This research was funded by grant no. 0723341 (to KGL) from the
National Science Foundation. We are also grateful for contributions
provided by the Schapiro Undergraduate Research Fellowship program and
the Psychology Department at Randolph&#x2013;Macon College. Finally, we
appreciate Craig Kinsley&#x2019;s collaborative contributions in this
research and Amanda Rzucidlo&#x2019;s help in preparing the R-MC laboratory
for this video article.

1. Identification of Animal Model
<ol>
	<li>The California Deer Mouse (Peromyscus californicus) is an ideal model to explore paternal responses. As you see, this mouse grooms the pups just as the moms do - they even crouch over them so that it looks like the pups are nursing. The behavior of the California mice is compared to a species of the same genus, the common deer mouse (Peromyscus maniculatus) which exhibits little interest in the pups, often trying to escape or even attack them. (See Figure 1 for pictures of these species interacting with pups.)</li>
	<li>Once the species model has been identified then the various groups need to be established. In this study, biological fathers, virgins with no parenting experience, and virgins with limited pup exposure (pup-exposed or foster fathers) of both species are used so that both predisposed and acquired paternal characteristics can be assessed. In this particular study we also exposed all groups to a toy mouse pup to assure that the social interactions were specific to a pup and not a representative response to anything placed in the cage (See Figure 2.)</li>
	<li>Before investigating neurobiological factors, it is important to confirm that the selected species exhibit clear differences in the behavior of interest. In this study, males are placed in a cage with a pup for five minutes and a paternal behavior ethogram (see Table 1 for example of ethogram) is used to score behaviors such as latency to approach the pup and amount of time spent in contact with the pup. If aggressive responses are observed, the pup is removed immediately. These sessions are often videotaped so a careful behavioral analysis can be subsequently conducted.</li>
</ol>
2. Preparing Brain Tissue
<ol>
	<li>Following a standard perfusion of each mouse (protocol attached), the brains are removed so that they can be sectioned in the specific area of interest (show brain).</li>
	<li>A standard mouse brain atlas (show atlas) is used to locate the paraventricular nucleus in the hypothalamus, an area known to be rich in cells producing the neuropeptide of interest in this study - vasopressin (AVP).</li>
	<li>The brain is then blocked and mounted on a freezing chuck so that the tissue will freeze before being sectioned with the microtome. The brain is sectioned in a trim mode until specific landmarks are identified, then the specific brain thickness, 30 microns in this case, is set for the brain sections used for analysis.</li>
	<li>The brain sections are carefully placed in well plates filled with phosphate buffer saline (PBS); (protocol for PBS attached; please note that this is only one example of a standard immunocytochemistry protocol).</li>
</ol>
3. Immunocytochemistry
<ol>
	<li>Throughout the various steps of this process (see attached protocol), it is important to have accurate pipetting skills to assure accurate measurements of chemicals and other ingredients important for this technique (show student pipetting).</li>
	<li>The first step in this process is washing the brains which involves replacing the PBS in the well plates three to five times; between each wash the well plates are placed on a rocker for 10 minutes (show student replacing PBS and placing well plates on rockers).</li>
	<li>The brain slices are then exposed to the primary antibody solution and stored at 4 &deg;C overnight on the rocker (show process).</li>
	<li>Following another wash, the brain slices are exposed to a secondary antibody for one hour on the rocker at room temperature, then washed again.</li>
	<li>Next the brains are exposed to an Avitin-Biotin Complex solution to prepare for the visualization of the neurochemical-positive cells.</li>
	<li>For the final visualization step, the brains are exposed to DAB. This is a hazardous product and should always be handled with caution. As seen here, the brain slices begin to get darker before your eyes (show process).</li>
	<li>Following the DAB exposure, the brains go through the last series of washes and are then carefully placed on subbed microscope slides and allowed to dry overnight (show the placement of a brain slice on a slide; see attachment for subbed slides protocol).</li>
	<li>The slides are then cleared through a series of distilled water and alcohol washes, finally being submerged in Citrosolv before being coverslipped and stored in a slidebox for safe keeping (show various aspects of this process; see attachment for clearing process).</li>
</ol>
4. Neuroquantification
<ol>
	<li>After the slides have dried, they can be assessed with specialized neuroquantification software. Here Bioquant software is used to quantify the vasopressin-positive cells and fibers in the paraventricular nucleus of the mouse brains (show software on screen...and microscope).</li>
	<li>The specific area of interest is identified using the measurement options of the software to establish the visual field for neuroquantification. It is important that a consistent visual field size is quantified for each animal (show student doing this).</li>
	<li>Here the darkly stained vasopressin-immunoreactive cell bodies and fibers are apparent. Because it is difficult to count or trace this tissue, a special feature of this software uses light thresholding to determine the total amount of positively stained tissue within the specified area of interest. This thresholding tells us how much of the specified area contained vasopressin-positive tissue (show the data value on the computer screen).</li>
</ol>
5. Behavioral Analysis
<ol>
	<li>If more than one observer will be scoring the videotapes, it is important to establish inter-rater reliability to assure that the observers are scoring the behavior in a consistent manner. For the actual scoring session, a behavior scoring sheet should be prepared to allow for easy scoring of behavior during observation sessions (see Table 2 for example of scoring spread sheet).</li>
	<li>For behaviors that aren't fast episodic responses, a microsequencing analysis can reveal subtleties about the progression of the specific responses. For example, grooming behavior consists of a chain of very fast responses. Although one option is to simply record the presence and duration of grooming, another is to document the chain of events accompanying this response. Using this microsequencing software, observers score the presence of a particular behavior every second, as prompted by the software (show software and video).</li>
	<li>Following data collection, the parameters of interest are determined and the appropriate behavioral scores are analyzed. Examples may be total duration of time spent in contact with the pup, or number of disruptions in the grooming sequence (show computer with spread sheet with data entered for each animal).</li>
	<li>Once the behavior is scored it is important to confirm that the two species exhibited different response strategies for the behavior of interest in the study. In this case the California deer mice should exhibit more paternal behavior than the common deer mice. Additionally, various behavioral measures can be correlated with brain measures to get a more informed view of relevant influences.</li>
</ol>
6. Representative Results:
<ol>
	<li>To validate the model, the data should clearly indicate that the two species performed differently with regard to the behavior of interest. Here you see that the P. californicus males spent more time grooming and crouching over the pups, two hallmarks of paternal responses (See Figure 4).</li>
	<li>In order to determine if the neurobiologial variable of interest is important in paternal responses, the amount of vasopressin (AVP)-positive tissue was quantified for several relevant brain areas; as can be seen, the paternal P. californicus animals had more immune-positive tissue in several of these brain areas. (see Figure 5).</li>
	<li>In a related study using the microsequencing analysis, it was hypothesized that paternal California mice would exhibit less anxiety than their virgin counterparts; accordingly, when exposed to a predator odor, the fathers exhibited fewer interruptions in the grooming sequence than the virgin animals (see Figure 6).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3173/3173fig1.jpg" /> 
Figure 1: (A) Male California mice exhibiting paternal responses toward conspecific alien pup. (B) Male deer mouse exhibiting a cautious approach (stretch attend) and avoidance response in the presence of a conspecific alien pup. Note: In these social interaction assessments, if a male exhibits aggressive responses toward the pup, the experimenters immediately hit the top of the cage to distract the male. At this time the session is terminated and the pup is removed, inspected for any wounds and returned to the mother. In our laboratory, this rarely is observed with P. californicus males but is occasionally observed with P. maniculatus; close observation and immediate intervention, however, prevent harm from occurring to the animals.
<img alt="Figure 2" src="/files/ftp_upload/3173/3173fig2.jpg" /> 
Figure 2: A California mouse interacting with a toy mouse; most tried to chew on these stimuli.
<img alt="Figure 3" src="/files/ftp_upload/3173/3173fig3.jpg" /> 
Figure 3: Experimental design of study; there were approximately 6 animals in each group for each species.
<img alt="Figure 4" src="/files/ftp_upload/3173/3173fig4.jpg" /> 
Figure 4: Graphic of immunocytochemistry process.
<img alt="Figure 5" src="/files/ftp_upload/3173/3173fig5.jpg" /> 
Figure 5. Paternal responses in the California and deer mice; more paternal responses (groom, crouch, faster approach) were observed in the California mice. More stretch attends (stress response) were observed in the deer mice.
<img alt="Figure 6" src="/files/ftp_upload/3173/3173fig6.jpg" /> 
Figure 6. Vasopressin immunoreactivity in various brain areas of both species. The California mice had more staining in the PVN cells and fibers.
<img alt="Figure 7" src="/files/ftp_upload/3173/3173fig7.jpg" /> 
Figure 7: Paternal California mice exhibited fewer disruptions in their grooming sequences in the presence of a predator odor than the pup-exposed and virgin groups.

<ol>
	<li>Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., Ogawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+in+the+presence+of+raffinose+and+glycerol.">Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.</a> J. Reprod. Fertil. 89, 511-516 (1990).</li><li>Yokoyama, M., Akiba, H., Katsuki, M., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+normal+young+following+transfer+of+mouse+embryos+obtained+by+in+vitro+fertilization+using+cryopreserved+spermatozoa.">Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa.</a> Jikken Dobutsu. 39, 125-128 (1990).</li><li>Sztein, J. M., Farley, J. S., Young, A. F., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+cryopreserved+mouse+spermatozoa+affected+by+temperature+of+collection+and+rate+of+thawing.">Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.</a> Cryobiology. 35, 46-52 (1997).</li><li>Thornton, C. E., Brown, S. D., Glenister, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+numbers+of+mice+established+by+in+vitro+fertilization+with+cryopreserved+spermatozoa:+implications+and+applications+for+genetic+resource+banks,+mutagenesis+screens,+and+mouse+backcrosses.">Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.</a> Mamm. Genome. 10, 987-992 (1999).</li><li>Sztein, J. M., Farley, J. S., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilization+with+cryopreserved+inbred+mouse+sperm.">In vitro fertilization with cryopreserved inbred mouse sperm.</a> Biol. Reprod. 63, 1774-1780 (2000).</li><li>Liu, L., Nutter, L. M., Law, N., McKerlie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+freezing+and+in+vitro+fertilization+in+three+substrains+of+C57BL/6+mice.">Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.</a> J. Am. Assoc. Lab. Anim. Sci. 48, 39-43 (2009).</li><li>Nakagata, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+and+in+vitro+fertilization.">Cryopreservation of mouse spermatozoa and in vitro fertilization.</a> Methods. Mol. Biol. 693, 57-73 (2011).</li><li>Mazur, P., Koshimoto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+intracellular+ice+formation+the+cause+of+death+of+mouse+sperm+frozen+at+high+cooling+rates.">Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates.</a> Biol. Reprod. 66, 1485-1490 (2002).</li><li>Jin, B., Yamasaki, C., Yamada, N., Seki, S., Valdez, D. M., Kasai, M., Edashige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+mouse+spermatozoa+are+injured+during+freezing.">The mechanism by which mouse spermatozoa are injured during freezing.</a> J. Reprod. Dev. 54, 265-269 (2008).</li><li>Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., Diekman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+signaling+pathways+involved+in+sperm+acquisition+of+fertilizing+capacity.">Novel signaling pathways involved in sperm acquisition of fertilizing capacity.</a> J. Reprod. Immunol. 53, 133-150 (2002).</li><li>Ostermeier, G. C., Wiles, M. V., Farley, J. S., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserving,+distributing+and+managing+genetically+modified+mouse+lines+by+sperm+cryopreservation.">Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.</a> PLoS ONE. 3, e2792-e2792 (2008).</li></ol>

No conflicts of interest declared.

This article discusses three essential components thought to be critical for the successful execution of behavioral neuroscience investigations: (1) a representative and valid animal model; (2) an accurate and sensitive immunocytochemistry protocol; and (3) the most analysis (observational and statistical) of both the behavioral and brain data. Mistakes in a single category will most certainly compromise the results of the entire study. Hence, after carefully selecting the appropriate animal model, considerable effort should be directed toward pilot testing the behavioral and histological procedures to assure that the behavior will be reliably observed in the study, followed by the successful processing of the brains.
As mentioned earlier, the use of comparative species to identify neurobiological mechanisms of a particular response is a valuable methodological approach because this technique doesn't require genetic engineering or painful surgical manipulations. Thus, this methodological approach utilizes less threatened, intact animals. The use of natural variations of animals, however, is strengthened by the incorporation of natural-like environments even if the animals are housed in the laboratory. Further, if laboratory studies can be extended to the field to further validate the authenticity of species differences in the behavior of interest, this next step is recommended. Although the comparative approach offers many advantages, a limitation is the correlative nature of the data; consequently, additional techniques such as pharmacological manipulations should be used to further validate the role of targeted neurobiological systems in the behavior of interest.
Once the experimenters feel confident about the behavioral, histological and statistical procedures, care should be taken so that the living conditions of the animals remain constant for all groups except, of course, for the experimental manipulation. Changes in variables such as light schedules, noise levels, caretakers, humidity levels and odors in the laboratory could have significant effects on the animals' neurobiological responses.
In this article the primary antibody was used for the detection of vasopressin but other primary antibodies can be used for a plethora of different neurochemicals of interest. If the experimenter is working with a new antibody, it is important to conduct titration studies to determine the optimal dilution of the new antibody. Many times too much antibody is used (as suggested by the vendor), resulting in tissue that is too dark to differentiate the signal; further, excessive use of the antibody is very expensive. 
Finally, if more than a single statistical analysis can be used to provide alternate views and interpretations of the data, they should be utilized. In this particular study, general linear models, correlational, and multidimensional scaling analyses were used to provide the most informative views of the data.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Binocular Compound Microscope</td>
			<td>Motic</td>
			<td>BA400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BIOQUANT Life Science Software</td>
			<td>BioQuant Image Analysis Corporation</td>
			<td>Version 8.40.20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microm</td>
			<td>HM525</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Masterflex Console Drive Easy-Load L/S (Perfusion Pump)</td>
			<td>Cole-Parmer Instrument Company</td>
			<td>7518-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>24 Well Cell Culture Cluster (tissue culture treated; non-pyrogenic polystyrene; sterile)</td>
			<td>Corning Costar</td>
			<td>3526</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25x75x1mm Microscope Slides</td>
			<td>Globe Scientific, Inc.</td>
			<td>1324W</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>22x50mm No.1 Cover Slip</td>
			<td>Globe Scientific, Inc.</td>
			<td>1414-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non-sterile 3mL Graduated Large Bulb Transfer Pipettes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70962-9</td>
			<td>&nbps;</td>
		</tr>
		<tr>
			<td>Alconox Detergent Powder</td>
			<td>Alconox</td>
			<td>1104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-1kg</td>
			<td>CAS 7647-14-5</td>
		</tr>
		<tr>
			<td>Monobasic Sodium Phosphate</td>
			<td>Spectrum</td>
			<td>SO130</td>
			<td>CAS 10049-21-5</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>30412</td>
			<td>CAS 10028-24-7</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>Spectrum</td>
			<td>TR135</td>
			<td>CAS 9002-93-1</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-500</td>
			<td>CAS 108-88-3</td>
		</tr>
		<tr>
			<td>Chromium potassium Sulfate</td>
			<td>Sigma</td>
			<td>C-5926</td>
			<td>CAS 7788-99-0</td>
		</tr>
		<tr>
			<td>CitriSolv</td>
			<td>Fisher Scientific</td>
			<td>22-143975</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol, High Den.Poly Bottles, 200 proof, 24 x 1 pint</td>
			<td>Pharmco-AAPER</td>
			<td>111000200CSPP</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Normal Goat Serum </td>
			<td>Vector</td>
			<td>S-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti vasopressin Analyte specific reagent</td>
			<td>Immunostar, Inc.</td>
			<td>20069</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotinylated anti-rabbit IgG (H+L) affinity-purified</td>
			<td>Vector</td>
			<td>BA-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elite Standard Vectastain ABC Kit</td>
			<td>Vector</td>
			<td>PK-6100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAB Peroxidase Substrate Kit</td>
			<td>Vector</td>
			<td>SK-4100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Original Unflavored Gelatin (4-count envelopes) 1-oz box</td>
			<td>Knox</td>
			<td>&nbsp;</td>
			<td>Purchase at local grocery store</td>
		</tr>
		<tr>
			<td>3% Hydrogen Peroxide </td>
			<td>CareOne</td>
			<td>&nbsp;</td>
			<td>Purchase at local pharmacy</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>&nbsp;</td>
			<td>Purchase at local grocery store</td>
		</tr>
	</tbody>
</table>

using a comparative species approach to investigate the neurobiology of paternal responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

Watch this Scientific Journal Video about Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses at JoVE.com

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

using-comparative-species-approach-to-investigate-neurobiology

Research

JoVE Journal

Neuroscience

12.5K Views.  Randolph-Macon College. Сравнительный подход видов позволяет поведенческих нейробиологов для изучения различных нейробиологических факторов, связанных с конкретными поведение рассматривается как характеристика конкретной модели животных. Воспользовавшись естественные различ?...

Video: Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

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 ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the &quot;Loser&quot; Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

A Visual Guide to Sorting Electrophysiological Recordings Using &#039;SpikeSorter&#039;

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...