Serotonin disruption at gestation alters expression of genes associated with serotonin synthesis and reuptake at weaning.

RATIONALE: Serotonin (5-HT) is a monoamine neuromodulator that plays a key role in the organization of the central nervous system. 5-HT alterations may be associated to the emergence of social deficits and psychiatric disorders, including anxiety, depression, and substance abuse disorders. Notably, disruption of the 5-HT system during sensitive periods of development seems to exert long-term consequences, including altered anxiety responses and problematic use of alcohol. OBJECTIVE: We analyzed, in mice, the effects of transient 5-HT depletion at gestation (a developmental stage when medial prefrontal cortex (mPFC) 5-HT levels depend exclusively on placental 5-HT availability) on 5-HT central synthesis and reuptake at weaning. We also explored if 5-HT disruption at the embryonic stage influences behavioral outcomes that may serve as a proxy for autistic- or anxiety-like phenotypes. METHODS: C57/BL6 male and female mice, born from dams treated with a 5-HT synthesis inhibitor (PCPA; 4-Chloro-DL-phenylalanine methyl ester hydrochloride) at gestational days (G)13.5-16.5, were subjected to a behavioral battery that assesses social preference and novelty, compulsive behavior, stereotypies, and ethanol's anti-anxiety effects, at postnatal days (P) 21-28. Afterwards, expression of the genes that encode for 5-HT synthesis (Tph2) and SERT (5-HT transporter) were analyzed in mPFC via real-time RT-PCR. Dopamine 2 receptor (D2R) expression was also analyzed via RT-PCR to further explore possible effects of PCPA on dopaminergic transmission. RESULTS: Transient 5-HT disruption at G13.5-16.5 reduced Tph2 expression of both male and female mice in mPFC at P23. Notably, female mice also exhibited higher SERT expression and reduced D2R expression in mPFC. Mice derived from 5-HT depleted dams displayed heightened compulsive behavior at P21, when compared to control mice. Alcohol anti-anxiety effects at early adolescence (P28) were exhibited by mice derived from 5-HT depleted dams, but not by control counterparts. No social deficits or stereotyped behaviors were observed. CONCLUSION: Transient 5-HT inhibition at gestation resulted in altered expression of genes involved in 5-HT synthesis and reuptake in mPFC at weaning, a period in which the 5-HT system is still developing. These alterations may exert lingering effects, which translate to significant compulsivity and heightened sensitivity to the anxiolytic effects of alcohol at early adolescence.

MeCP2 deficiency exacerbates the neuroinflammatory setting and autoreactive response during an autoimmune challenge.

Rett syndrome is a severe and progressive neurological disorder linked to mutations in the MeCP2 gene. It has been suggested that immune alterations may play an active role in the generation and/or maintenance of RTT phenotypes. However, there is no clear consensus about which pathways are regulated in vivo by MeCP2 in the context of immune activation. In the present work we set to characterize the role of MeCP2 during the progression of Experimental Autoimmune Encephalomyelitis (EAE) using the MeCP2308/y mouse model (MUT), which represents a condition of "MeCP2 function deficiency". Our results showed that MeCP2 deficiency increased the susceptibility to develop EAE, along with a defective induction of anti-inflammatory responses and an exacerbated MOG-specific IFNγ expression in immune sites. In MUT-EAE spinal cord, we found a chronic increase in pro-inflammatory cytokines gene expression (IFNγ, TNFα and IL-1ß) and downregulation of genes involved in immune regulation (IL-10, FoxP3 and CX3CR1). Moreover, our results indicate that MeCP2 acts intrinsically upon immune activation, affecting neuroimmune homeostasis by regulating the pro-inflammatory/anti-inflammatory balance in vivo. These results are relevant to identify the potential consequences of MeCP2 mutations on immune homeostasis and to explore novel therapeutic strategies for MeCP2-related disorders.

Normal mitral cell dendritic development in the setting of Mecp2 mutation.

Rett syndrome (RTT) is an autism spectrum disorder caused by mutation in the gene encoding methyl CpG binding protein 2 (MECP2). Evidence to date suggests that these disorders display defects in synaptic organization and plasticity. A hallmark of the pathology in RTT has been identified as decreased dendritic arborization, which has been interpreted to represent abnormal dendritic formation and pruning during development. Our previous studies revealed that olfactory axons display defective pathfinding and targeting in the setting of Mecp2 mutation. In the present work, we use Mecp2 mutant mouse models and the olfactory system to investigate dendritic development. Here, we demonstrate that mitral cell dendritic development proceeds normally in mutant mice, resulting in typical dendritic morphology at early postnatal ages. We also failed to detect abnormalities in dendritic inputs at symptomatic stages when glomeruli from mutant mice appear smaller in area than the wild type (WT) (6 weeks postnatally). Collectively, these findings suggest that the initial defects in glomeruli impairment seen with Mecp2 mutation do not result from abnormal dendritic development. Our results using the olfactory system indicate that dendritic abnormalities are not an early feature in the abnormalities incurred by Mecp2 mutation.

Inhibitory role of diazepam on autoimmune inflammation in rats with experimental autoimmune encephalomyelitis.

Glutamate and GABA are the main excitatory and inhibitory neurotransmitters in the CNS, and both may be involved in the neuronal dysfunction in neurodegenerative conditions. We have recently found that glutamate release was decreased in isolated synaptosomes from the rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. In contrast to control animals where GABA induced a decrease in the evoked glutamate release, which was abolished by picrotoxin (a GABA(A) antagonist), synaptosomes from EAE rats showed a loss in the inhibition of the glutamate release mediated by GABA with a concomitant diminution of the flunitrazepam-sensitive GABA(A) receptor density. We have presently further evaluated the relevance of the GABAergic system in EAE by treating rats challenged for the disease with the GABA agonist diazepam. Administration of diazepam during 6 days starting at day 6 or 11 after EAE active induction led to a marked decrease of the disease incidence and histological signs associated with the disease. Cellular reactivity and antibody responses against the encephalitogenic myelin basic protein were also diminished. Beyond the effects of diazepam on the autoimmune, inflammatory response, we report also a positive effect on neurotransmission. Treatment with diazepam inhibited the previously described reduction in glutamate release in the frontal cortex synaptosomes from EAE animals. These data suggest that an endogenous inhibitory GABAergic system within the immune system is involved in the diazepam effect on EAE and indicate that increasing GABAergic activity potently ameliorates EAE.

Time course of IgM antibodies which block anti-myelin basic protein IgG antibodies associated with development of experimental autoimmune encephalomyelitis in rabbits.

Several authors have demonstrated the presence in normal sera of antibodies that inhibit binding of a variety of autoantibodies. These inhibitory or blocking antibodies are generally considered to play a role in humoral self-tolerance. We examined sera from normal rabbits and from rabbits with experimental autoimmune encephalomyelitis (EAE), in search for antibodies capable to inhibit reactivity of autoantibodies directed to myelin basic protein (MBP). Rabbits injected with bovine myelin in complete Freund's adjuvant (EAE rabbits) or with adjuvant alone (control rabbits) were bled at various intervals post-injection. Sera were subjected to chomatography on a protein A-Sepharose column, retained and nonretained fractions were collected, and ability of these fractions to block reactivity of affinity-purified anti-MBP IgG-antibodies was analyzed by immunoblot technique. Protein A nonretained fraction from control rabbits inhibited anti-MBP IgG reactivity to the same degree at all intervals tested, whereas the same fraction from EAE animals showed an increase in inhibitory activity after induction of the disease. This inhibitory activity declined with the onset of clinical symptoms, and remained low in rabbits that did not recover from the disease. In contrast, the inhibitory activity remained at maximum value in EAE rabbits with spontaneous remission of clinical symptoms. We showed that the inhibitory activity is due to IgM-antibodies, and discussed the role of these antibodies in the development of EAE.

Passive transfer of experimental autoimmune encephalomyelitis in Wistar rats: dissociation of clinical symptoms and biochemical alterations.

We have used passive transfer of myelin-reactive lymphocytes in the Wistar rat model of experimental autoimmune encephalomyelitis (EAE) to investigate the nature of the central nervous system immunopathological alterations induced by these cells. Mononuclear cells from lymph nodes or spleen from sick myelin/complete Freund's adjuvant-immunized donors did not transfer clinical disease. However, depending on the previous treatment of the transferred cells, recipients develop central nervous system biochemical and histological alterations. Fresh cells from lymph nodes immediately transferred after procurement from the sick EAE donor rat were capable of inducing the most significant diminution in the content of myelin basic protein, sulfatides, and 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, with concomitant inflammatory infiltrations of white matter, principally in spinal cord and cerebellar lobules. Similar alterations were observed when animals were injected with spleen mononuclear cells activated in the presence of a nonspecific mitogen as concanavalin A. However, antigen-specific activated spleen cells generated by culturing in the presence of bovine myelin induced alterations to a lesser degree. Results point to a dissociation of the clinical disease from the central nervous system biochemical and histopathological lesions occurring in the EAE-transferred Wistar rats and indicate that these alterations in EAE are induced principally by T cells activated in vivo rather than by cells activated in vitro by myelin antigens. Therefore, these findings suggest a possible participation of lymphocytes unlike the encephalitogenic T cells in the induction of the described alterations and provide a useful model to explore further the subclinical responses to this experimental disease.

Suppression of acute experimental allergic encephalomyelitis by intraperitoneal administration of synaptosomal antigens.

The effect of a synaptosomal fraction isolated from bovine brain was examined on acute experimental allergic encephalomyelitis (EAE) in Wistar rats. Intraperitoneal administration of the animals with low doses of saline-soluble synaptosomal antigens 10 and 3 days previous to the active induction of the disease was an effective way of suppressing EAE. This treatment diminished the incidence and severity of EAE, reverted the appearance of central nervous system histological and biochemical alterations, and produced changes in the autoimmune humoral response against the encephalitogenic myelin basic protein. The phenomenon observed by treatment with synaptosomal fraction is similar to the previously described suppression mediated by myelin antigens. Taking into account that affinity-purified antibodies and T lymphocytes specific for myelin basic protein can also recognize several neuronal proteins, among them the specific synaptosomal protein synapsin I, can be suggested that antigen-driven bystander suppression could be a mechanism by which synaptosomal proteins suppress the response against myelin antigens.

Time course of biochemical and immunohistological alterations during experimental allergic encephalomyelitis.

A comprehensive biochemical, immunological and histological study was undertaken during different stages of experimental allergic encephalomyelitis (EAE). Wistar rats with EAE induced by sensitization with bovine myelin showed a maximum decrease of body weight 14-16 days post-inoculation (dpi), coincident with the appearance of the paralysis symptom (acute period). Quantitation of some brain components indicated a temporal dissociation among the alterations observed. The higher diminution of myelin basic protein (MBP) occurred at 6 dpi and then increased to reach 21 dpi, a normal value. Also, the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase was reduced by 40% with respect to control animals only at 6 dpi. The total lipid content was normal; however, among the individual lipids, sulfatides were principally degraded during the acute stage but the amount of cerebrosides was decreased during the recovery period (29-40 dpi). Free cholesterol was similar in both groups of animals, whereas cholesterol esters were detected in EAE animals from 14 to 40 dpi. Central nervous system meningeal and parenchymal infiltration with mononuclear cells was recognized principally at 14 dpi, but some of cells were still present at 40 dpi. Deposits of immunoglobulins in the infiltrated regions as well as in spinal cord motor neurons were observed among 14-29 dpi. Total circulating antibodies to MBP began to increase at 14 dpi, reaching a plateau at 21 dpi and then maintaining this value until 40 dpi. However, the population of anti-MBP antibodies that also recognizes the neuronal protein synapsin was only present at 14 dpi. The present results suggest that the neurological symptoms can be related to some early changes in the myelin membrane followed by alterations involving neuronal structures. The existence of immunological factors against some epitopes in MBP that also recognize a synaptosomal protein might account, at least in part, for the axonal damage and disruption of the normal interneuronal activity in EAE and lead together with the alterations in some specific myelin constituents and the concomitant CNS inflammatory process to the observed hindlimb paralysis.