Neuroendocrine Correlates of Behavioral Polymorphism: Gonadotropin-Releasing Hormone
¹Jessica I. Lake, Henry S. Lange, Laura D. Mayer and Donna L. Maney
²Sara O'Brien
¹Department of Psychology, Emory University, Atlanta, GA;
²Department of Biology, University of Washington, Seattle, WA

Introduction

• The white-throated sparrow exhibits a behavioral polymorphism that correlates with a plumage phenotype. Those individuals with a white stripe (WS) on the crown exhibit more aggressive behavior, such as singing, territorial defense, and mate-seeking, than their tan striped (TS) counterparts. The TS morph utilizes a more parental approach, feeding their young more (Kopachena and Falls, 1993).

• Neuroendocrine differences between the morphs have also been described. For example, WS males have higher testosterone levels than TS males. Few such studies, however, have been conducted on females.

• Spinney et al. (2006) reported that TS females have higher levels of luteinizing hormone (LH) than WS females, suggesting morph differences in the regulation of the Hypothalamo-Pituitary-Gonadal (HPG) axis in females.

• The purpose of this study was to (1) replicate the morph difference in LH, and (2) look for morph differences in the HPG axis that might explain this difference. We focused on gonadotropin-releasing hormone (GnRH), a hypothalamic peptide that controls the release of LH from the pituitary.

Methods and Materials

1. Photostimulation
• 16 female white-throated sparrows (9 WS, 7 TS) were collected during the fall of 2005 and held on short days (10 hours of light, 14 hours of dark) for several months. They were then exposed to long days (15 hours of light, 9 hours of dark) for 30 days in order to stimulate the HPG axis.

2. Collection and Processing of Tissue
• The birds were deeply anesthetized with isoflurane.
• A blood sample was taken from the jugular vein.
• Brains were harvested from the skulls and immersed in fixative
• Brains were washed, frozen, and cut into 50 µm sections with a microtome.

3. Quantification of LH
• Blood samples were sent to U of Washington in Seattle, WA for LH radioimmunoassay.

4. Immunocytochemistry
• Brain sections containing the preoptic area (POA) were incubated with antibodies against GnRH.
• GnRH protein was visualized using an ABC-DAB method.

5. Alignment
• Brain sections were aligned using the intersection of the decussatio supraoptica ventralis (DSV) and the quintofrontal tract (QF) as a standard landmark (Stokes et al., 1974). This section was defined as C1 and successive caudal sections as C2, C3, etc. The section immediately rostral to C1 was defined as R1 and the preceding rostral sections R2, R3, etc.

6. Quantification
• GnRH neurons were counted at 40x magnification in alternate sections containing the POA.
• For each bird, the section containing the highest number of GnRH neurons was chosen for the quantification of soma size and optical density (OD) of staining.

Images were acquired at standard light levels using Leica software.
• Soma size was calculated using ImageJ software (NIH) by tracing the perimeters of nonoverlapping neurons.



• The OD of background staining was calculated for each section by averaging the OD of 5 randomly selected areas near, but not containing, GnRH labeling. This value was subtracted from the OD of each cell quantified in that section to give a “corrected OD”.

Data Analysis
• Results were analyzed using a single MANOVA with morph as a between-subjects variable.
• Post-hoc t-tests were used to look for effects of morph on plasma LH, cell number, soma size, and OD.

Results

1. LH levels in TS females were higher than those in WS females.


2. TS females had more GnRH neurons than did WS females.



3. GnRH somata were larger in WS females than in TS females.


4. The intensity of GnRH staining did not depend on morph (p = 0.3413).

Conclusions and Future Studies

As reported by Spinney et al. (2006), TS females had higher circulating LH than did WS females. This difference in plasma LH might be explained by differences in the GnRH system:

• TS females had more GnRH neurons than WS females, which could have represented greater GnRH production and thus more LH secretion;
• WS females had larger GnRH somata, which has been associated in some species with decreased secretory activity (e.g., Tsai & Jones, 2005).

In other words, more GnRH accumulation inside the soma may be associated with less release. This would be supported by morph differences in GnRH fiber innervation of the median eminence.

Resources

We are indebted to Henryk Urbanski for supplying the GnRH antibody and to Rashidat Ayantungi, Tulasi Ghimirey, and Marsha Howard for expert animal care. This work was supported by the National Science Foundation, the Center for Behavioral Neuroscience, and the Program for Scholarly Research and Inquiry at Emory.

References

Kopachena JG, Falls JB (1993). Re-evaluation of morph-specific variations in parental behavior of the white-throated sparrow. Wilson Bull. 105: 48-59.
Stokes TM, Leonard CM, Nottebohm F (1974). The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates. J. Comp. Neurol. 156: 337-374.
Spinney LH, Bentley GE, Hau M (2006). Endocrine correlates of alternative phenotypes in the white-throated sparrow (Zonotrichia albicollis). Horm Behav. 50: 762-771.
Tsai P, Jones JT (2005). Steroid-induced changes in the morphology of GnRH neurons in the male leopard frog, Rana pipiens: correlation with plasma gonadotropin and gonadal size. Gen Comp Endocrinol. 141: 152-160.