Genetic variation in total number and locations of GnRH neurons identified using in situ hybridization in a wild-source population

Abstract

The evolution of brain function in the regulation of physiology may depend in part upon the numbers and locations of neurons. Wild populations of rodents contain natural genetic variation in the inhibition of reproduction by winter-like short photoperiod, and it has been hypothesized that this functional variation might be due in part to heritable variation in the numbers or location of gonadotropin releasing hormone (GnRH) neurons. A naturally variable wild-source population of white-footed mice was used to develop lines artificially selected for or against mature gonads in short, winter-like photoperiods. We compared a selection line that is reproductively inhibited in short photoperiod (Responsive) to a line that is weakly inhibited by short photoperiod (Nonresponsive) for differences in counts of neurons identified using in situ hybridization for GnRH mRNA. There was no effect of photoperiod, but there were 60% more GnRH neurons in total in the Nonresponsive selection line than the Responsive selection line. The lines differed specifically in numbers of GnRH neurons in more anterior regions, whereas numbers of GnRH neurons in posterior areas were not statistically different between lines. We compare these results to those of an earlier study that used immunohistochemical labeling for GnRH neurons. The results are consistent with the hypothesis that the selection lines and natural source population contain significant genetic variation in the number and location of GnRH neurons. The variation in GnRH neurons may contribute to functional variation in fertility that occurs in short photoperiods in the laboratory and in the wild source population in winter.

Figure 1.

Representative coronal sections at a position equivalent to Plate 19 in Paxinos and Watson (‘86) showing GnRH neurons identified by in situ hybridization in the nonresponse and responsive selection line. The lower panel shows a negative sense-probe control with a lack of specific staining. Abbreviations: MPOA, medial preoptic area; OC, optic chiasm; 3V, third ventricle. Scale bar: 300μm.

Figure 2.

Estimated paired testes volume in males raised in long photoperiod (LD) or short photoperiod (SD) from lines artificially selected to be photoperiod nonresponsive (NR) or responsive (R). Sample sizes are shown in bars at the base of each bar. Statistical significance is indicated for selection line (Line) and photoperiod (Phot) (Mean +/− SEM).

Figure 3.

Estimated numbers of GnRH neurons, after adjustment for potential over-counting (see Methods section) labeled using in situ hybridization (ISH) in lines artificially selected to be photoperiod nonresponsive (NR) or responsive (R) to short photoperiod: (A) total number of neurons, (B) neurons in more anterior regions of the brain, and (C) neurons in more posterior regions of the brain (see Methods section for region boundaries). Statistical significance levels are indicated for differences between selection lines; there were no statistically significant effects of photoperiod. Individual values are plotted as either open diamonds (long photoperiod) or closed circles (short photoperiod) (Mean +/− SEM).