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The results are represented graphically as mean + standard error mean (sem).
Differences were considered to be statistically significant when P-value <0.05.
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marked at eight weeks of age; NE (51%), 5-HT (47%) and 5-HIAA (34%).The ontogenic profile of DOPAC levels differed between Mecp2-null and wt mice through age (as given by an interaction age x genotype, DOPAC: F3,27=7.67; p=0.010); we observed an increase in the DOPAC levels from three to eight weeks of age in the wt group that was not accompanied by the Mecp2-null mice group, that showed a decrease of DOPAC levels (figure 4.3A).
The 5-HT turnover was assessed by the ratio of 5-HIAA to 5-HT, which was significantly increased in this region in the Mecp2-null as compared to wt mice, both at three and eight weeks of age (figure 4.3B; F3,29=5.80, p=0.023). The DA turnover was also analyzed, as given by the ratios of DOPAC+HVA to DA and of each one of its metabolites to DA (DOPAC/DA and HVA/DA) between the Mecp2-null and wt controls and no differences were found (figure 4.3B).
In the MCx, comparisons revealed that the levels of NE and 5-HT were decreased, in the Mecp2-null mice, at both three (26% and 32%, respectively) and eight (33% and 28%, respectively) weeks of age, as compared to wt mice (figure 4.4A; NE: F3,27=7.26, p=0.012; 5-HT: F3,27=6.45, p=0.017). No differences were detected in DA or its metabolites, and no differences were detected in the 5-HIAA levels between genotypes.
The turnover of 5-HT showed an increase in the MCx in the Mecp2-null as compared to wt mice (figure 4.4B; F3,27=18.73, p=0.000). No differences were detected between genotypes in the metabolism of DA in this brain region.
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Figure 4.3. Neurochemical analysis of the prefrontal cortex region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolite and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean + sem. *, genotype effect; &, age effect and #, age x genotype interaction; all P<0.05).
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Figure 4.4. Neurochemical analysis of the motor cortex region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect, & age effect; all P<0.05).
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In the CPu, Mecp2-null mice showed levels of HVA and 5-HIAA lower than the wt levels, both at three (34% and 27%, respectively) and at eight weeks of age, when it was even more notorious (45% and 37%, respectively) (figure 4.5A; HVA: F3,29=8.99, p=0.006;
5-HIAA: F3,29=7.09, p=0.013). No other differences were noticed in the levels neurotransmitters or its metabolites.
The decrease observed in the DOPAC+HVA/DA, HVA/DA and 5-HIAA/5-HT ratios in the Mecp2-null as compared to wt mice showed that the metabolism of both DA and 5-HT was altered (figure 4.5B; DOPAC+HVA/DA: F3,29=7.54, p=0.010; HVA/DA: F3,29=16.66, p=0.000; 5-HIAA/5-HT: F3,29=4.41, p=0.044) in the CPu.
In the hippocampus, no significantly different levels of the neurotransmitters or their metabolites were observed between Mecp2-null and wt mice (figure 4.6A). However, at eight weeks of age Mecp2-null mice showed a decrease, although not statistically significant, in the levels of NE and 5-HT (42% and 41%, respectively) as compared to wt.
Interestingly, the ontogenic profile of HVA and 5-HT in this region was significantly different between wt and Mecp2-null mice, which evolved differently from three to eight weeks of age (figure 4.6A; HVA: F3,23=7.989, p=0.010; 5-HT: F3,26=7.74, p=0.031).
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Figure 4.5. Neurochemical analysis of the caudate-putamen region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect, & age effect; all P<0.05).
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Figure 4.6. Neurochemical analysis of the hippocampus region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect and & age effect; all P<0.05.)
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The nuclei of dopaminergic neurons are localized in the midbrain, arranged in the substantia nigra and ventral tegmmental areas, here dissected as ventral mesencephalon.
Interestingly, the levels of DA, its metabolites (DOPAC and HVA) and the DA turnover in the area of origin (SN-VTA) did not differ between Mecp2-null and wt male mice. Also, no other differences were found in the levels of the other neurotransmitters or metabolites between genotypes (figure 4.7A).
The only significant difference that we found between Mecp2-null and wt mice in this brain region was in the 5-HIAA/5-HT ratio, that was decreased in the Mecp2-null relative to wt mice (figure 4.7B; F3,22=4.83, p=0.039).
The D/MRN is the region of origin of the serotonin-producing cell bodies. These neurons project their axons to virtually all brain region, including the frontal regions of the brain. No differences at all were found in this brain region in the levels of monoamines and their metabolites or their turnover (figure 4.8A-B).
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Figure 4.7. Neurochemical analysis of the ventral mesencephalon region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect and & age effect; all P<0.05).
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Figure 4.8 Neurochemical analysis of the D/MRN region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect and & age effect;
all P<0.05).
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In the vestibular area, the only difference found between Mecp2-null and wt mice was a reduction of 36% in the levels of NE in the Mecp2-null as compared to wt mice, already from three and which was maintained at eight weeks of age (figure 4.9A;
F3,23=7.41, p=0.012).
Figure 4.9. Neurochemical analysis of the vestibular region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect and & age effect; all P<0.05).
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In the cerebellum, an interaction between age and genotype was observed for most neurotransmitters, which means that the response variable (neurotransmitter/metabolite) did not respond linearly to the variation of the factors (age and genotype). This means, for example, that for a given genotype, the change from one age to another generates a response whereas for the other genotype the response is much different (increasing or decreasing).
This age x genotype interaction was noticed for NE, 5-HT and 5-HIAA in the cerebellum. At three-weeks of age Mecp2-null NE, 5-HT and 5-HIAA levels all showed an increase of approximately 30% of the wt levels. However, at eight weeks of age the NE, 5-HT and 5-HIAA levels were 61%, 55% and 35% decreased, respectively, as compared to the wt levels (figure 4.10A; NE: F3,26=13.682, p=0.001; 5-HT: F3,26=7.830, p=0.010; 5-HIAA: F3,26=8.821, p=0.006).
Regarding all the other amines, the ontogenic profiles of Mecp2-null and wt mice were similiar. In the cerebellum, Mecp2-null mice showed levels of DA lower than the wt levels, both at three (39%) and at eight weeks of age, which was even more notorious (60%) (figure 4.10A; DA: F3,26=4.957, p=0.003). No other differences were noticed in the levels of neurotransmitters or its metabolites.
The turnover ratio DOPAC/DA was increased in the Mecp2-null as compared to wt (figure 4.10B; DOPAC/DA: F3,24=4.640, p=0.041).
At three weeks of age, cerebellar levels of HVA were not detectable by HPLC/EC in a considerable number of samples. Therefore, in this brain region, only the levels of DA and DOPAC were considered in the analysis.
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Figure 4.10. Neurochemical analysis of the cerebellum region of wt and Mecp2-null mice at three and eight weeks of age. (A) Concentration of each neurotransmitter and metabolites and (B) neurotransmitter (DA and 5-HT) turnover. (wt, wild type; ko, Mecp2-null. All values are mean+sem. * genotype effect, & age effect and # age x genotype interaction; all P<0.05).
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Serotonergic innervation
Given that in Mecp2-null mice at three weeks of age the levels of 5-HT were reduced in both the PFCx and the MCx, two brain regions highly innervated by serotonergic fibres, we explored whether the observed reduction in the levels of the neurotransmitter was associated with a reduction in the number of serotonergic fibres in these regions.
The serotonergic innervation was evaluated in the PFCx (ko, n=4; wt, n=3) and MCx (n=4 for each genotype) areas of three-week-old Mecp2-null and wt mice by counting the number of serotonergic fibre intersections with L-cycloids. Overall, the analysis of the 5-HT imunohistochemical sections failed to show any significant difference in the density of 5-HT fibres between Mecp2-null and wt groups (figure 4.11; t-test; PFCx, p=0.275 and MCx, p=0.488).
Figure 4.11. Serotonergic innervation in the motor cortices of Mecp2-null mice and their wt littermate controls at 3 weeks of age. (A-B) Representative photomicrograph of 5-HT imunohistochemistry reactivity in the MCx region. (C) Quantification of the serotonergic innervation in the PFCx and MCx brain areas. Values represent mean+sem, wt, wild-type; ko, Mecp2-null; N/A, number of fibre intersections per area; PFCx, prefrontal cortex; MCx, motor cortex.
A
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mRNA expression levels of NE and 5-HT receptors and transporters
We then assessed, by qRT-PCR, the levels of mRNA expression of several noradrenergic and serotonergic receptors and transporters in the PFCx and MCx of three- and eight-week-old Mecp2-null and wt animals. The receptors in study were selected based on their expression in these brain areas and on the way they mediate the effects of the neurotransmitter (role in behaviour).
We found that in the PFCx the mRNA expression levels of NET and of the adrenergic receptor Adrα2a, both at three and eight weeks of age, were reduced in the Mecp2-null as compared to wt mice (figure 4.12; NET: F3,18=9.76, p=0.006; Adrα2a:
F3,20=8.392, p=0.009). A trend was observed as the levels of Adrα2a in both wt and Mecp2-null mice behave differently at the two ages, as given by the almost significant age and genotype interaction (figure 4.12; Adrα2a: F3,20=4.237, p=0.053). The serotonergic receptors Htr2a and Htr3a were also altered in this brain region of Mecp2-null mice.
Interestingly, the ontogenic profile of Htr2a expression was significantly different between wt and Mecp2-null mice, which evolved differently from three to eight weeks of age (figure 4.12; Htr2a: F3,23=4.32, p=0.05). In the wt animals there was an increase of the Htr2a mRNA levels, which did not happen in the Mecp2-null mice. Additionally, the levels of Htr3a mRNA, were reduced in the Mecp2-null mice, at both timepoints, as compared to wt levels (Htr3a: F3,20=4.91, p=0.038). No differences were detected in the levels of expression of the other genes studied.
In the MCx differences were found in the serotonergic receptors Htr2a and Htr3a.
Relative to wt values, the levels of expression of Htr2a receptor in the Mecp2-null mice were reduced (figure 4.12; Htr2a: F3,18=7.690, p=0.013). The ontogenic profile of Htr3a receptor expression was significantly different between wt and Mecp2-null mice, which evolved differently from three to eight weeks of age, as given by an interaction between age and genotype for the expression levels of this receptor (figure 4.12; Htr3a: F3,20=6.86, p=0.016).
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Figure 4.12. Expression levels of NE and 5-HT receptors and transporters in the Mecp2-null mice at 8 weeks of age. (n>5; wt, wild type; ko, Mecp2-null. Values represent mean+sem. &, age effect; *, genotype effect; #, age x genotype interaction; all P<0.05. Data analysed by 2-way ANOVA).
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