emitting diode (LED) lights and an infrared (IR) camera installed within nest boxes. The setup enables simultaneous acquisition of video recordings, including audio, which allows researchers to assess effects on behaviors and vocalizations. Great tits utilize nest boxes for breeding, and sleep in the nest boxes between November and March. Females also sleep inside the nest boxes during the breeding season12. The system has also been used to a lesser extent to study effects of ALAN on blue tits (Cyanistes caeruleus). The first difficulty, involving knowing light levels encountered by the animal, is mitigated in that, given that an individual is willing to enter the nest box (or is already in the nest box in the case of immobile nestlings), light levels can be precisely determined by the researcher. The second difficulty, involving correlations to confounding variables, can be controlled by using nest boxes in similar environments, and/or measuring the levels of confounding variables near nest boxes. In addition, in cavity-nesting birds, adopting an experimental approach is powerful because nest boxes or natural cavities can shield nestlings and adults from ALAN13, which may explain why some correlative studies find little effect of ALAN (or anthropogenic noise)14, whereas experimental studies more often find clear effects (see below). Moreover, a repeated measures experimental design can be adopted in which individuals serve as their own control, which further increases statistical power, and the probability of detecting meaningful biological effects. The sections below: (1) explain the details of the design and implementation of the system, (2) summarize the important results that have been thus far derived using the system, and (3) propose future research directions that could be pursued, both in tits and other animals.

<table><tbody><tr><td>Broad spectrum; 15 mm x 5 mm; LED headlight</td><td>RANEX; Gilze; Nederlands</td><td>6000.217</td><td>A similar model could also be used</td></tr><tr><td>Battery</td><td>BYD</td><td>R1210A-C</td><td>Fe-battery 12 V 120 Wh ( lithium iron phosphate battery)</td></tr><tr><td>Dark green paint</td><td></td><td></td><td>Optional. To color nest boxes/electronic enclosures</td></tr><tr><td>Electrical tape</td><td></td><td></td><td>For electronics</td></tr><tr><td>Homemade timer system</td><td>Amazon</td><td>YP109A 12V</td><td>A similar model could also be used</td></tr><tr><td>Infrared camera</td><td>Koberts-Goods, Melsungen, DE</td><td>205-IR-L</td><td>Mini camera; a similar model could also be used</td></tr><tr><td>Light level meter</td><td>ISO-Tech ILM; Corby; UK</td><td>1335</td><td>To calibrate light intensity</td></tr><tr><td>Mini DVR video recorder</td><td>Pakatak, Essex, UK</td><td>MD-101</td><td>Surveillance DVR Recorder Mini SD Car DVR with 32 GB</td></tr><tr><td>Passive integrated transponder (PIT) tags</td><td>Eccel Technology Ltd, Aylesbury, UK</td><td>EM4102</td><td>125 Kh; Provides unique electronic ID</td></tr><tr><td>Radio frequency identification (RFID) Reader</td><td>Trovan, Aalten, Netherlands</td><td>GR-250</td><td>To scan PIT tags and determine bird identity</td></tr><tr><td>Resistor</td><td>RS Components</td><td></td><td>Value depending on voltage battery and illumination</td></tr><tr><td>SD card</td><td>SanDisk</td><td></td><td>64 GB or larger</td></tr><tr><td>SongMeter</td><td>Wildlife Acoustics; Maynard, MA</td><td></td><td>Optional. Provides a means of monitoring vocalizations outside of nest boxes</td></tr><tr><td>TFT Color LED Portable Test Monitor</td><td>Walmart</td><td></td><td>Allows verification that the camera is on and recording the image correctly</td></tr><tr><td>Wood</td><td></td><td></td><td>To construct nest boxes/electronic encolsures</td></tr></tbody></table>

an experimental approach to investigating effects of artificial light at night on free-ranging animals: implementation, results, and directions for future research

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from human infrastructure and recreational activity. Artificial light at night (ALAN) has the potential to have widespread effects on animal behavior, physiology, and fitness, which can translate into broader-scale effects on populations and communities. Understanding the effects of ALAN on free-ranging animals is non-trivial due to challenges such as measuring levels of light encountered by mobile organisms and separating the effects of ALAN from those of other anthropogenic disturbance factors. Here we describe an approach that allows us to isolate the effects of artificial light exposure on individual animals by experimentally manipulating light levels inside nest boxes. To this end, a system can be used consisting of light-emitting diode (LED) light(s) adhered to a plate and connected to a battery and timer system. The setup allows exposure of individuals inside nest boxes to varying intensities and durations of ALAN while simultaneously obtaining video recordings, which also include audio. The system has been used in studies on free-ranging great tits (Parus major) and blue tits (Cyanistes caeruleus) to gain insight into how ALAN affects sleep and activity patterns in adults and physiology and telomere dynamics in developing nestlings. The system, or an adaptation thereof, could be used to answer many other intriguing research questions, such as how ALAN interacts with other disturbance factors and affects bioenergetic balance. Furthermore, similar systems could be installed in or near the nest boxes, nests or burrows of a variety of species to manipulate levels of ALAN, evaluate biological responses, and work towards building an interspecific perspective. Especially when combined with other advanced approaches for monitoring the behavior and movement of free-living animals, this approach promises to yield ongoing contributions to our understanding of the biological implications of ALAN.

The peer-reviewed research articles published using this system are summarized in Table 2. Several other manuscripts are in progress. These studies address three major suites of research questions. First, the system has been used to study the effects of light exposure on sleep behavior and activity levels in adults. To this end, a repeated measures experimental design was employed, in which the same individual was first recorded sleeping under natural conditions and subsequently recorded sleeping in a li...

Watch this Scientific Journal Video about An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Res

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera. Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on organisms.

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from ...

an-experimental-approach-to-investigating-effects-artificial-light-at

Research

JoVE Journal

Biology

2.3K Views.  University of Antwerp. Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera. Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on organisms.

Video: An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson&#8217;s disease.

Exercise may alleviate behavioral impairments associated with neurodegenerative diseases. Studies of the mechanisms underlying exercise-mediated effects have increased in disease models, but the neurobiological background accompanying these diseases, specifically aging, is likely not considered. Here, we illustrate measures to increase the likelihood of a successful exercise paradigm in aged rats.

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these ...

Behavior

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Neuroscience

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive sampling of flying insects have a poor temporal resolution because it is time-consuming, costly and/or logistically difficult to perform. Effective sampling of flying insects attracted to artificial light at night (ALAN) requires sampling at user-defined time points (nighttime only) across well-replicated sites resulting in major time and labor-intensive survey effort or expensive automated technologies. Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate, making it a viable option for studies that require temporal sub-sampling across multiple sites. The trap can be used to address a wide range of other ecological questions that require a greater temporal and spatial scale than is feasible with previous trap technology.

To study the impacts of artificial light at night (ALAN) on nocturnal flying insects, sampling needs to be confined to nighttime. The protocol describes a low-cost automated flight intercept trap that allows researchers to sample at user-defined periods with increased replication.

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive ...

Environment

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Measuring Light-Switching Behavior Using an Occupancy and Light Data Logger

Due to discrepancies between self-reported and observed pro-environmental behavior, researchers suggest the use of more direct measures of behavior. Although direct behavioral observation may increase the external validity and generalizability of a study, it may be time-consuming and be subject to experimenter or observer bias. To address these issues, the use of data loggers as an alternative to natural observation can allow researchers to conduct broad studies without interrupting the participants&#39; naturally occurring behaviors. This article describes one of such tools&#8212;the occupancy and light data logger&#8212;with its technical description, deployment protocol, and information about its possible applications in psychological experiments. The results of testing the reliability of the logger in comparison to human observation is provided alongside an example of the gathered data during a 15-day measurement in public restroom (N = 1,148) that includes: 1) room occupancy changes; 2) indoor light changes; and 3) room occupancy time.

This article describes a procedure for using and deploying an occupancy and light data logger which allows collecting data about light-switching behavior of participants in field settings.

Due to discrepancies between self-reported and observed pro-environmental behavior, researchers suggest the use of more direct measures of behavior. ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

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Chapters in this video

Light/dark Transition Test for Mice

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Measuring Light-Switching Behavior Using an Occupancy and Light Data Logger

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals