Quantification of neurons expressing androgen receptor and volume estimation of the basolateral nuclear group of the canine amygdaloid body

A protocol was developed for the stereological quantification of neurons expressing androgen receptor (AR) in the basolateral nuclear group (BNG) of the canine amygdaloid body. The Cavalieri method was used to estimate the BNG volume and the physical disector technique was applied for assessing the numerical densities and total numbers of both ordinary and AR-positive BNG neurons. The overall number of BNG neurons and the BNG volume were assessed on Nissl-stained sections, while AR was visualised using indirect immunohistochemistry. The morphological differentiation between neurons, astrocytes, and oligodendrocytes in these immunohistochemical sections was hampered by the cytoplasmic localisation of AR in these cells. Therefore, an additional criterion was developed based on the nuclear diameters of these cells. With the cutoff value of 7.4 μm, a sensitivity of 97.7% and specificity of 97.6% were obtained. A negative correlation was found between the BNG volume and the numerical density of its neurons, implicating that a large BNG will not necessarily have a higher number of neurons. Therefore, the numerical density or BNG volume should always be assessed in addition to the total number of neurons, justifying the use of the physical disector instead of the fractionator technique in the present study. However, higher coefficients of error were obtained for the total number of neurons with the physical disector method because of the indirect measurement of cell numbers. Therefore, the precision of the estimates must be high enough when using the disector method to compensate the precision loss caused by this indirect calculation of the total cell number.