We are grateful to anonymous reviewers for comments and suggestions that greatly improved this manuscript. We thank R. Bowes, R. Carreno, and T. Panhuis for assistance in collecting the fish. We also thank M. Lee for assistance with male preference trials. We are grateful to the Ohio Wesleyan University Department of Zoology for helpful advice and suggestions throughout this study and Arizona State University (McGraw Lab) for software advice.

All experiments were conducted with the approval of Ohio Wesleyan University&#39;s Institutional Animal Care and Use Committee.
1. Recording UV Reflectance of Fish using a Spectrophotometer
<ol>
	<li>Calibrate a spectrophotometer and light source with a known white standard over the range of wavelengths to be measured according the recommendations of the instrument or software.</li>
	<li>Anesthetize fish by placing in a 0.5% solution of ethyl 3-aminobenzoate methane sulfonic acid salt (MS-222) buffered with an equal quantity of sodium bicarbonate until fish becomes unresponsive. Once unresponsive, immediately place the fish on a black, non-reflective background. Note: MS-222 is potentially toxic, and gloves should be worn at all times.</li>
	<li>Take all measurements in a darkened room to minimize excess light beyond that of the spectrometer light source.</li>
	<li>Measure reflectance using a fiber optic probe held against the body of the organism. Make sure to form a consistent 45&#176; angle between the fiber optics and the body, as is built into some probe designs. Note: For measurement consistency, it may be desirable to average across multiple scans (2 - 5) per body region.</li>
	<li>Record reflectance data with appropriate reflectance analysis software. Note: The software used here was set to: integration time = 2,000 sec, average scans = 2, and pixel smoothing = 5. Data was copied and pasted to a spreadsheet.</li>
	<li>Repeat on multiple body areas to determine presence or absence of UV reflectance and range of phenotypic variation across organisms. Note: Preliminary scans may be necessary to determine which, if any, body areas consistently show UV markings.</li>
	<li>Return the fish to a well-oxygenated holding aquarium until fully recovered, as evidenced by normal operculum beats and swimming behaviors. Note: A commercial protectant to restore the mucous coating to the body may be added to aid in skin recovery.</li>
	<li>Repeat across multiple individuals, including both sexes if desired.</li>
	<li>On spreadsheet where all data have been copied, plot data using a scatterplot, with reflectance on the Y-axis and wavelength on the X-axis. Amplitude peaks in the UV range (300 - 400 nm) indicate the presence of UV reflectance on the fish.</li>
</ol>
2. Dichotomous Mating Trials
NOTE: Perform control observations, in which focal fish, UV filters, and opaque filters are in place. In these trials, when the opaque filters are removed, focal fish will be exposed to parts of the tank displaying the presence and absence of UV light, but lacking potential mating partners. For details on the validity of dichotomous choice tests in this experiment see Palmer and Hankison (2015)10, although note that these tests also have drawbacks11.
<ol>
	<li>Use sexually receptive individuals as focal organisms. Use female P. latipinna &#60; 48 hr postpartum5 and males isolated for at least 24 hr prior to testing to improve the likelihood of sexual motivation.</li>
	<li>Place a single focal individual into a neutral area adjacent to two choice compartments. For P. latipinna, use a 75.7 L test aquarium (30.5 cm x 30.5 cm x 75 cm; Figure 1) divided into three sections by transparent Plexiglas partitions, forming a central area for the focal individual flanked by two equal-sized choice compartments. Ensure that no water or olfactory cues are shared between individual tank sections. Repeat for each individual to be observed.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54453/54453fig1.jpg" /> 
Figure 1. Experimental Aquarium Setup. A rectangular aquarium divided into three sections: a central area that held the focal individual and two end compartments that held object pairs (the figure shows a female in the middle with object males at the ends). Interchangeable filters that either blocked or permitted UV reflectance could be placed over the glass partitions dividing the male and female sections of the aquarium. This figure is modified from Palmer and Hankison (2015) 10. <a href="http://cloudfront.jove.com/files/ftp_upload/54453/54453fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Under full-spectrum lighting conditions (lighting whose spectrum extends into the UV-A range), isolate one potential mating partner in each end compartment (choice compartment) of the test aquarium. Ensure that potential mating partners resemble one another as closely as possible in size, coloration and display characteristics, so that the main difference between the two isolated mating partners is whether they appear behind a filter that blocks or transmits UV light. 
	NOTE: It may be preferable to record reflectance in the visible spectrum to enable the best possible color matching, although here matching was done by eye.</li>
	<li>Acclimate individuals under visual isolation (opaque filters) for 15 min. Ensure that UV + and - filters are in place at this time. Following acclimation, remove the opaque filters. To ensure that side biases are not present (inherent preference for one side of the tank regardless of object), randomly assign the UV+ and UV- sides of the tank for each each trial.</li>
	<li>Record the amount of time that the focal individual spends within specific preference zones (areas near the choice compartments, in this case approximately two standard body lengths) by using stopwatches. For female fish, this is the amount of time it spends near each of two males, and vise versa for male focal fish. 
	NOTE: Event recorder software can also be used for recording. Fifteen minutes trials were used in this experiment and fish that did not visit each preference zone at least once were excluded from the results as being unresponsive.</li>
	<li>Record and analyze the time that fish spend near empty UV+ and UV- compartments to ensure that there is not an inherent bias towards a particular lighting environment that may influence the mating choice of the fish. Use paired t-tests to determine if individuals prefer a particular UV condition.</li>
</ol>

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The authors have nothing to disclose.

Spectrophotometry was successful in identifying UV markings on P. latipinna. Both sexes of P. latipinna possess UV markings along their sides. In addition, some males had UV markings on their dorsal fins, traits previously found to be important in female mating preferences7.
We recommend using UV spectrophotometry as a mechanism to detect the presence of UV markings. Further testing could determine its role in social interactions, including mating preference (as de...

Understanding the cues and signals used in animal social interactions allows us to comprehend the phenotypic variation both within and among species. This variation plays an important role in evolutionary processes such as population divergence, sexual selection, and speciation. Often, however, researchers are limited to exploring the cues most obvious to human sensory systems, especially those within the visual or auditory realms. Use of spectrophotometry, however, allows us to expand our investigations beyond the human visible spectrum and into wavelengths that may be important in social interactions in other species.
In particular, the short-range communication afforded by ultraviolet (UV; 300 - 400 nm) sensitivity has the potential to be highly advantageous during mate choice1. Many visually-hunting predators of birds and fishes, for example, are unable to detect UV radiation. In systems where males display elaborately to females, these males would reduce their risk of predation while maintaining their ability to attract mates by exploiting the UV spectrum rather than developing cues detectable in the visible spectrum2,3.. If one fails to consider the possibility that organisms are communicating with each other using these &#34;private communication channels&#34;, significant drivers of behavior and evolution may be missed.
This protocol outlines an investigation into the use of UV cues for mate choice in the sailfin molly, Poecilia latipinna, a polygamous fish that has no previously known ability to detect UV or utilize UV markings. This fish species has a close phylogenetic proximity to other UV-sensitive live-bearing fishes4 and there is microspectroscopic evidence that P. latipinna, along with other molly species such as P. mexicana and P. formosa, possess a class of cones (photoreceptor cells responsible for color vision) that are most sensitive for UV wavelengths5. In this sexually dimorphic species, female choice has played a strong role in the evolution of the males&#39; brightly colored and enlarged fins6-9. This methodology allows us to explore whether UV is an additional medium by which females assess male quality.
The detection and measurement of UV markings on P. latipinna using a spectrophotometer with a fiber optic probe is detailed here. Further, whether receptive female mollies differentially associate with males viewed through an optical filter transmitting full-spectrum light including UV-A ([UV+]; 320 - 700 nm) and males viewed through an UV-blocking filter ([UV-]; 400 - 700 nm) is discussed. This method has broad applications for discovering UV sensitivity and color patterns in fishes and other organisms, allowing research into a variety of questions involving UV and its role in behavior.

<table border="0" cellpadding="0" cellspacing="0" width="1044">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Spectrophotometer, P1000</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td style="width:351px;">newer models are availabe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">DT 1000 xenon UV light source</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td>newer models are availabe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ocean Optics Overture Software</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td>newer software is available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">R200-Angle-UV bifurcated fiber-optic probe (Guided Wave)</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td>newer models are availabe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Certified reflectance standard, white</td>
			<td style="width:217px;">labsphere</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">75.7 L Aquarium, divided</td>
			<td style="width:217px;">Experimental Builder</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Full Spectrum Bulb</td>
			<td style="width:217px;">Nature&#39;s Sunlight</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">UV blocking sheet</td>
			<td style="width:217px;">GAM UV Sheet</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

assays to detect uv-reflecting structures and determine their importance in mate preference using the sailfin molly poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Figures 1 and 2 show the mating preference aquarium set up and UV measurement sites for our experiments.
Measuring UV reflectance allowed for the determination that P. latipinna do possess UV characteristics, especially along the sides of their bodies (Figure 3), in addition to individual variation in these traits. Once UV traits were found, testing revealed that female...

Watch this Scientific Journal Video about Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna at JoVE.com

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

assays-to-detect-uv-reflecting-structures-determine-their-importance

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6.5K Views.  Ohio Wesleyan University. This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Video: Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...