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Bird Song System - FAQ

Frequently Asked Questions about the Bird Song System module.

Q1:  Should I be able to measure something in every image provided?

Q2:  I can't find any Area Xs in some animals.  Why is this?

Q3:  Some of the birds only seem to have images for one hemisphere.  Why is this and what can be done about it?

Q4:  How many birds per condition would we need to obtain significant results?

Q5:  The position of HVC seems to keep shifting in different photographs.  Why does this happen?

Q6:  The tissue is broken/torn and the relevant structure is affected.  How can I deal with this?

Q7:  One student is getting numeric results that are why out of range from all the other students, even when they are quantifying birds from the same treatment.  Is this to be expected?

 


 

Q1: Should I be able to measure something in every image provided?

No.  Depending on who was capturing the images, some pictures were taken of sections before the nucleus was visible, and some were taken of sections after the nucleus had disappeared.  This was done to ensure that the entire region of interest was captured.

We chose to keep such images (that lack a structure) in our collection to help maintain student experimental blindness.  Subsequently, images lacking a structure to draw/quantify help to control for derelict imagination.  Furthermore, some of our subjects do not possess some of the regions of interest, such as Area X.  See Q2 below for more explanation.

 

Q2: I can't find any Area Xs in some animals.  Why is this?

Students feel compelled to measure something on every last image, even when they are told that females don't have an Area X and that many of the birds in the database are female.

Control females lack an Area X, and it is rarely quantifiable on most females treated with 5-μg estradiol.  Some females treated with 15-μg estradiol may have a small Area X, but it is still possible that it is not quantifiable as well.

To maintain student experimental blindness, subjects lacking a quantifiable structure (such as Area X) are still given a set of images capturing the homologous region of the striatum in which the structure (Area X) would normally occur.

Students are usually asked to begin by quantifying the control males (although they are not told this), then move onto females of increasing estradiol dosage (5-, 15-, and 50-μg, respectively).  This sequence orients students well to the task.

 

Q3:  Some of the birds only seem to have images for one hemisphere.  Why is this and what can be done about it?

When preparing tissue for mounting onto slides, the tissue can easily broken/torn.  Sometimes the tissue is so badly damaged in one hemisphere that it becomes unquantifiable.  As a result, images of the tissue were not captured for that hemisphere.

In this database, three males have a quantifiable HVC in only one hemisphere (BK8, Y60, and Y63), and two males have a quantifiable RA in only one hemisphere (PK64 and Y60).

To acquire data for subjects with a single-sided image set, students should follow the normal instructions to quantify the volume of the structure, and then use that value (un-averaged) to represent the structure in the brain.

 

Q4:  How many birds per condition would I need to obtain significant results?

We haven't systematically eliminated data in order to determine this, but students can certainly obtain the desired sex difference with n=4 per group.

More subtle differences will be harder to statistically establish as the sample size goes down because the statistical power will also go down.

 

Q5:  The position of HVC seems to keep shifting in different photographs.  Why does this happen?

The sequence of images is presented rostral to caudal.  As you move caudally through the brain, HVC becomes more medially situated.


Q6:  The tissue is broken/torn and the relevant structure is affected.  How can I deal with this?

In the instance in which tissue is damaged, but all the pieces of a given structure are still present, students can measure what remains of the structure.  Sometimes, students can extrapolate across a gap, or they can ignore the missing portion if it is seemingly small.  The latter does add to the error variance, but the effect of estradiol treatment and sex differences are so robust that they are still likely to obtain significant differences.

 

Q7:  One student is getting numeric results that are way out of range from all the other students, even when they are quantifying birds from the same treatment.  Is this to be expected?

If a student's data are discrepant from his peers by ten-fold or more, then there is a problem.  Such data are usually the result of at least one of the following oversights:

  • failing to calibrate each image series (by changing the Set Scale values in Image J) as detailed in the instructions (Area X, HVC, and RA all have different values)
  • failing to multiply the total area quantified (in mm2) by 0.12-mm in order to obtain the corresponding volume (in mm3)
  • failing to average the volumes of both hemispheres (in double-sided image sets)
  • poor handling of data within or organization of a spreadsheet

If the discrepancy is less than ten-fold, then the data isn't questioned because the study will still work favorably.

 

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