C-fos down-regulation inhibits testosterone-dependent male sexual behavior and the associated learning

Abstract

Environmental stimulation results in an increased expression of transcription factors called immediate early genes (IEGs) in specific neuronal populations. In male Japanese quail, copulation with a female increases the expression of the IEGs zenk and c-fos in the medial pre-optic nucleus (POM), a key nucleus controlling male sexual behavior. The functional significance of this increased IEG expression that follows performance of copulatory behavior is unknown. We addressed this question by repeatedly quantifying the performance of appetitive (learned social proximity response) and consummatory (actual copulation) sexual behavior in castrated, testosterone-treated males that received daily intra-cerebroventricular injection of an antisense oligodeoxynucleotide targeting c-fos or control vehicle. Daily antisense injections significantly inhibited the expression of copulatory behavior as well as the acquisition of the learned social proximity response. A strong reduction of the proximity response was still observed in antisense-treated birds that copulated with a female, ruling out the indirect effect of the absence of interactions with females on the learning process. After a 2-day interruption of behavioral testing but not of antisense injections, birds were submitted to a final copulatory test that confirmed the behavioral inhibition in antisense-injected birds. Brains were collected at 90 min after the behavioral testing for quantification of c-fos-immunoreactive cells. A significant reduction of the number of c-fos-positive cells in the POM but not in other brain regions was observed following antisense injection. Taken together, the data suggest that c-fos expression in the POM modulates copulatory behavior and sexual learning in male quail.

Keywords: Japanese quail; appetitive sexual behavior; c-fos antisense; medial pre-optic nucleus; sexual learning.

Figure 1

Experimental design: Animals were castrated 4 weeks post-hatching and implanted with a stereotaxic injection-cannulae at 8 weeks post-hatching. After a 2 weeks recovery period, animals were subcutaneously implanted with a testosterone (T)-filled Silastic^™ capsule. Starting seven days after the testosterone implantation animals were daily ICV injected with either 2 μg of antisense (AS), 2 μg of scrambled oligodeoxynucleotides (SC) or vehicle. On the second day of ICV injections animals were pretested for gross behavioral effects of the treatment in an empty test arena. On third, fourth, fifth and sixth days of ICV injections animals were tested for appetitive and consummatory sexual behavior. Animals were allowed to rest on seventh and eighth day of ICV injections. On the ninth day of ICV injections animals were tested one last time for appetitive and consummatory sexual behavior and killed for brain collection 90 minutes after the behavioral tests.

Figure 2

Schematic drawings of coronal sections through the quail brain illustrating the areas where c-fos expression was quantified (dark areas). Panels A to D represent coronal sections through the quail brain presented in a rostral to caudal order. Aq: aqueductus mesencephali; BNST, bed nucleus of the stria terminalis; CA, commissura anterior; Cb, cerebellum; CO: chiasma opticum; CP, commissura posterior; CPa: commissural pallii; FPL, fasciculus prosencephali lateralis; GLv: nucleus geniculatus lateralis, pars ventralis; mPOA: medial preopic area; Hp, hippocampus; LaM, lamina mesopallialis; M, mesopallium; N, nidopallium; PAG, substantia grisea centralis; SL, nucleus septalis lateralis; SM, nucleus septalis medialis; TeO: tectum opticum; TnA, nucleus taeniae of the amygdala; TSM: tractus septopallio-mesencephalicus; VL, ventriculus lateralis; VLT: nucleus ventro-lateralis thalami; VT, ventriculus tecti mesencephali.

Figure 3

Alignment of the sequence obtained from the Japanese quail with the chicken c-fos (NM_205508). The start codon ATG is in bold. The sequence upstream of the ATG (92 nucleotides) showed 99% identity with the chicken sequence. The sequence in the box is targeted by the antisense LNA is fully identical to the corresponding chicken sequence

Figure 4

Inhibitory effect of c-fos down-regulation by daily AS LNA injections in the third ventricle at the level of the POM on one measure of the appetitive male sexual behavior (learned social proximity response) in castrated male quail treated with exogenous testosterone. Mean (±SEM) time that the subjects stood looking through the window is presented. The line graphs summarize the social proximity response during the acquisition phase, while the bar graphs represent the social proximity response during the final test. *=p<0.05, ***=p<0.001 versus CTRL by Bonferroni post-hoc or t-test.

Figure 5

Inhibitory effect of c-fos down-regulation by daily AS LNA injections in the third ventricle at the level of the POM on two measures of the male consummatory sexual behavior, the frequencies of Mount Attempts (MA; A) and Cloacal Contact Movements (CCM; B) in castrated male quail treated with exogenous testosterone. Mean (±SEM) number of MA or CCM produced by the subjects of the AS or CTRL group are presented. The line graphs summarize the behavior frequencies during the acquisition phase, while the bar graphs represent the frequencies during the final test. *=p<0.05 and **=p<0.01 versus CTRL by Bonferroni post-hoc or t-test.

Figure 6

Inhibitory effect of c-fos down-regulation by daily AS LNA injections in the third ventricle at the level of the POM on one measure of the appetitive male sexual behavior (learned social proximity response) in castrated male quail treated with exogenous testosterone. The results concern exclusively subjects that showed at least one MA during the first three days of behavioral testing. Mean (±SEM) time the subjects stood looking through the window is presented. The line graphs summarize the social proximity response during the acquisition phase, while the bar graphs represent the social proximity response during the final test. **=p<0.01 versus CTRL by Bonferroni post-hoc test.

Figure 7

Quantification (means ± SEM) of FOS-ir cells in the brain of CTRL and AS-treated subjects. Brains were collected 90 min following a test trial consisting of 5 min social proximity response testing and 5 min of consummatory sexual behavior testing where subjects were able to freely interact with a sexually mature female. *=p<0.05 versus CTRL by t-test.

Figure 8

A. Quantification (means ± SEM) of FOS-ir cells in the medial part of the POM of CTRL and AS-treated subjects as a function of whether they did or did not display cloacal contact movements (CCM) or mount attempts (MA) during the final tests before brain collection. B. Representative photomicrographs illustrating FOS-ir cells in the POM of the 4 sub-groups of birds as defined in panel A. Magnification bar= 500 μm; AC: anterior commissure, the * indicates the position of the third ventricle.