Gender-dependent modulation of brain monoamines and anxiety-like behaviors in mice with genetic serotonin transporter and BDNF deficiencies

Abstract

1. Brain-derived neurotrophic factor (BDNF) supports serotonergic neuronal development and our recent study found that heterozygous mice lacking one BDNF gene allele interbred with male serotonin transporter (SERT) knockout mice had greater reductions in brain tissue serotonin concentrations, greater increases in anxiety-like behaviors and greater ACTH responses to stress than found in the SERT knockout mice alone. 2. We investigated here whether there might be gender differences in these consequences of combined SERT and BDNF deficiencies by extending the original studies to female mice, and also to an examination of the effects of ovariectomy and tamoxifen in these female mice, and of 21-day 17-beta estradiol implantation to male mice. 3. We found that unlike the male SERTxBDNF-deficient mice, female SERTxBDNF mice appeared protected by their gender in having significantly lesser reductions in serotonin concentrations in hypothalamus and other brain regions than males, relative to controls. Likewise, in the elevated plus maze, female SERTxBDNF-deficient mice demonstrated no increases in the anxiety-like behaviors previously found in males. 4. Furthermore, female SERTxBDNF mice did not manifest the approximately 40% reduction in the expression of TrkB receptors or the approximately 30% reductions in dopamine and its metabolites that male SERTxBDNF did. After estradiol implantation in male SERTxBDNF mice, hypothalamic serotonin was significantly increased compared to vehicle-implanted mice. These findings support the hypothesis that estrogen may enhance BDNF function via its TrkB receptor, leading to alterations in the serotonin circuits, which modulate anxiety-like behaviors. 5. This double-mutant mouse model contributes to the knowledge base that will help in understanding genexgenexgender interactions in studies of SERT and BDNF gene polymorphisms in human genetic diseases such as anxiety disorders and depression.

Fig. 1.

Concentrations of 5-HT in different brain regions were significantly reduced in sb double-mutant mice compared to SB and Sb mice (pg/mg protein, mean ± SEM, n=5–6): (A) Hippocampus −79%, (B) Hypothalamus −80%, (C) Brain stem −79%, (D) Striatum −69%. A significant further serotonin reduction of 37% in hippocampus and 43% in hypothalamus was observed in sb mice (A and B.^# p<0.01) compared to sB mice. In addition, male compared to female sb mice had a significant reduction of 5-HT concentrations in hippocampus and striatum, ^††† p<0.001; in hypothalamus,^†† p<0.01; in brain stem, ^† p<0.04. Furthermore, both genders of sB mice had significant reductions in all four brain regions (^*** p<0.001) relative to SB mice. Sb mice compared to SB controls had significant reductions of 5-HT in only the hippocampus (^§§ p<0.008) but not in other brain regions.

Fig. 2.

Concentrations of 5-HIAA were also significantly reduced in corresponding brain regions of sb double-mutant mice compared with SB and Sb mice: (A) Hippocampus −65%, (B) Hypothalamus −60%, (C) Brain stem −48%, (D) Striatum −55% compared to SB controls. A significant further reduction of 5-HIAA 33% in hippocampus and hypothalamus was demonstrated in male sb mice (^### p<0.001) compared to sB male mice. In contrast, the sb female double-mutant mice showed significantly different 5-HIAA concentrations in hippocampus, hypothalamus, and striatum (^††† p< 0.001), and brain stem (^†† p<0.01) compared to sb male mice. Again, both genders of sB mice had significant reductions in all four brain regions (^*** p<0.001) relative to SB mice. Sb mice were only significantly reduced in striatum (p<0.04) but not in other brain regions compared to SB controls.

Fig. 3.

(A) Striatal dopamine concentrations were significantly reduced by 32% (^*** p<0.0001) only in male sb double- mutant mice compared to SB, Sb, and sB mice. DA concentrations in female sb mice were significantly different compared to sb male mice (^††† p<0.0004). (B) and (C) DOPAC and HVA concentrations were also significantly reduced by 32% (^*** p<0.001) and 30% (^*** p<0.001) only in male sb mice compared to SB, Sb, and sB mice. In contrast, DOPAC (^††† p<0.001) and HVA (^†† p<0.01) concentrations in female sb mice were significantly different compared to male sb mice.

Fig. 4.

Male sb double-mutant mice showed heightened anxiety-like behavior in the elevated plus-maze test relative to SB controls. (A) sb double-mutant male mice spent less time on the open arms (^** p<0.006) and (B) made fewer open arm entries (^* p<0.003) compared to SB or Sb controls. Female sb double-mutant mice were not different from their littermate controls, but female sb double-mutant mice were significantly different from male sb mice in percent open time (^†† p<0.001). (C) Male sb double-mutant mice spent more time on the closed arm than male SB mice (p<0.05). In addition, sb male mice were significantly different from female sb mice (p<0.02). (D) sb male mice exhibited a significant genotype difference in closed arm entries relative to SB controls. Data in Figs. 4 and 5 are means ± SEM, n=12–15 males and n=10–12 females per genotype.

Fig. 5.

Effects of 17-β estradiol (0.5 mg×21 days) treatment on 5-HT concentrations in the hypothalamus. As indicated, male sb mice had a significant increase in 5-HT concentrations in hypothalamus after estradiol treatment compared to vehicle controls (^** p<0.02). For comparison, 5HT concentrations from mutant male mice (Fig. 1B) are also presented.

Fig. 6.

Dopamine concentrations in striatum in female sb mice with ovariectomy plus implanted tamoxifen. The results showed that significantly (p<0.001) reduced dopamine concentration are found following ovarectomy plus implanted tamoxifen (n=15) mice compared to placebo (n=15) or tamoxifen treatment only (n=15).

Fig. 7.

(A) Reductions of 5-HT and BDNF affect the development of neuronal dendritic branches in sb mice. The morphology of brain neuronal hippocampal near dentrate gyrus dentrities and spines was evaluated in 20 fields (scale bars=10 μm). (B) The quantity of dendrites in brain sections with Golgi impregnation. Both genders had significant reductions in sb mice (p<0.0001) compared to SB mice using two-way ANOVA test.

Fig. 7.

(A) Reductions of 5-HT and BDNF affect the development of neuronal dendritic branches in sb mice. The morphology of brain neuronal hippocampal near dentrate gyrus dentrities and spines was evaluated in 20 fields (scale bars=10 μm). (B) The quantity of dendrites in brain sections with Golgi impregnation. Both genders had significant reductions in sb mice (p<0.0001) compared to SB mice using two-way ANOVA test.

Fig. 8.

(A) Expression of BDNF protein was examined in both genders of three genotypes (n=8). (B) The blots were scanned and the relative abundance of the 30 kDa protein was estimated by quantitative analysis of the Western blots. The results were expressed as density means. BDNF protein expression was calculated as a percent of the mean values, with significant reductions in the hypothalamus of −47% in Sb and −60% in sb mice compared to SB controls (^*** p<0.001).

Fig. 9.

(A) Expression of TrkB protein was evaluated in both genders of the four genotypes (n=8). (B) The blots were scanned and the relative abundance of the 145 kDa protein was estimated by quantitative analysis of the Western blots. Sb −34% (^§§ p<0.001), sB −41% (^*** p<0.001), and sb double-mutant mice −67% (^*** p<0.001) of both genders had significantly reduced TrkB protein expression. In addition, the male sb mice had significant further reduction in TrkB protein compared to sb female mice (^††† p<0.001). There was no TrkB gender difference in SB, Sb, and sB genotypes.