Patricia Varela

Electroacupuncture reverses ethanol-induced locomotor sensitization and subsequent pERK expression in mice

Extracellular signal-regulated kinase (ERK) plays a role in neuronal changes induced by repeated drug exposure. Given that electroacupuncture reverses locomotor sensitization induced by ethanol, we investigated whether this effect is parallel to ERK signalling. Mice received daily ethanol (2 g/kg i.p), for 21 d. Electroacupuncture was performed daily, during four (subsequent) days of ethanol withdrawal. The stimulus of 2 Hz or 100 Hz was provided in combinations of two acupoints: Ea1 (ST-36/Zusanli and PC-6/Neiguan) or Ea2 (Du-14/Dazhui and Du-20/Baihui). The specificity of acupoint effects were assessed by the inclusion of additional groups: Ea3 (ST-25/Tianshu--acupoint used for other non-related disorders), Sham1 or Sham2 (transdermic stimulation near the respective acupoints). The control group was only handled during withdrawal and the saline group was chronically treated with saline and handled similarly to controls. At day 5 of withdrawal, each group was divided in two subgroups, according to the presence or absence of ethanol challenge. The animals were perfused and their brains processed for pERK immunohistochemistry. Only Ea1 at 100 Hz (Ea1_100) and Ea2 at 2 Hz (Ea2_2) reversed locomotor sensitization induced by ethanol. Ethanol withdrawal decreases pERK in the dorsomedial striatum. This decrease is not abolished by electroacupuncture. Conversely, ethanol challenge increases pERK in the dorsomedial striatum, infralimbic cortex and central nucleus of amygdala. The specificity of acupoint stimulation to reverse these increases was seen only for Ea2_2, in the infralimbic cortex and dorsomedial striatum. Therefore, behavioural effects of Ea2_2 (but not Ea1_100) depend, at least in part, on ERK signalling.

Electroacupuncture inhibits CB1 upregulation induced by ethanol withdrawal in mice

CB1R play a role in alcohol withdrawal and in some effects of acupuncture. Interestingly, acupuncture has been used to alleviate alcohol withdrawal. Here, we investigated electroacupuncture (EA) effects during ethanol withdrawal on CB1R immunoreactivity. Male Swiss mice were daily injected with ethanol (2g/kg, i.p) (EtOH group), for 21 days. EA was performed daily during 4 days of ethanol withdrawal. The stimuli of 2 or 100 Hz were provided in two acupoints combination: Ea1 [(ST-36/Zusanli) and (PC-6/Neiguan)] or Ea2 [(DU-14/Dazhui) and (DU-20/Baihui)]. The specificity of the acupoints were assessed by the inclusion of three additional groups, Ea3 [(ST 25/Tianshu - acupoints used to other non-related disorders)], Sham1 and Sham2 (transdermic stimulation nearly to the respective acupoints). EtOH group were only handled during withdrawal and Saline group was chronically treated with Saline and handled similarly to EtOH group. One day after withdrawal the animals were perfused and their brains processed for immunohistochemistry. There was an increase of CB1R in the prefrontal cortex, striatum, hippocampus, amygdala and ventral tegmental area. The procedures used in the 2HzEa1 and 100HzEa2 groups were the most effective and specific to inhibit this CB1R upregulation. Therefore, EA inhibits CB1R upregulation seen in ethanol withdrawn mice. The specificity of acupoints stimulation depends of the encephalic nuclei, acupoints association and frequency of stimulation.

Effects of ethanol on hippocampal neurogenesis depend on the conditioned appetitive response

Neurogenesis in the subgranular layer of the dentate gyrus (DG) has been suggested to underlie some forms of associative learning. The present study was undertaken to determine whether there was also a role of neurogenesis in the ethanol (EtOH)‐induced conditioned place preference (CPP). Outbreed Swiss mice were conditioned with EtOH (2.0u2003g/kg) in one compartment of a non‐biased place preference chamber and saline in the other compartment. This procedure produced three groups of mice: some developed a conditioned preference (EtOH_Cpp), others developed a conditioned avoidance (EtOH_Cpa) and still others demonstrated indifference to the context previously paired with ethanol (EtOH_Ind). BrdU (40u2003mg/kg, i.p.) was administered 4 hours after each session comprising the conditioning phase. When measured 24 hours following the CPP test, there was no effect of EtOH on doublecortin (DCX) expression or Fluoro Jade B staining. However, there were decreases in the number of BrdU+ and Ki‐67+ cells in the EtOH_Cpa and EtOH_Ind groups, but not in the EtOH_Cpp group. Most of BrdU+ cells were co‐labeled with DCX. Similarly, in another experiment, in that the perfusion was done 28 days after CPP test, most BrdU+ cells were co‐localized with NeuN. These results suggest that conditioned appetitive response is able to maintain normal levels of neurogenesis in DG and might counteract ethanol‐produced decreased cell proliferation/survival rate.

Targeted Next-Generation Sequencing in Brazilian Children With Nephrotic Syndrome Submitted to Renal Transplant

Background The aims of this study were to identify the genetic mutations profile in Brazilian children with nephrotic syndrome (NS) and to determine a genotype-phenotype correlation in this disease. Methods Next-generation sequencing and mutation analysis were performed on 24 genes related to NS in a cross-sectional study involving 95 children who underwent kidney transplantation due to NS, excluding congenital cases. Results A total of 149 variants were identified in 22 of 24 sequenced genes. The mutations were classified as pathogenic, likely pathogenic, likely benign and benign per the chance of causing the disease. NPHS2 was the most common mutated gene. We identified 8 (8.4%) patients with hereditary NS and 5 (5%) patients with probably genetically caused NS. COL4A3-5 variants were found as well, but it is not clear whether they should be considered isolated FSGS or simply a misdiagnosed type of the Alport spectrum. Considering the clinical results, hereditary NS patients presented a tendency to early disease onset when compared with the other groups (P = 0.06) and time to end stage renal disease (ESRD) was longer in this group (P = 0.03). No patients from hereditary NS group had NS recurrence after transplantation. Conclusions This is the first study in children with steroid-resistant NS who underwent kidney transplantation using next-generation sequencing. Considering our results, we believe this study has shed some light to the uncertainties of genotype-phenotype correlation in NS, where several genes cooperate to produce or even to modify the course of the disease.

Reconsolidation and update of morphine-associated contextual memory in mice

Drug addiction can be viewed as a pathological memory that is constantly retrieved and reconsolidated. Since drug abuse takes place in different contexts, it could be considered that reconsolidation plays a role in memory updating. There is consistent evidence supporting the role of reconsolidation in the strength and maintenance of contextual memories induced by drugs of abuse. However, this role is not well established in memory update. The purpose of the current study was to assess the reconsolidation process over memory update. C57BL6 mice were subjected to a morphine-induced, conditioned place preference (CPP) paradigm. Based on CPP results, animals were divided into distinct experimental groups, according to the contextual characteristics of the re-exposure and a second CPP Test. Re-exposure in the original context was important for memory maintenance and re-exposure under discrete contextual changes resulted in memory updating, although original memory was maintained. Interestingly, cycloheximide, an inhibitor of protein synthesis, had different outcomes in our protocol. When the re-exposure was done under discrete contextual changes, cycloheximide treatment just after re-exposure blocked memory updating, without changes in memory maintenance. When re-exposure was done under the original context, only two subsequent cycloheximide injections (3 and 6h) disrupted later CPP expression. Considering the temporal window of protein synthesis in consolidation and reconsolidation, these findings suggest that re-exposure, according to the contextual characteristics in our protocol, could trigger both phenomena. Furthermore, when new information is present on retrieval, reconsolidation plays a pivotal role in memory updating.

Novel Complex ABCA4 Alleles in Brazilian Patients With Stargardt Disease: Genotype-Phenotype Correlation

PurposenTo analyze the presence of complex alleles of the ABCA4 gene in Brazilian patients with Stargardt disease and to assess the correlation with clinical features.nnnMethodsnThis was an observational cross-sectional study. Patients with a diagnosis of Stargardt disease who presented three pathogenic variants of the ABCA4 gene or who had variants previously described as complex alleles were included. The relatives of these probands were evaluated in the segregation analysis. The patients were evaluated based on age at symptom onset and visual acuity, and the clinical characteristics were classified according to the findings observed on autofluorescence examination.nnnResultsnAmong the 47 families analyzed, approximately 30% (14/47) presented complex alleles. The segregation analysis in 14 families with cases of Stargardt disease identified three novel complex alleles and one previously described complex allele. The known complex allele p.[Leu541Pro; Ala1038Val] was identified in two families. The novel complex alleles identified were p.[Leu541Pro; Arg1443His] in five families, p.[Ser1642Arg; Val1682_Val1686del] in seven families, and p.[Pro1761Arg; Arg2106Cys] in one family. Furthermore, four new variants (p.Lys22Asn, p.Asp915Asn, p.Glu1447Val, and p.Pro1761Arg) were identified in the second allele of the ABCA4 gene.nnnConclusionsnSegregation analysis is important in order to confirm the molecular diagnosis of patients with Stargardt disease, given the frequency of complex alleles in the ABCA4 gene. The various pathogenic variation combinations observed in this study were associated with different phenotypes.

Chronic light deprivation inhibits appetitive associative learning induced by ethanol and its respective c-Fos and pCREB expression.

To address the role of mixed anxiety/mood disorder on appetitive associative learning, we verify whether previous chronic light deprivation changes ethanol-induced conditioned place preference and its respective expression of c-Fos and pCREB, markers of neuronal activity and plasticity. The experimental group was maintained in light deprivation for 24xa0h for a period of 4xa0wk. Subsequently, it was adapted to a standard light-dark cycle for 1xa0wk. As a control, some mice were maintained in standard cycle for a period of 4xa0wk (Naïve group). Then, all animals were submitted to behavioral tests to assess emotionality: elevated plus maze; open field; and forced swim. After that, they were submitted to ethanol-induced conditioned place preference. Ninety minutes after the place preference test, they were perfused, and their brains processed for c-Fos and pCREB immunohistochemistry. Light deprivation induced anxiety-like trait (elevated plus maze), despair (forced swim), and hyperlocomotion (open field), common features seen in other animal models of depression. Ethanol-induced conditioned place preference was accompanied by increases on c-Fos and pCREB in the hippocampus, prefrontal cortex and striatum. Interestingly, mice previously submitted to light deprivation did not develop either acquisition and/or expression of ethanol-induced conditioned place preference or increases in c-Fos and pCREB. Therefore, chronic light deprivation mimics several behavioral aspects of other animal models of depression. Furthermore, it could be useful to study the neurochemical mechanisms involved in the dual diagnosis. However, given its likely deleterious effects on appetitive associative memory, it should be used with caution to investigate the cognitive aspects related to the dual diagnosis.

The Challenge of Diagnosis and Indication for Treatment in Fabry Disease

Fabry disease, caused by deficient alpha-galactosidase A lysosomal enzyme activity, remains challenging to health-care professionals. Laboratory diagnosis in males is carried out by determination o...