The overall goal of this procedure is to use a comparative species approach. To learn more about the brain areas and neurochemicals associated with paternal behavior, the first step is to identify two closely related species that differ significantly in the expression of the behavior of interest. Next, both species are allowed to interact with pups and observe for behavior within the parental response.
Eth echogram. These behaviors are videotaped for further analysis. The next step of the procedure is to collect brain samples and analyze them for the presence of vasopressin immunoreactive tissue in the target area.
Neuroimaging software is then used to determine species specific differences in targeted brain areas. Ultimately, results can be obtained that show species related differences in paternal responses and vasopressin immunoreactivity in specific brain areas. The primary advantage of using the comparative species approach as opposed to other existing methods, such as pharmacologically manipulated or genetically engineered species, is that it allows you to utilize naturally occurring differences between closely related species as opposed to using more artificially induced behaviors.
Thought this method can provide insight into paternal behaviors. It can also be applied to other behaviors such as aggression, sexual behavior, or grooming Before testing. Mice from two closely related species are separated into test groups of biological fathers virgins with no parenting experience and virgins with limited pup exposure.
First, prepare the cage and the video recording system. Next place a pup in the middle of the cage. Once everything is prepared, introduce the test mail into the cage with the pup and record behavior for five minutes.
If aggressive responses are observed immediately remove the pup. The California deer mouse grooms the pups just as the moms do crouching over them so that it looks like the pups are nursing. In contrast, the common deer mouse exhibits little interest in the pups and will often try to escape or attack them.
All groups are also exposed to a toy mouse to assure that the observed social interactions are specific to a pup and not a general response. After the observation period, prepare the behavior scoring sheets. If more than one observer will be scoring the videotapes.
It is important to establish interrater reliability. The paternal behavior rogram seen here is used to score behaviors. Examples include crouching over the pups, grooming the pups, or exhibiting anxiety behaviors such as stretch attend responses.
Micro sequencing software can be used to reveal the subtleties between certain behaviors. Here, data from cacus mice that we're exposed to a predator odor is used as an example. First, combine the behavior into binary states IE grooming versus non-G grooming, then produce a time series.
Finally, calculate the degree of fluctuation. Using a predetermined formula, Many important behaviors can be missed. Using common observation techniques, it is best to videotape the behaviors so that responses can be viewed in slow motion.
For more accurate viewing, specialized software enables researchers to analyze micro sequences of behaviors, providing data that would be missed with a human high in real time Time. This method can help answer key questions in the behavioral neuroscience field, such as which neurochemical systems mediate specific behaviors observed in the animals Following pup exposure. Use a standard perfusion protocol to isolate the brain and freeze it before sectioning section the brain on trim mode until specific landmarks are identified.
A standard mouse brain atlas is used to locate the paraventricular nucleus in the hypothalamus, an area known to be rich in cells, producing the neuropeptide vasopressin or a VP.Once the pair of ventricular nucleus is reached, take 30 micron sections for further analysis. Carefully place the brain sections into well filled plates with phosphate buffered saline or PBS first. Wash the brain sections with PB S3 to five times placing the plates on a rocker for 10 minutes after the addition of fresh PBS.
Next, add a solution containing the primary antibody solution to the plates and store them at four degrees Celsius overnight with rocking the following day. Wash the slices before adding the secondary antibody. Leave the samples to rock for one hour at room temperature After a final wash, expose the brain slices to an Aden biotin complex solution, which aids in the visualization of the neurochemical positive cells.
The last step in the visualization procedure is to carefully add DAB following DAB exposure. Submit the brain slices to a last series of washes. Carefully place them on subbed microscope slides and allow them to dry overnight.
Once dried, clear the slides through a series of distilled water and alcohol washes after washing submerge the slice in citrus solve. Finally apply cover slips and store the slides in a box for safekeeping. There are many methods for the immunochemistry process.
The key to success is to create and follow a clear protocol. The slides are now ready to be analyzed using specialized neuro quantification software.Here. Bio quant software is used to quantify the vasopressin positive cells and fibers in the paraventricular nucleus of the mouse.Brains.
Use the measurement option to identify the specific area of interest and establish the visual field for neuro quantification. Here, the darkly stained VA suppressant, immunoreactive cell bodies and fibers are apparent. Use the light thresholding feature of the software to determine the total amount of positively stained tissue within a specific area.
This thresholding tells us how much vasopressin positive tissue is present. As seen here, the Californicus males spent more time grooming and crouching over the pups two hallmarks of paternal responses compared to a similar species. Micro sequencing analysis reveals that paternal California mice exhibit fewer interruptions in their grooming sequence compared to virgin animals.
When exposed to predator odor to determine if neurobiological variables are important in paternal responses, the amount of vasopressin positive tissue was quantified for several relevant brain areas. As seen here, the paternal californicus animals have more immune positive tissue in several of these brain areas. In closing, the comparative species approach allows you to identify key neurobiological systems associated with a certain behavior of interest.
However, the nature of the data is correlational, so it's important to add additional methodological techniques to further confirm the role of the neurobiological system and the behavior of interest.
