Rett syndrome (RTT, MIM #312750) is a severe neurological disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene 1. Female patients are affected with an incidence of 1/15000 live births 23 and develop normally from birth to 6-18 months of age before the onset of deficits in autonomic, cognitive, motor functions and autistic features 45. Several studies shed light on the involvement of catecholaminergic and serotonergic disturbances as a major contributor of the disease in RTT patients and Mecp2 mouse models. Neurochemical analyses of biogenic amines in Mecp2-null male (Mecp2^-/y) were previously performed on whole brain homogenates showing that the concentration of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) was reduced after birth 67. Moreover, the absence of Mecp2 impairs several functions that are regulated by bioaminergic nuclei or peripheral ganglia where such neurochemical deficits occur 89101112. However, these studies are discordant for several parameters. Here, we evaluated the level of DA, NE and 5-HT and their catabolites homovanillic Acid (HVA), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA), using high performance liquid chromatography, combined with electrochemical detection (HPLC/EC). We performed our analyses on micropunched brain samples from the cortex, caudate-putamen, hypothalamus, midbrain (Substantia Nigra + Ventral Tegmental Area) and the hindbrain (Brainstem + Pons) (Figure 1) of Mecp2^-/y mice and their respective age-matched wild-type (WT) littermates from postnatal day 35 (P35, disease onset) and 55 (P55, severe phenotype).

Mecp2^-/y cortical neurons exhibit several alterations, ranging from reduced activity to abnormal dendritic spine morphology 1819. These defects are influenced by Bdnf dosage 20. Bdnf plays a key role in neuronal survival, differentiation and synaptic plasticity 21. Bdnf metabolism was shown to be involved in the development and function of catecholaminergic neurons, affected in Mecp2^-/y 22232425. Recently, several studies identified NE reductions in the piriform cortex of Mecp2-null mouse at P35 9 and in the prefrontal and motor cortex at both 3 (P21) and 8 (P56) weeks of age 11. We were not able to reproduce these findings. One possible explanation for this divergence could be differences in the sampling procedure. We performed micropunches of the motor cortex. Different cortical territories receive monoaminergic inputs from different nuclei, some areas being less densely innervated than others 26. Serotonergic dosage in Mecp2^-/y brains were also reported and discrepancies are existing. Isoda and collaborators did not to identify any modifications of 5-HT and its main catabolite 5-HIAA in the prefrontal cortex from P14 to P56 10. However, another laboratory argued for a reduction in the same structure of 5-HT only, at 3 and 8 weeks of age 11. As Isoda and coauthors measured, we did not found significant reductions of 5-HT or 5-HIAA. Another group reported a 5-HT deficit in whole brain extracts in the same developmental window 7. Unfortunately, samples used in this study came from pooled 6 to 8 weeks Mecp2^-/y animals, two ages that are know to be phenotypically different according to the postnatal development of the Mecp2^-/y mouse pathology 27. These results thus combine 5-HT contents from both mildly and severely affected animals.

It is generally believed that catecholamines play a role in hypothalamic nuclei neurotransmission and that their release is modulated by several factors including Bdnf and leptin 17. Here, we report a postnatal alteration of hypothalamic NE and 5-HT contents that follows a biphasic scheme. These biogenic amines were found significantly decreased at P35 in Mecp2^-/y animals whereas their levels are not significantly different from WT at P55. A recent study showed that specific deletion of Mecp2 in Sim1-positive neurons in the paraventricular nucleus (PVN) of the hypothalamus results in higher serum level of leptin 13. Since leptin was proposed to lower NE-dependant oxytocin release in the PVN 17 and to reduce firing of serotonergic neurons 28 it was tempting to assess the level of bioamines in this brain area. Our results at P35 could reflect a leptin-dependant bioaminergic reduction. However, Fyffe and coauthors did not identify alterations at 6 weeks (P42) but noticed a significant increase 42 weeks. It is important to stress that Mecp2-deletion is not restricted to hypothalamic neurons in the model we used. Moreover, mice used in one study were of mixed 129/FVB genetic background 13 while we used C57Bl6 Mecp2^-/y mice 29.

In agreement with Taneja and co-workers 9, we found a reduction of DA and HVA in our hindbrain samples containing both the brainstem and the pons at P35. However, we did not found DOPAC reduction. Taneja et al. used a different Mecp2^-/y strain (i.e. generated by Jaenisch laboratory; 30). It would be interesting to investigate possible difference in the activity/modulation of DA degradation enzymes in these two models. In the Bird mouse 29 no difference was found in monoamine oxydase (MAO-A and MAO-B) activity in the cortex 11. It remains to be determined if MAO and/or catechol-O-methyl transferase (COMT) levels are impaired elsewhere. Interestingly, the caudal structures are the most affected with dopaminergic disturbances at P35 when the Mecp2 animals display a mild phenotype, and a global monoaminergic deficit at P55 affecting DA, NE, 5-HT and their degradation products. This is in good agreement with several studies arguing for a brainstem and pontic basis for the Mecp2^-/y phenotype and RTT symptoms in humans 3132. The observed increase in the serotonergic ratio (Table 1) at P55 in the pons and brainstem supports a possible alteration of 5-HT projections innervating this structure and modulating the autonomic functions altered in the absence of Mecp2 33.

Mecp2^-/y mice display a progressive and postnatal alteration in their motor behavior 1134. Interestingly, mice with a targeted deletion of Mecp2 in Th-expressing neurons show a deficit in motricity among other dysregulations 7. We recently described in mice harboring a constitutive deletion of Mecp2 progressive alterations of the SNpc and the nigrostriatal dopaminergic pathway, a key component of the brain motor circuitry 12. Moreover, some of the behaviors impaired were ameliorated by L-Dopa, arguing for a strong, although clearly non-unique, involvement of the DA disturbances in Mecp2^-/y motor phenotype. In complement to this previous study, we here pursued our neurochemical investigations for NE and 5-HT in the SNpc and its striatal target. NE, provided from the Locus Coeruleus, A1 and A2 (from brainstem) is implicated in several brain functions and its action on midbrain DA neurons was shown to modulate motivational behaviors 3536. The absence of NE and 5-HT disturbances in the midbrain and the caudate-putamen confirm previous findings 1011.

The consequence of Mecp2-deficiency on the integrity of dopaminergic and serotonergic nuclei could be indexed on the HVA + DOPAC/DA and 5-HIAA/5-HT ratios respectively since a toxic alteration of dopaminergic and serotonergic systems induces counteradaptative processes that impact on this values 3738. The calculated ratios are reported in Table 1. Results suggest dopamine turnover was left unaffected in all the structures investigated at both P35 and P55 in Mecp2^-/y compared to their WT littermates. Conversely, hypothalamus and caudate-putamen dosage revealed a significant increase of 5-HIAA/5-HT ratio at P35 but not P55. Values from the pons and brainstem indicate a clear increase in the serotonergic turnover in Mecp2^-/y at P55. However, this index was not modified in the cortex and the midbrain at P35 and P55. Alteration of the serotonergic innervation lead to an increase in the turnover 37. Interestingly, a reduction in 5-HT immunoreactive fibers was reported in the hippocampus of Mecp2^-/y animals 10. It remains to be elucidated if such phenomenon contribute to serotonergic deficits described here.

Our results indicate that the level of catecholamines and serotonin is differentially affected in Mecp2^-/y brain areas in a time-dependent fashion (Figure 4). Considering behavioral and physiological function altered in the context of Mecp2-deficiency and the positive impact of drugs targeting this system, monoamine neurochemical dosage could help identifying therapeutics resetting central bioaminergic deficits in different brain areas of the Mecp2-deficient mouse.

LV and JCR conceived and designed the experiment. NP and AG performed the experiments. NP and JCR analyzed the data. NP and JCR wrote the paper. All authors read and approved the final manuscript.